JP2001512702A - Denaturing multi-ion polynucleotide chromatography for detecting mutations - Google Patents

Abstract

  (57) [Summary] The present invention is directed to an improved method for detecting mutations in DNA using denatured matched ion polynucleotide chromatography (DMIPC). The present invention includes the following aspects: analyzing the PCR amplification products to identify factors that affect the degree of PCR replication regeneration; designing PCR primers; selecting the optimal temperature for performing DMIPC; Column preparation and maintenance; and preparation of polynucleotide samples prior to chromatographic analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

【Technical field】

 The present invention relates to improved methods for detecting mutations in nucleic acids.

BACKGROUND OF THE INVENTION The ability to detect mutations in double-stranded standard polynucleotides, and especially in DNA fragments, is of great importance in medicine as well as in natural and social sciences. Human Genome Project is providing genetic information enormous amount of setting a new standard for evaluating the relationship between the disease mutation and human (Guyer et al., Pro C.Natl.Acad.Sci.USA 92: 10841 (1995)). The ultimate cause of the disease is described, for example, by a genetic code different from the wild type (Cotton, TIG 13:43 (1997)). Elucidating the genetic basis of the disease is the starting point for treatment. Similarly, determining differences in the genetic code can provide a powerful and possibly definitive insight into evolution and population studies (Cooper et al., Human Genetics vol. 69: 201).
(1985)). Knowledge of these and other issues regarding the genetic code is based on the ability to identify abnormalities, ie, mutations, in DNA fragments relative to wild type. Thus, there is a need for a technique for detecting mutations in an accurate, reproducible, and reliable manner.

[0003] DNA molecules are polymers comprising sub-units called deoxynucleotides. The four deoxynucleotides found in DNA comprise the customary cyclic sugar, deoxyribose, which is the four bases adenine (purine), hereinafter referred to as A, G, C and T, respectively. ), Guanine (purine), cytosine (pyrimidine) and thymine (pyrimidine).
A phosphate group links the 3'-hydroxyl of one deoxynucleotide with the 5'-hydroxyl of another deoxynucleotide to form a polymeric chain. In double-stranded DNA, the double strands are arranged in a helical structure by so-called hydrogen bonding between complementary bases. Base complementarity is determined by their chemical structure. In double-stranded DNA, each A is paired with a T, and each G is paired with a C, ie, a purine is paired with a pyrimidine. Ideally, DNA is replicated in exact copies by DNA polymerase during cell division in the human body or other organisms. DNA strands can also be replicated in vitro by the polymerase chain reaction (PCR).

[0004] Occasionally, exact replication is not possible, resulting in incorrect base pairing, which, after replication of a new strand, also results in a double-stranded strand containing a heritable difference in the base sequence from the original base sequence. This produces a derivative of DNA. Such heritable changes in the base pair sequence are called mutations.

[0005] In the present invention, double-stranded DNA is referred to as "duplex". A duplex is called a homoduplex when the base sequence of one strand is completely complementary to the base sequence of the other strand. When a duplex contains at least one non-complementary base pair, the duplex is called a heteroduplex. Heteroduplexes are formed when errors are made by the DNA polymerase enzyme and non-complementary bases are added to the replicating polynucleotide strand.
In addition, heteroduplex replication ideally produces a heterozygous homoduplex,
That is, such a homoduplex will have an altered sequence as compared to the original DNA strand. When the original DNA has a sequence that predominates in the natural population, it is commonly referred to as "wild-type."

[0006] Many different types of DNA mutations are known. Examples of DNA mutations include, but are not limited to, "point mutations" or "single base pair mutations", where incorrect base pairing occurs. The most common point mutations comprise "transitions" in which one purine or pyrimidine base is replaced by another, and "transversions" in which purines are replaced by pyrimidines (and vice versa). Point mutations also comprise mutations in which bases are added or deleted from the DNA strand. Such "insertions" or "deletions" are also known as "frameshift mutations". Although they occur less frequently than point mutations, larger mutations affecting multiple base pairs also occur and are important. A more detailed discussion of mutations can be found in US Pat. No. 5,459,039 to Modrich (1995) and US Pat. No. 5,698,400 to Cotton (1997). All of these references and the references contained therein are incorporated herein by reference.

[0007] The sequence of base pairs in DNA encodes protein production. In particular, the DNA sequence of the exon part of the DNA chain encodes the corresponding amino acid sequence in the protein. Thus, mutations in the DNA sequence can cause changes in the amino acid sequence of the protein. Such changes in the amino acid sequence may be completely benign, or inactivate the protein, or alter the function of the protein to be life-threatening or lethal. Absent. On the other hand, mutations in the intron portion of the DNA strand would not be expected to have a biological effect because the intron segment does not contain the code for protein production. Despite this, the detection of mutations in intron segments is important, for example, in forensics.

[0008] The detection of mutations is therefore of great interest and importance in diagnosing disease, elucidating the pathogenesis and developing potential treatments. Mutation detection and DNA
The identification of similarities or differences in samples can also be used to increase global food supply by developing disease resistant and / or higher yielding crop species, forensics, evolution and population studies, and it is very important in the general scientific research (Guyer et al., Pro c.Natl.Acad.Sci.USA 92: 10841 (1995 ); Cotton, TIG 13:43 (1997)). All of these references and the references contained therein are hereby incorporated by reference.

[0009] Changes in a DNA sequence that do not produce benign or bad results are often referred to as "polymorphisms." In the present invention, whether a change in the DNA sequence has or does not have a bad result is referred to as "mutation". It should be understood that the methods of the present invention have the ability to detect mutations regardless of biological effect or their loss. For simplicity, the term “mutation” is used throughout this specification to
It is used to mean a change in the base sequence of the NA chain. In the context of the present invention, the term "mutation" should be understood to include "polymorphism" or other similar or equivalent technical terms.

[0010] There is a need for an analytical method for detecting accurate and reproducible mutations that is easy to implement. There is also a strong need for such a method that is automated and that allows an operator to provide high sample screening throughput with minimal care.

[0011] The analysis of DNA samples has historically been performed using gel electrophoresis. Capillary electrophoresis was used to separate and analyze a mixture of DNA. However, these methods cannot distinguish point mutations from homoduplexes having the same base pair length.

As used herein, the term “heteroduplex site separation temperature” refers to the temperature at which one or more base pairs are denatured, ie, separated, at a site of base pair mismatch in a heteroduplex DNA fragment. means. Since at least one base pair is not complementary in a heteroduplex,
Separation of bases at that site requires less energy than a completely complementary base pair analog in a homoduplex. This results in a lower melting temperature of the heteroduplex compared to the homoduplex. Partial denaturation generally results in so-called "bubbles" at sites of base pair mismatch. These bubbles distort the structure of the DNA fragment as compared to a perfectly complementary homoduplex of the same base pair length. This structural distortion under partially denaturing conditions has previously been used to separate heteroduplexes and homoduplexes by denaturing gel electrophoresis and denaturing capillary electrophoresis. However, such techniques are difficult to operate and require highly skilled technicians. In addition, the analysis is long-running and very time-consuming to prepare. Denaturing capillary gel electrophoresis analysis of 90 base pair fragments takes more than 30 minutes and denaturing gel electrophoresis analysis takes more than 5 hours. The long analysis times of the gel technique are further exacerbated by the fact that the movement of DNA fragments in the gel is inversely proportional to the length of the fragments.

In addition to the disadvantages of the denaturing gel method described above, these techniques are not always reproducible or even accurate because the gel preparation and single run run vary from operator to operator. Absent.

Recently, matched ion polynucleotide chromatography (Matched Ion Po
A chromatographic method called lynucleotide Chromatography (MIPC) has been introduced to effectively separate mixtures of double-stranded polynucleotides, generally, and especially DNA, where the separation is based on base pair length (Bonn ( U.S. Pat.
236 Pat; Huber et al., Chromatographia 37: 653 (1993) ; Huber et al., Anal.Bioche m .212: 351 (1993 )). All of these references and the references contained therein are incorporated herein by reference. MIPC is not limited by any disadvantages associated with gel-based separation methods.

“Matched ion polynucleotide chromatography” as used herein
The term refers to a method for separating single and double stranded polynucleotides using a non-polar separation medium, wherein the method releases the polynucleotides from the counter-ionic agent and the separation medium. Use an organic solvent to perform MIPC
Separation is completed in less than 10 minutes, and often in less than 5 minutes. The MIPC system (WAVE ™ DNA fragment analysis system, Transgenomic, Inc., San Jose, CA) has a computer controlled oven surrounding the column and column inlet area.

[0016] Using and understanding the developed MIPC, the MIPC has a partial denaturation temperature,
Performed at a temperature sufficient to denature the heteroduplex at the mismatched site of the base pair
It has been shown that homoduplexes can be separated from heteroduplexes with the same base pair length.
(Hayward-Lester et al.Genome Research 5: 494 (1995); Underhill et al., Proc.Natl.Acad.Sci.USA 93: 193 (1996); Doris et al.DHPLC Workshop, Stanfo
University of Ward, (1997)). All of these references and the references contained therein,
The specification is incorporated herein by reference. Thus, the use of DHPLC has been applied to mutation detection (Underhill et al.,Genome Research 7: 996 (1997); Liu et al.Nucleic Aci ds Res ., 26: 1396 (1988)).

DHPLC can separate heteroduplexes that differ by as little as one base pair. However, the separation of homoduplexes and heteroduplexes cannot be fully resolved. Artifacts and impurities may interfere with the interpretation of the DHPLC separation chromatogram in the sense that it may be difficult to distinguish between putative mutations as artifacts or impurities (Underhill et al., Genome Res . 7: 996. (1997)). The presence of the mutation may be completely overlooked (Liu et al., Nucleic Acids Res. 26: 1396 (1988)
). All references cited and the references contained therein are hereby incorporated by reference.

The accuracy and reproducibility of DHPLC-based mutation detection assays have been compromised for two main reasons: system-related problems with DHPLC and P
Issues related to CR.

When used under partially denaturing conditions, MIPC is defined herein as denatured matched ion polynucleotide chromatography (DMIPC).

Samples to be analyzed for the presence or absence of a mutation often contain too little material to be detected. Therefore, the first step in the mutation detection assay is
Amplification of a sample using the PCR method. PCR amplification comprises steps such as selection of primer design, DNA polymerase enzyme, number of amplification cycles and reagent concentration.
Each of these steps, as well as other steps involved in the PCR method, will affect the purity of the amplified product. Factors that affect the degree of PCR and the reproducibility of replication and product purity are well known in the field of PCR, but have been addressed in the context of detecting mutations using MIPC. Never before. As a result, PCR-induced mutations, where non-complementary bases are added to the template, are often formed during sample amplification. Such PCR-induced mutations obscure the results of mutation detection because it is not clear whether the detected mutation was present in the sample or formed during the PCR procedure. I do. Unfortunately, many people in the field of PCR and mutation detection make the wrong assumption that PCR replication is perfect or near-perfect, and in PCR mutation analysis, Generally not considered. This approach produces false positives. Applicants have emphasized the importance of optimizing PCR sample amplification to minimize PCR-induced mutation formation and to ensure accurate and unambiguous analysis of samples containing putative mutations. Recognized. The use of MIPC is being reviewed by the applicant in the detailed description to identify and optimize factors that affect the degree of reproduction of PCR replication.

[0021] Other aspects of mutation detection by MIPC, which have not been addressed hitherto, are the treatment of chromatographic components and the materials comprising them, and the treatment of separation media and the materials comprising them, the methods of the methods. Includes solvent pre-selection to minimize development time, optimal temperature preselection to perform partial denaturation of the heteroduplex in MIPC, and automated high-throughput mutation detection screening assays Become. These factors are essential to achieve clear, accurate and reproducible mutation detection using MIPC.

It is necessary to identify and optimize all aspects of the MIPC method in order to minimize artifacts in the sample suspected of containing the mutation and remove ambiguity from the analysis.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to be able to obtain accurate, ie, not incorrect, results (eg, “false positives”), convenient to use, and quick to obtain results, It is to provide a method for detecting mutations in nucleic acids that is reliable, simple, convenient and inexpensive to operate.

Another object of the present invention is to provide a method for detecting mutations using chromatographic methods to separate polynucleotides with improved and predictable separation and efficiency.

It is a further object of the present invention that the nucleic acid (eg, DNA or RN
It is to provide an improved sample preparation method of A).

It is yet another object of the present invention to provide a method for optimizing PCR for use in detecting mutations.

Yet another object of the present invention is to provide an improved method of selecting temperature for performing a chromatographic separation of nucleic acids to detect mutations.

[0028] It is a further object of the present invention to provide an improved method for determining the optimal mobile phase for eluting nucleic acids in mutation screening.

Yet another object of the present invention is to provide a method that can be automated.

A further object of the present invention is to provide a method that can be used for basic investigations to test for unknown mutations and that can be used to rapidly screen large numbers of samples for known mutations. To provide.

[0031] These and other objects will become apparent from the following specification made in accordance with the present invention.

In one aspect, the invention is an improved method for separating a sample mixture of polynucleotides by matched ion polynucleotide chromatography, wherein the polynucleotides (eg, double-stranded DNA) in the sample mixture are separated. Is below the determined threshold concentration (eg, the lower limit of detection of polynucleotides). This improvement includes adding the sample to the column, which causes the polynucleotide to accumulate on the column. In a preferred embodiment, the method comprises adding the sample in a mobile phase having an organic solvent concentration below that required to elute the polynucleotide in the mixture. The mobile phase preferably also contains a counter-ionic agent. In a particular embodiment, the method further comprises adding the mixture to a matched ion polynucleotide chromatography column and flowing the column under isocratic conditions with an aqueous mobile phase, where impurities are removed from the mixture. If the sample mixture is added to the column in an aliquot greater than 10 μL, the solvent mixture preferably includes a counter-ionic agent.

In an important aspect, the invention is a method for preparing a double-stranded DNA fragment for the detection of a mutation and also for detecting a mutation in a double-stranded DNA fragment, wherein each method comprises the steps of: Use nucleotide chromatography (DMIPC). DMIPC is M
IPC, but performed at a temperature that causes denaturation at any mutation site (ie, a base pair mismatch site) without denaturing another portion of the sample sequence. For each of these methods, the double-stranded DNA fragment is a wild-type double-stranded DN having a known nucleotide sequence.
Corresponds to fragment A. The steps of this method include: (a) analyzing the sequence of the wild-type double-stranded DNA fragment to convert the double-stranded DNA fragment to a sample sequence of nucleotides having a melting point range of less than about 15 ° C. (eg, a constant melting domain) ), Each sample sequence has first and second ends at opposite positions; (b) such a sequence is obtained by PCR using a set of primers adjacent to the first and second ends of the sample sequence. Amplifying one of the sample sequences; and (c) analyzing the amplified sample by MIPC. PCR amplification, dGT
Analogs of P can be included, for example, 2,6-aminopurine, and can include a GC clamp of up to 40 bases in the primer. In a preferred embodiment, a mixture of the amplified sample sequence and the corresponding wild-type double-stranded DNA segment is subjected to a hybridization procedure, wherein the mixture is heated to a temperature at which the strands are completely denatured, and Cool until annealing, which forms a mixture comprising two homoduplexes and two heteroduplexes if the sample sequence contains a mutation.

A double-stranded DNA fragment is prepared for detection of a mutation by denaturing matched-ion polynucleotide chromatography in which the double-stranded DNA fragment corresponds to a wild-type double-stranded DNA fragment having a known nucleotide sequence. In another embodiment, the method comprises analyzing the sequence of a wild-type double-stranded DNA fragment to convert the double-stranded DNA fragment into nucleotides having a high melting domain and a low melting domain in which the mutation site is located. Dividing into sample sequences: and amplifying one of the sample sequences by PCR using a set of primers flanking the first and second ends of the sample sequence.

A double-stranded DNA fragment is prepared for detection of a mutation by denaturing matched-ion polynucleotide chromatography in which the double-stranded DNA fragment corresponds to a wild-type double-stranded DNA fragment having a known nucleotide sequence. In yet another aspect for
This method analyzes a wild-type double-stranded DNA fragment and adds the double-stranded DNA to a sample sequence of nucleotides whose mutation site is within 25 percent of the total number of bases from the end of the fragment.
And amplifying one of the sample sequences by PCR using a set of primers flanking the first and second ends of the sample sequence.

In another aspect, the present invention relates to a method for determining whether a PCR method induces a mutation.
Provides an evaluation method for the law. This method includes: (a) amplifying the polynucleotide by performing a plurality of PCR cycles to obtain a PCR amplification product;
Analyzing the R amplification product by MIPC to obtain a PCR amplification product profile, including the profile of any mutations created by the PCR generated mutations. An example of such a profile is an elution profile obtained from a denatured matched ion polynucleotide chromatography method. In a preferred embodiment, the product profile is compared to a reference profile to determine the presence of a PCR-induced mutation in the PCR amplification product. In a related aspect, the invention is a method for identifying a bias in a PCR method from a predetermined reference profile. The steps of this method include amplifying the polynucleotide by performing multiple PCR cycles to obtain a PCR amplification product, and analyzing the PCR amplification product by MIPC to obtain any PC
A PCR amplification product profile is obtained, including the profile of the R-induced mutation. The profile of the PCR amplification product can be compared to a reference profile to identify biases in the PCR reaction product, including PCR-induced mutations, from the predetermined reference profile. In a preferred embodiment, PCR-induced mutations are detected by hybridizing the reaction after the last cycle and analyzing the reaction by MIPC.

In an important aspect, the invention is a method of reducing PCR-induced mutations, comprising: (a) amplifying a polynucleotide by performing multiple PCR amplification cycles to produce a first PCR amplification product; (B) analyzing the first PCR amplification product by MIPC to obtain a PCR amplification product profile, (c) comparing the PCR amplification product profile to a reference profile to determine the presence of a PCR-induced mutation, and (D) amplifying the polynucleotide by performing a plurality of PCR amplification cycles with adjusting one or more method variables to form a second PCR amplification product having reduced PCR-induced mutations.

The method comprises analyzing the PCR reaction product obtained in step (d) by MIPC to obtain a second reaction product profile, followed by (f) combining the second reaction product profile with a set of standards. Determining the bias of the PCR method from the predetermined standard in comparison with the predetermined profile; and (g) performing multiple PCR cycles by adjusting one or more method variables to determine the bias from the predetermined standard. An additional step of forming a third PCR product with reduced PCR bias. Examples of method variables include magnesium concentration, dNTP concentration, enzyme concentration, temperature and source of DNA polymerase. For example, a non-proofreading DNA polymerase can be replaced with a proofreading polymerase. Analysis of PCR products can be used to evaluate primers and redesign primers to minimize artifacts such as primer dimer formation.

Evaluation of the PCR method by MIPC can also be used to increase product yields and minimize by-products. The PCR product profile is compared to a predefined standard profile. PCR is repeated with increasing concentrations of one or more nucleotides, magnesium ions or enzymes, or decreasing temperature, or a combination thereof. Further improvements in PCR can be made by reducing the number of PCR cycles when excessive levels of by-products are observed.

Deviations from the predefined standard profile are due to the MIPC
Can be further reduced by analyzing the second reaction product profile of the PCR reaction obtained using The second profile is compared to a set of standard profiles to determine the bias of the PCR method from predetermined standards. Then another set of PCR
The cycle is performed adjusting one or more method variables to obtain a third PCR reaction product profile with reduced bias from predetermined standards in the PCR product.

In another preferred embodiment of this aspect of the invention, PCR products can be separated from reaction impurities and collected during MIPC analysis of the reaction. In this manner, the purified PCR product can be amplified in another series of PCR cycles. Purified PCR products can also be amplified by cloning in a host system.

In yet another important aspect, the invention provides a method for determining a heteromutant site separation temperature. The method comprises the steps of (a) heating a mixture of a sample double-stranded DNA segment and a corresponding wild-type double-stranded DNA segment to a temperature at which the strands are completely denatured; Cooling until the strands have completely annealed, thereby forming a mixture comprising two homoduplexes and two heteroduplexes if the sample segment contains a mutation; (c) the heteromutant Determining the site separation temperature; (d) analyzing the product of step (b) at the heteromutant site separation temperature using MIPC to identify the presence of the component in which the heteromutant site was separated. Comprising the steps of: In one embodiment, given the sequence of the normal double-stranded DNA, the heteromutant site separation temperature is determined by the following equation: T
(hsst) = X + m · T (w), where T (hsst) is the heteromutant site separation temperature and T (w) is the temperature calculated by software or determined experimentally. At this temperature, a selected equilibrium between the denatured and non-denatured state of normal double-stranded DNA (eg, a 50/50 or 25/75 denaturation to non-denaturation ratio) is selected, where m is And X is the DMIPC detector. In a related embodiment, the heteromutant site isolation, temperature, as described above, is performed by subjecting the product of step (b) to a series of incremental MIPC by MIPC.
Separation is determined by analyzing in the mutation segregation temperature range, with each successive segregation either observing a mutation segregation profile or observing the absence of a mutation segregation profile in the mutation segregation temperature range. Have a higher temperature than the previous separation, where the mutation separation profile identifies the presence of the mutation, and the absence of the mutation separation profile indicates the absence of the mutation in the sample. Similarly, the heteromutant site separation temperature can be determined by performing a series of incremental MIPC separations over the mutation separation temperature range, with a mutation separation profile observed or suddenly Has a lower temperature than the previous segregation until the absence of a mutation segregation profile in the mutation segregation temperature range, where the mutation segregation profile identifies the presence of a mutation and Absence indicates that no mutation is present in the sample. In a preferred embodiment, the determination of T (hsst) by MIPC is computer controlled and automated, even if a series of MIPC separations are performed at incrementally higher or incrementally lower temperatures.

In a further aspect of the present invention, there is provided a preferred method for detecting a gene mutation in DNA, comprising: (a) a calculating step for obtaining a calculated heteromutant site separation temperature; (B) a prediction step to obtain an expected heteromutant site separation temperature; (c) a mixture of the sample double-stranded DNA segment and the corresponding wild-type double-stranded DNA segment Heating to the mutant site isolation temperature; (d) analyzing the product of step (c) using MIPC at the expected heteromutant site isolation temperature where the heteromutant site was isolated Identifying the presence of the component. In a preferred embodiment, the calculating step comprises calculating a calculated heteromutant site separation temperature according to a first mathematical model. In a further preferred embodiment, the predicting step comprises adjusting the heteromutant site separation temperature calculated according to the second mathematical model. This second mathematical model can be based on a comparison between the empirically determined heteromutant site separation temperature and the calculated heteromutant site separation temperature. The calculated heteromutant site separation temperature can be calculated using a first mathematical model. In a preferred embodiment, the determination of T (hsst) by MIPC is computer controlled and automated.

In another important aspect of the present invention, a mixture of heteroduplex and homoduplex DNA molecules, including first and last eluting DNA molecules, is used in a heteroduplex DNA molecule. Provides a chromatographic method for separating under mutating conditions that selectively denatures the mutation site present in: (a) adding the mixture to a matched ion polynucleotide chromatography column; Eluting the molecules of the mixture using a mobile phase comprising a concentration range of an ionic reagent and a pre-selected fragment in an organic solvent, the range comprising the initial concentration of the organic solvent and The final concentration comprises up to the amount required to elute the first eluting DNA molecule in the mixture, and the final concentration Finally elute the DNA molecule in the mixture comprises an organic solvent in a concentration sufficient to elute.

In a preferred embodiment, the range of batches of preselected fragments is obtained from references regarding the organic solvent concentration required to elute DNA molecules of various base pair lengths and base pair lengths. In certain embodiments, a reserve organic solvent concentration capable of eluting a specific base pair length of DNA molecule is referred to as a reference to the relevant organic solvent concentration required to elute various base pair lengths and base pair length DNA molecules. From and use preliminary solvent concentrations to select the area to bracket the fragments. Heteroduplex and homoduplex molecules can be the same base pair length. A heteroduplex molecule can be composed of at least two different heteroduplexes, and a homoduplex molecule can be at least two different homoduplexes. After elution from the column, such molecules are detected (eg, by UV absorption). The organic solvent used in this aspect of the invention is selected from the group consisting of methanol, ethanol, acetonitrile, ethyl acetate and 2-propanol. A preferred organic solvent is acetonitrile. In this aspect of the invention, the counter ionic agent is selected from the group consisting of lower alkyl primary, secondary and tertiary amines, lower trialkyl ammonium salts and lower quaternary ammonium salts. Examples of counter ionic agents include octyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate, pyridinium ammonium acetate, cyclohexylammonium acetate,
Diethylammonium acetate, propylethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate,
Tetramethylammonium acetate, tetraethylammonium acetate,
Tetrapropylammonium acetate, tetrabutylammonium acetate, dimethyldiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, carbonate, phosphate, sulfate, nitrate, Propionate, formate, chloride, bromide,
And mixtures of one or more of the foregoing. However, the most preferred counter-ionic agent is triethylammonium acetate.

In a related aspect, immediately prior to step (a) above: (a) D of various base pair lengths
Obtain the relationship between the organic solvent concentrations in the mobile phase required to elute the NA molecule from the column as a function of base length, and (b) batch up the preselected fragments of the organic solvent from this obtained relationship And a preliminary step of determining a preliminary organic solvent concentration.

An important aspect of the present invention is a method for treating a matched ion polynucleotide chromatography column for improving the resolution of a double-stranded DNA fragment separated by the column, which method uses a polyvalent cation binding agent. Flowing the containing solution through a column, wherein the solution has a temperature of about 50C to 90C. A suitable temperature is about 70
C. to 80C. In a preferred embodiment, the multivalent cation binding reagent is a coordination compound,
Examples include water-soluble chelators and crown ethers. Specific examples include acetylacetone, alizarin, aluminone, chloranilic acid, kojic acid, morin, rhodizonic acid, thionalide, thiourea, α-furyldioxime, nioxime, salicylaldoxime, dimethylglyoxime, α-furyldioxime, Cupron, α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN, SPADNS, glyoxal-bis (2-hydroxanyl), murexide, α-benzoinoxime, Mandelic acid, anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, thionalide, triethylenetetraamine, EDTA, metalphthalein, arsonic acid, α, α'-bipyridine, 4-hydroxybenzothiazole, β-hydroxyquinaldine, β -Hydroxyki Norin, 1,10-phenanthroline, picolinic acid, quinaldiic acid, α, α ', α''-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol, rhodizonic acid, salicylald Includes xime, salicylic acid, thyrone, 4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid, sodium diethyldithiocarbamate and zinc dibenzyldithiocarbamate. However, the most preferred chelating agent is EDTA. In this aspect of the invention, the solution preferably contains an organic solvent such as exemplified by alcohols, nitriles, dimethylformamide, tetrahydrofuran, esters and ethers. The most preferred organic solvent is acetonitrile. In one aspect, the solution can include a lower primary, secondary and tertiary amine, and a counter ionic agent such as a lower trialkyl ammonium salt or a quaternary ammonium salt.

More specifically, the counter ionic agent is octyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate, pyridinium ammonium acetate, cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, butyl ethyl ammonium acetate, methyl hexyl ammonium Acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethyldiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethyl It can be ammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, bromide and mixtures of any one or more of the foregoing. However, the most preferred counter-ionic agent is triethylammonium acetate.

In a further aspect, the present invention provides a method for storing a matched ion polynucleotide chromatography column to improve the resolution of double-stranded DNA fragments separated on the column. A preferred method involves flowing the solution containing the multivalent cation binding agent through the column before storage of the column. In a preferred embodiment, the multivalent cation binder is a coordination compound, examples of which include water-soluble chelators and crown ethers. Specific examples include acetylacetone, alizarin, aluminone, chloranilic acid, kojic acid, morin, rhodizonic acid, thionalide, thiourea, α-furildioxime, nioxime, salicylaldoxime, dimethylglyoxime, α-furyldioxime, cupperon, α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN,
SPADNS, glyoxal-bis (2-hydroxanyl), murexide, α-benzoin oxime, mandelic acid, anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, thionalide, triethylenetetraamine, EDTA, metalphthalein, arsonic acid, α, α'-bipyridine, 4-hydroxybenzothiazole, β-hydroxyquinaldine, β-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldiic acid, α, α ', α''-terpyridyl, 9-methyl-2 , 3,7-Trihydroxy-6-fluorone, pyrocatechol, rhodizonic acid, salicylaldoxime, salicylic acid, thyrone, 4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid , Sodium diethyldithiocarbamate and dibenzyldithiocarbamate Including the over door zinc. However, the most preferred chelating agent is EDTA. In this aspect of the invention, the solution preferably contains an organic solvent such as exemplified by alcohols, nitriles, dimethylformamide, tetrahydrofuran, esters and ethers. The most preferred organic solvent is acetonitrile. In one aspect, the solution can include a lower primary, secondary and tertiary amine, and a counter ionic agent such as a lower trialkyl ammonium salt or a quaternary ammonium salt. More specifically, the counter ion reagent is octyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate, pyridinium ammonium acetate, cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, butyl ethyl ammonium acetate, methyl hexyl ammonium acetate, tetra Methylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate,
Tetrabutylammonium acetate, dimethyldiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate,
Tetrapropylammonium acetate, tetrabutylammonium acetate, carbonate, phosphate, sulfate, nitrate, propionate,
It can be formate, chloride, bromide and a mixture of any one or more of the above. However, the most preferred counter ion reagent is triethylammonium acetate.

DETAILED DESCRIPTION OF THE INVENTION In its most general form, the subject of the present invention is to use MIPC to produce a mixture of predominantly homoduplex and heteroduplex DNA fragments having the same base pair (bp) length. And improved methods for separation. Such separations are performed under partial denaturing conditions, i.e., at elevated temperatures sufficient to denature the heteroduplex with bp mismatches, and this separation method is referred to herein as denaturing matched ion polynucleotide chromatography. (DMIP
Call it C).

A separation method called “denaturing HPLC (DHPLC)” uses heteroduplexes with the same bp length (
(Resulting from the presence of mutations) and homoduplexes have been used to detect mutations. This separation is based on the fact that heteroduplexes have a lower melting temperature (Tm) than homoduplexes. When DHPLC is performed at a partial denaturation temperature (ie, a temperature sufficient to denature the heteroduplex at the site of the base pair mismatch), the homoduplex has a heteroduplex having the same base pair length. can be separated from (Hayward-Lester, et al., Genome Research, 5: 494 ( 1995); Underhill et al., Pr oc.Natl.Acad.Sci.USA 93: 193 (1996 ); Doris , et al., DHPLC Workshop, Sutanfo chromatography De University, (1997)). All of these references and references cited therein are hereby incorporated by reference. Thus, the use of DHPLC has been applied to mutation detection (Underhill et al., Genome Research 7: 966 (1977); Liu et al., Nucleic Acids Res ., 26: 1396 (1998)).

DHPLC can separate heteroduplexes that differ by as little as one base pair under certain conditions. However, the separation of homoduplex and heteroduplex cannot be separated well. Artifacts and impurities also interfere with the interpretation of DHPLC separation chromatograms in the sense that it may be difficult to distinguish between putative artifacts or impurities (Underhill et al., Genome Research 7: 966 (1
977)). For these and other reasons that will soon become apparent, the presence of the mutation may be completely overlooked (Liu et al., Nucleic Acids Res ., 26: 1396 (1998)). For example, if the mutation is located in the high melting domain of the DNA fragment, it may not be possible to detect the mutation using known techniques. All references cited above and the references cited therein are hereby incorporated by reference.

Applicants have discovered that when chromatography is carried out at a non-denaturing temperature, typically below 50 ° C., a matched ionic polysaccharide capable of separating DNA fragments comprising 10-500 base pairs based on fragment size. A method called nucleotide chromatography (MIPC) has been found. "Matched ion polynucleotide chromatography"
The term is defined herein as a method of separating single- and double-stranded polynucleotides using a non-polar separation medium, wherein the method comprises separating the counter-ionic agent and the polynucleotide from the separation medium. Use the organic solvent that releases. When MIPC is carried out at a partial denaturation temperature (ie, a temperature sufficient to denature the heteroduplex at the site of base pair mismatch), the method is referred to herein as "denaturing matched ion polynucleotide chromatography (denaturing). DMIPC) ". DMIPC can be used to detect mutations that differ by only one base pair from the wild type. Applicants have found many aspects of the previously unrecognized analysis of mutations by MIPC, and by handling them distinguished their mutation detection methods from DHPLC.

MIPC is a silica medium having a non-polar surface comprising an organic polymer, a coated or covalently bonded organic polymer or a covalently bonded alkyl and / or aryl group, a continuous non-polar separation medium, Using a unique non-polar separation medium comprising a so-called monolith or rod column comprising polar silica gel and an organic polymer. The separation medium used for MIPC can be porous or non-porous. A detailed description of MIPC separation methods, MIPC separation media and MIPC systems is provided in US Pat. No. 09 / 058,337 filed on Apr. 10, 1998: No. 09 / 065,913 filed on Apr. 24, 1998: 199
No. 09 / 081,040 filed on May 18, 1998: No. 09/08 filed on May 18, 1998
1,039: and 09 / 080,547, filed May 18, 1998. MIPC systems and separation media are commercially available (Transgenomic,
San Jose, California).

The quality of MIPC separation of DNA fragments is very sensitive to the presence of polyvalent cations throughout the solvent and sample flow path. Therefore, the MIPC separation media is washed with acid before column packing. Further, the newly packed column is washed with a 0.1 M EDTA solution at least 50 ° C., and preferably at least 70 ° C., to remove traces of polyvalent ions from the separation medium and the interior of the column. The columns and solution contact surfaces of the MIPC system comprise materials that do not release polyvalent cations, such as coated stainless steel, titanium, polyetheretherketone (PEEK), or any combination thereof. To ensure longer column life and effective separation even after multiple uses, the column and sample should have a resin that captures polyvalent cations between the solvent reservoir and the column and / or inlet. Placing guard cartridges that include in-line provides additional protection from accidental multivalent cations.

Surprisingly, when using MIPC for the detection of mutations, the Applicant has found that this method is more sensitive to the purity of the separation medium, the presence of trace levels of polyvalent cations and other separation parameters. Was found. Columns that work well with MIPC cannot be operated with DMIPC without further cleaning; this cleaning method involves flushing with organic solvents and / or chelating agents to remove contaminants. The requirements for preventing multivalent cation contamination are even more stringent than for using DMIPC to detect mutations.

Samples analyzed for the presence or absence of a mutation often contain undetectable amounts of material that are too small. A first step useful in mutation detection assays is therefore amplification of a sample using PCR methods. PCR amplification involves steps such as selection of primer design, DNA polymerase enzyme, number of amplification cycles and reagent concentration.
Each of these steps, as well as other steps involved in the PCR method, affects the purity of the amplification product. Factors and product purity that affect PCR and replication reproducibility are well known in PCR technology, but these factors have been addressed in the past in connection with the detection of mutations using MIPC. It never happened. As a result, PCR-induced mutations (where non-complementary bases are added to the template) are often formed during sample amplification. Such PCR-induced mutations obscure the detection of mutations because it is not clear whether the detected mutation was present in the sample or generated during PCR. Unfortunately, many people in the field of PCR and mutation detection find that PCR replication has an inherently “perfect” degree of reproduction, and that PCR-induced mutations are generally They make the wrong assumption that they are not taken into account in mutation detection analysis. This method produces false positives. Applicants have recognized that optimization of PCR sample amplification is important to minimize PCR-induced mutations and ensure accurate and unambiguous analysis of samples containing putative mutations. Applicants to identify and optimize factors affecting the reproducibility of PCR replication
Consider the use of MIPC below.

Other aspects of MIPC mutation detection that have not been addressed previously include improved processing of materials comprising the components of a chromatography system, improved processing of separation media, and development time of the method. Using a solvent preselection method to minimize DNA, an optimal temperature pre-selection method for performing partial denaturation of the heteroduplex in DMIPC, and an automated high-throughput mutation detection screening assay Comprising optimization for rapid DMIPC analysis. Another important finding by the Applicant takes advantage of MIPC's unique mechanism of concentrating polynucleotides in a sample by multiple loadings on a MIPC column. This new method avoids the need to concentrate the sample by solvent evaporation, which may cause sample degradation or introduction of contaminants.

Applicants have therefore devised a new and straightforward protocol that addresses the above-mentioned problems of the prior art. This protocol comprises all steps necessary to ensure the accuracy, reproducibility and speed of mutation detection using MIPC. A straightforward approach to detecting such mutations using MIPC has not been described before. For clarity, various aspects of the protocols of the present invention are set forth in their respective headings. Optimal embodiments of the present invention include the implementation of all aspects described herein to achieve clear, accurate and reproducible mutation detection results using MIPC.

The components are divided into the following sections: sample preparation; primer design; PCR optimization; temperature selection; mobile phase selection; column preparation and maintenance.

The analysis of polynucleotides is often undetectable because the concentration of the polynucleotide is too low or the sample volume is too large to be disturbed by diluted samples.
The sample is subjected to a volume reduction step until the polynucleotide concentration is sufficient to be detected. One example is to evaporate the solution with or without heating. Alternatively, the sample can be treated with a precipitant, for example, ethanol, acetonitrile or other organic solvents. They also include ion exchangers (eg, commercially available from Qiagen, Valencia, Calif.), Silica gel (eg, sold by CPG, Lincoln Park, NJ), and polymers (eg, Hamilton: (Sold by Hamilton, Inc., Reno, Nevada). Such methods for concentrating a sample are inconvenient and time consuming, and subject the sample to possible inaccurate collection, contamination, degradation or accidental loss.

Therefore, a rapid sample concentration method is needed. Applicants have surprisingly found that in matched ion polynucleotide chromatography (MIPC) the polynucleotide binds to the solid phase and that the organic solvent concentration in the mobile phase is sufficient to release fragments of the corresponding base pair length. When, all are released in a strong band at once, in order of base pair length only. The term "release" is not what is commonly used in liquid chromatography. This term means that the conditions under which the DNA binds to the separation medium and is completely dissolved in the mobile phase are (1) sufficiently limited and (2) small in terms of the conditions required to separate different fragment lengths. Used for MIPC because it has differences.
For example, the change in the concentration of bulk acetonitrile from complete adsorption of a 102 bp DNA fragment to a separation medium to complete desorption is less than 2%. Larger fragments require a larger range, but for the separation of DNA in the 100-600 bp range, the entire range of acetonitrile change is 7.5% acetonitrile, which can be achieved with a 5 minute gradient.

While not wishing to be bound by any theory, it is important that D
It is believed that NA is emitted from the top of the column. This release is gradual,
As the concentration of acetonitrile is increased during the gradient elution step, the fragments will move faster until they move at the linear velocity of the mobile phase. Based on experiments comparing 1 cm and 5 cm long columns, the release method using the conditions reported in these examples has a release length of 1 cm or less. That is, fragment separation is mostly based on 20% of the top of the 5 cm column, and especially on the thinner section of the top of the column bed. This means that in order to perform high-resolution separation, the completeness or uniformity of the column bed vertex is more important than the column length. When changing elution conditions to small gradients or single separation conditions, it can be said that the length of the column is important.

As used herein, “matched ion polynucleotide chromatography”
The term is defined as a method of separating single and double stranded polynucleotides using a non-polar separation medium, wherein the method comprises releasing a polynucleotide from the counter ionic agent and the separation medium. Use an organic solvent. MIPC separates polynucleotides based on size under non-denaturing conditions, ie, at temperatures below about 50 ° C. As used herein, the concentration of organic solvent in the mobile phase sufficient to elute a polynucleotide of known base pair length can be predetermined from references relating to organic solvent concentration and base pair length. it can. For a selected concentration of organic solvent in the mobile phase, only one base length of polynucleotide (and all short polynucleotides) will elute in a strong band from the column. As an explanation, 100bp and 400
When a polynucleotide mixture containing bp fragments is added to a MIPC column, the column uses a mobile phase containing an organic solvent concentration sufficient to elute the 100 bp fragment, but not enough to elute the 400 bp fragment. When eluting, the latter remains at the top of the column, even after passing a large volume of mobile phase or multiple injections through the column. The result is that the amount of organic solvent in the mobile phase sufficient to elute one mixture component generally elutes partially the other desired mixture component, and the slowly acting customary reverse Very different from phase chromatography. In such cases, the later eluting components will generally be broad and poorly defined peaks. In contrast, mixed components that later elute in MIPC immediately elute as sharp peaks when sufficient organic solvent concentration is added to the mobile phase.

In a main aspect of the present invention, Applicants advantageously use the properties of this MIPC method to circumvent conventional processing of highly diluted samples prior to chromatographic analysis. The present invention is an improved method for separating a sample mixture of polynucleotides by MIPC, wherein the polynucleotide concentration in the sample mixture is contained in a large volume where the sample concentration is less than the determined threshold concentration. . An example of such a threshold is the detection limit of a UV absorption signal that is below the background signal. A specific example is about 0.3ng
Injection of 3 μL containing less than DNA.

The improvement comprises adding the sample to the MIPC column in more than one aliquot or a larger aliquot (eg, greater than about 20 μL), whereby the sample accumulates and is concentrated on the column. A polynucleotide sample, typically a double-stranded DNA, is added in a solvent or mobile phase having a concentration of organic components lower than that required to elute the polynucleotide from the MIPC column. Polynucleotides added to the column from multiple aliquots simply accumulate and concentrate at the top of the column because the organic solvent concentration in the mobile phase is not sufficient to elute the polynucleotide. This improvement avoids the need to concentrate the sample by evaporation and thus eliminates steps where the sample can be degraded. This is extremely important since eliminating steps that can degrade the sample also eliminates sources of analytical ambiguity.

For very large injection volumes (ie, greater than about 20 μL), use a counter ionic agent.
It may be necessary to add (eg, TEAA) to the sample before injection.

In a preferred embodiment, multiple sample aliquots are automatically added to a MIPC column by a sample injector. In another preferred embodiment, a large sample (eg, greater than about 20 μL) is injected and pre-concentrated on the column. The sample contains a counter-ionic agent, such as TEAA, that facilitates binding of the sample to the column.

In another aspect of the invention, when multiple aliquots of the sample are added, the column contains a fixed concentration of organic solvent that is insufficient for the aqueous mobile phase to elute the polynucleotide of interest. It can be subjected to a washing step under a single condition. This step is
Wash out impurities such as salts, nucleotide bases, buffers and other debris,
The polynucleotide sample is left in a concentrated band at the top of the column.

The mobile phase is preferably a counter ionic agent and acetonitrile, ethanol,
Comprising an organic solvent selected from the group consisting of methanol, 2-propanol and ethyl acetate. The preferred organic solvent in the mobile phase is acetonitrile. The concentration of acetonitrile in a single mobile phase is preferably greater than 2%.

The counter ionic agent in the mobile phase is selected from the group consisting of lower alkyl primary, secondary and tertiary amines, lower trialkyl ammonium salts, and lower quaternary ammonium salts. A preferred counter ionic agent is triethylammonium acetate due to its volatility.

After the addition and single elution of the multiple sample aliquots described above, the samples are separated using gradient elution, wherein the concentration of organic solvent in the mobile phase elutes the longest polynucleotide fragment in the sample Increase to a final concentration sufficient to Suitable solvents and gradient conditions for DNA fragments in a particular size range can be preselected as described herein. Example 1 and FIGS. 1, 2 and 3 illustrate the concept and application of this aspect of the invention. Figure 1 shows the MI of a standard pUC18 DNA- Hae III digest.
It is a PC chromatogram. A 5 μL sample containing 0.22 μg of DNA was added to the column and the mixture was separated using gradient elution.

In another experiment, another 5 μL of the pUC18 DNA- Hae III digest sample was added to the MIPC column, and a single addition using 35% B (where B was 25% acetonitrile plus 0.1 M TEAA). Washed in one mode for 10 minutes. FIG. 2 shows that no DNA fragments eluted, as indicated by the flat baseline in the chromatogram. In FIG.
A second 5 μL pUC18 DNA- Hae III digest was injected onto the same MIPC column and the column was eluted with 35% B followed by the gradient described in connection with FIG. 1 above. As can be seen from FIG. 3, the peak had essentially the same retention time and twice the height as the reference chromatogram shown in FIG. The fact that there was no shift in retention time or broadening of the peaks indicates that the first sample injection remained strong at the top of the column, despite the 10 minute single wash and subsequent addition of the second sample in FIG. Demonstrate that.

The method could be used for samples contained in volumes too small to be accurately injected into the MIPC column, eg, less than about 1 μL. In such a situation, the sample can be diluted and injected in multiple aliquots as described above. Large volumes of sample can also be added to the MIPC column as one continuous injection (eg, by using a pump or syringe).

The detection of unknown mutations requires sensitive, reproducible and accurate analytical methods. The design of polymerase chain reaction (PCR) primers used to amplify DNA samples that are analyzed for the presence of a mutation is an important factor contributing to the accuracy, sensitivity, and reliability of mutation detection. The design of special primers for the purpose of enhancing and optimizing mutation detection by MIPC has not been previously reported and is an important feature of the present invention.

In general, fragments such as exons contain sample sequences with different melting temperatures, but the variability within any one sample sequence is small. The sample sequence can be about 150-450 base pairs. It is possible to detect single base mutations in long fragments (eg 1.5k bases). However, if the mutation in such a fragment is in a sample sequence having a high melting point (eg, a GC-rich region), the mutation site will need to be partially denatured at elevated temperatures, and all other Low melting sequences may be undetectable because they denature first.

In one embodiment of the present invention, despite the fact that MIPC was used to detect single base mutations in the 1.5 kb fragment, applicants have determined that the degree of accuracy required is for exons. It has been found that this is best achieved by dividing preferably into 150-600 bp segments, and more preferably into 150-400 bp segments.

In one aspect of the invention, Applicants have determined that mutation detection of dsDNA using MIPC is more reliable and accurate if the mutation is located in a sample sequence having a narrow melting point range. I found something. A range of less than about 20 ° C. is preferred in the present invention, ie, any one base in the sample sequence has a melting point within about ± 10 ° C. of any other base in the sequence. In a more preferred embodiment, this range is about 15 ° C. An example of a sample sequence is the constant melting domain described by Lerman et al.
n) ( Meth. Enzymol. 155: 482 (1987)).

At the partial denaturation temperature, heteroduplexes that have base pair mismatches in the sample sequence will denature at the site of the mismatch, while the rest of the sample sequence will remain intact. Partially denatured heteroduplexes can be separated and detected using DMIPC.

In another aspect, the invention is a method of preparing a sequence of a normal dsDNA fragment into a segment, ie, partitioning the dsDNA fragment into a sample sequence of nucleotide sequences having a melting point in the range of less than about 15 ° C. Each sample sequence has opposing first and second ends. The selected sample sequence is amplified by PCR using both front and reverse primers flanking the first and second ends of the sequence.

In an important aspect of the present invention, when the sequence of a DNA fragment to be amplified by PCR is known, either the whole fragment or any sample sequence within the fragment is analyzed using commercially available software. It can be used to design the resulting primer.
The melting profile of the fragment was determined using MacMeltTM (BioRad Laboratories: BioR
ad Laboratories, Hercules, Calif . , MELT (Lerman et al . , Meth. Enzymol . 155: 482 (1987)) or software such as WinMelt ™ (Bio-Rad Laboratories).

In still another aspect, the present invention is a method for analyzing a sequence (amplicon) amplified by PCR using MIPC. Prior to analysis by MIPC, the sample is mixed with a standard, such as wild-type homoduplex DNA, and the mixture is heated and reannealed to form a high duplex that forms homoduplexes and heteroduplexes. Provide for the hybridization method.

In yet another aspect, the present invention relates to improved primer design methods for detecting mutations by MIPC. The design of the overall design process consists of both long-range and short-range primer designs. For long range primer design, the purpose is to design primers that yield good quality PCR products.
A “good quality” PCR product herein refers to a PCR product that is produced in high yield and has a low amount of impurities such as primer dimers and PCR-induced mutations. Good quality PCR can also be influenced by other reaction parameters such as the enzyme used, the number of PCR cycles, the concentration and type of buffer used, the thermal cycling procedure of the temperature and the quality of the genomic template. Methods for preparing good quality PCR products are described in Eckert et al., ( PCR : A Practical Approach ), McPherson, Qui
edited by rke and Taylor, IRL Publishing, Oxford, Vol. 1, pp. 225-244, 19
91). This reference and all references therein are incorporated herein by reference.

A short range primer design must meet two requirements. First, it must meet all the requirements of a long range primer design and give good quality PCR products. In addition, a fragment must be generated that allows detection of the mutation or polymorphism regardless of the location of the mutation or polymorphism in the fragment amplified by the MIPC method. For example, a large DNA fragment with up to several thousand base pairs
It can be amplified by PCR. If the purpose of amplification is only to replicate the desired fragment, there is great latitude in designing primers that can be used for this purpose. However
If the purpose of the PCR amplification is to generate a DNA fragment for analysis of mutation detection by DMIPC, the primers should detect the fragment generated by the PCR method and generate a signal when analyzed by DMIPC. Must be designed to be able to In a preferred embodiment of the invention, the fragment length is between 150 and 600 bp. In a most preferred embodiment, the fragment length for DMIPC mutation detection analysis is 150-400 bp.

The short range primer design has two purposes. One purpose of primer design is when the assay is used as a "screening" test. Another purpose is in analysis for research or diagnostic purposes. “Screening” is defined herein as the study or analysis of DNA fragments in which the fragment contains a mutation (polymorphism) in the population and the mutation is associated with a disease. In the context of the present invention, the term "mutation" is considered to also include polymorphism. When DMIPC is used as a screening technique, an important aspect of the present invention is a method of designing primers that generate fragments in which putative mutations can be detected, regardless of where the mutation site is located within the fragment. It is. On the other hand, if the mutation is known, primer design further refines the analysis so that it is optimized, i.e., maximizing the homoduplex and heteroduplex resolution peaks. be able to. By improving the resolution of the analysis of known mutations, accuracy of the analysis is achieved. Improved resolution is needed for diagnostic mutation applications. With even higher resolution, automatic identification of the presence of positive mutations is easier to perform using appropriate software and algorithms to overlay, compare and measure peaks of wild-type and mutant DNA samples it can.

In an important aspect, the method of the present invention determines whether the amplified fragment contains a region in which the putative mutation is difficult to detect. Applicants have found that mutations can be detected by DMPIC, even if the mutation is located in a fragment that is otherwise difficult to detect, such as in the middle of the fragment or in the high melting domain. Mutations so located can be detected by DMIPC in three ways. In one aspect of this aspect of the invention, a "peak overlap" method can be used, wherein the wild-type standard peak is overlapped with the peak of a sample containing a partially dissociated mutation. Subtract the area of the standard peak from the area of the sample peak. If the area difference is greater than 10% of the standard, the sample is considered to contain the mutation. In a second aspect of this aspect of the invention, if the fragment contains a region with high melting, DMIPC analysis can be performed at two or more temperatures, each temperature being associated with a different melting domain, as further described below. Corresponding. Surprisingly, Applicants have found that for multidomain fragments, changing the choice of primers has a dramatic effect on the melting profile of the amplified sequence predicted by the software program. This observation is advantageously used in the third aspect of the present invention,
Here the primers are reselected to change the melting profile of the fragment of interest to reduce the differences between the Tm's of the domains in the fragment. As mentioned above, there are two situations where a short range of primers is selected. One is when the mutation is used to screen for mutations in the genome. Another is a diagnostic or clinical application in which the presence of a particular mutation in a set of samples is determined. The following summarizes suitable short-range primer selection options in each of these situations.

[0087] Screening applications require the ability to detect mutations, whether or not they are located in fragments. In this situation, the mutation may be located in the middle or higher melting domain of the fragment and is difficult to detect in both cases. The range of melting variation of the fragments is preferably 10 ° C or less, and most preferably the range of variation is 5 ° C or less.

Another primer design method for screening applications is to design primers so that the region of interest is the lower melting domain in the fragment. In this case, the primers are preferably designed so that the fragment to be measured overlaps the region of interest when the analysis is performed moving down exons. In this case, the temperature difference between the higher and lower melting domains is greater than 5 ° C, and most preferably greater than 10 ° C.

Once the mutation of interest has been identified, the primers can be redesigned for R & D diagnostic or clinical applications. In such cases, the mutation is preferably located within 25% of the terminus or 25 bases and is near the terminus in any case. The other end of the fragment contains a high melting domain that is preferably 5 ° C, more preferably 10 ° C, and most preferably 15 ° C higher than the lower domain where the mutation is located. If the choice of primer does not result in a high melting domain on the other side of the fragment, a GC clamp can be applied. The size of this clamp can be up to 40 bp, but can be as small as 4-5 bp, and 10-20 bp is most preferred.

If it is not possible to generate a domain with a constant melting range or domain close enough to the Tm in the fragment during PCR amplification, in order to successfully detect the mutation by DMIPC, It may be necessary to lower Tm. This can be accomplished by replacing dGTP with 7-deaza-2'-dGTP, an analog known to effectively lower the melting temperature of GC base pairs (Dierick et al., Nucleic Acids Res. 21). : 4427 (1993)). If it is necessary to increase the Tm of the domain,
-Aminopurine can be used instead of dGTP.

[0091] Once the mutation of interest has been identified, it can be redesigned for research and development for diagnostic or clinical applications. In a preferred embodiment of the invention, a primer is selected to generate a fragment having a mutation near one of the termini. This is within about 25 bases at one end for fragments having a length similar to the examples described herein, or
Or it can be within 25% of the total length from either end. Also, in a preferred embodiment of the invention, a primer is selected to generate a fragment having a domain having a Tm at least 5 ° C. higher at the end opposite to the end containing the mutation.

In a most preferred embodiment, the primers are chosen such that the mutation is located in the “low melting” domain of the fragment. However, mutations may occur if the high melting domain does not have a melting temperature that is too different from the other domains in the fragment, or if a higher column temperature optimized for the higher melting domain of the fragment is used, It can be detected by DMIPC in the high melting domain of the fragment.

The method of the present invention for designing primers is described in Example 2. The p53 DNA template containing the mutation was amplified by PCR using three different sets of primers. Each primer set was designed to generate amplicon fragments with mutations located in different melting domains. The melting profiles calculated using WinMelt ™ show the melting domains of three amplicon fragments of the same size and the relative positions of the mutations within the fragments shown in FIG. FIGS. 5, 6 and 7 show the effect of primer design on the results of DMIPC mutation detection analysis of three selected amplicon fragments. The highest separation of homoduplexes and heteroduplexes showing four distinct peaks can be seen in FIG. 5, where the mutation indicates fragment 1 located near the end of the fragment in the constant melting domain. The worst separation showing a broad peak is seen in FIG. 7 and the mutation indicates fragment 3 located near the middle of the fragment.

Example 3 further illustrates the use of the method of the present invention. Primer designs in which the mutation was located within about 20% of either end gave the best separation in DMIPC analysis. Fragment 1 with the mutation located in the constant melting domain (FIG. 8) gave the best resolution (FIG. 9), but the worst resolution was when the mutation was located near the middle of the fragment (FIG. 8). Fragment 4) (FIG. 12).

If it is not possible to design a primer that generates a high melting domain at the opposite end of the fragment in a PCR amplification, an embodiment of the invention employs a GC clamp to increase the melting temperature at the desired end. (Myers et al., Nucleic Acids Res. 13: 3111
(1985)). GC clamping is a technique to include an additional G or C base at the 5 'end of one or both primers. The polymerase enzyme extends on such added bases to include them in the amplified fragment, thereby raising the melting temperature at the end (s) of the fragment to near the mutation. The size of the GC clamp can be up to 40 bp, and only 4 or 5 bp. The most preferred GC clamp for detecting mutations by DMIPC is 10-20 bp.

In denaturing gradient gel electrophoresis, a GC clamp is required for mutation analysis of almost all fragments (Sheffield et al., Proc. Natl. Acad. Sci. USA 86: 232 (1989)).
, DMIPCs rarely require GC clamps. The exception is probably when the mutation is in the middle of the fragment and is less than 100 bp in length and the melting profile is flat, or mutations in the high melting region of the fragment and higher melting regions are actually It is a GC clamp. In such cases, selection of appropriate primers will result in a fragment in which the mutation can be detected.

The above-described long-range primer design can be further refined by partial primer design, where several other factors should be considered. Primers without non-template tails, such as universal sequencing primers or the T7 promoter, should be avoided. Preferred primers have a Tm of about 56 ° C. The difference in Tm between the front and reverse primer is preferably about 1 ° C. The difference in Tm between the primer and the template is preferably about 25 ° C. The 3′-pentomer of each primer should be more stable than ΔG ゜ = −6 kcal / mol (ie, more negative). Any possible primer dimer is at least 5 kcal / mole less stable than the 3'-pentomer (ie, 5 kcal or more positive). Any primer self-annealing group should have a Tm of less than 12 ° C. Primers should be of high purity with no incorrect sequences. To avoid degradation, storage in a Tris-HCl (pH 8.0) buffer is preferred over pure water.

In some cases, it is more convenient to directly screen long fragments, up to 1.5 kb, eg, exons, for mutations. Such long fragments generally contain a number of melting temperature domains. Double-stranded DNA fragments melt in a series of discrete steps because the different regions have different thermal stability, denaturing in response to elevated temperatures. These different thermostable regions are termed "domains" and each domain is about 50-30
It is 0 bp long. Each domain has a unique respective Tm and will exhibit the thermodynamic behavior associated with each Tm. Base mismatches in the domain destabilize it, resulting in a decrease in the Tm of the domain in the heteroduplex relative to the fully hydrogen bonded pair found in the homoduplex. Generally, the presence of base mismatch reduces Tm by about 1-2 ° C. FIG. 13 illustrates the melting of a theoretical three domain fragment in schematic form.

As described above, each DNA fragment consists of one or more independent thermostable regions or domains. The Tm of a domain serves as a thermodynamic signature and determines the thermodynamic behavior of the domain. As illustrated in FIG. 13, as temperature increases, domain A will first denature because its Tm is lower than that of B or C. Domain B has an intermediate Tm and then melts, and C will melt last because the domain has the highest Tm within this fragment.

Rather than progressively “opening the zipper” from one end to the other, base pairs in the domain melt in unison over a very narrow temperature range. Denaturation of the domain is characterized by a sigmoid profile (FIG. 14) that shows "cooperativity" between the base pairs comprising the domain. The midpoint of the inflection (slope) is the Tm, and the domain corresponds to the temperature at which equilibrium exists between the single- and double-stranded states. Increasing the temperature above the Tm will rapidly convert all domains to a completely single-stranded structure.

In the three domain molecule described in FIG. 13, the putative point mutation may be in any domain: A, B or C. To establish a high probability of detecting a polymorphic mutation or a mutation in a previously uncharacterized DNA fragment,
It is necessary to carefully select one or more temperatures at which fragment analysis is performed by DMIPC.

The MIPC system can automatically profile the melting behavior of DNA fragments by performing a series of separations with incremental increases in temperature over an overall preferred denaturation range (eg, 50 ° -70 ° C.). it can.

FIG. 14 depicts the homo- and hetero-duplex forms of the four related DNA fragments (homoduplexes are represented by dashed lines). These melting profiles explain how heteroduplex inflection shifts to the left, lower Tm and MI compared to homoduplexes.
This indicates that elution is fast from the PC column. The Tm of the heteroduplex is about 1
It is also clear that the temperature is lower than ゜ -2 ° C. homoduplex.

FIG. 15 shows the melting profile of the 230 bp restriction fragment designated sY81. Any domains present in this fragment are represented here by a sigmoid curve extending between about 54 ° -59 ° C. The temperature at the midpoint of this inflection is the Tm, or Tm _homo , of the melting profile of the homoduplex. The determination of Tm _homo from the melting profile
It is necessary to select an appropriate temperature for performing the mutation screen. Since the presence of base mismatch reduces the Tm of the corresponding heteroduplex domain examined to about 1 ° C to 2 ° C, a fairly accurate prediction of the Tm of each heteroduplex fragment, _Tmhetero , can be made. _Yes (where Tm _hetero = Tm _homo -1 ° C).

As indicated above, the appearance of the melting profile indicates that the Tm _homo is about 56 ° C. Therefore, the preferred temperature for screening for mutations in this fragment is Tm _hetero = Tm _homo -1 ° C, ie 55 ° C. However, given the sharpness of the slope created by the inflection of both domains, and the closeness of the Tm of the two domains, it can be seen that any domain present within this fragment will be partially denatured at this temperature. When the Tm of two different domains is within 5 ° C of one, it is possible to simultaneously screen for mutations in both domains by selecting one analysis temperature. However, the temperature chosen must be below the Tm of the domain with the lower Tm. If an intermediate value is chosen, the low Tm domain in both heteroduplex and homoduplex fragments will be denatured and the ability to detect mutations in the domain will be lost. If a DNA fragment melts in a temperature range greater than 5 ° C., more than one temperature must be used to screen the fragment.

For example, if the DNA fragment contains three domains A, B and C with Tm of 55 ° C., 60 ° C. and 65 ° C. respectively, the slope of the melting profile extends over a range of 10 ° C., as shown in FIG. It will be wider than the profile shown. This indicates that more than one screening temperature must be used to extensively screen all domains in this fragment for the presence of the mutation. Domains A and B can be screened simultaneously at a temperature of 54 ° C, and domains B and C can be screened simultaneously at 59 ° C.
However, there is no one temperature that allows simultaneous screening of all three domains. FIG. 16 shows the theoretical melting profiles for three domain fragments with Tm of 55 ° C., 60 ° C. and 65 ° C.

When the melting profile extends over a temperature range greater than about 5 ° C., a wide range of mutation screens as shown in FIG. 16 can be performed using the following steps. 1. Divide the slope of the inflection into four parts. 2. Subtract 1 ° C from temperature at 0.25 and 0.75. 3. Perform the first analysis at 1 ° C. below the temperature corresponding to the 0.25 position. 4. Perform a second analysis at a temperature 1 ° C. below the temperature corresponding to the position of 0.75.

The polymerase chain reaction (PCR) described in US Pat. No. 4,683,202 to Mullis was an innovative invention in the field of biotechnology. PCR utilizes the amplification (replication) of a small sample of DNA or other polynucleotide of any base pair length (size) using a highly sensitive enzyme called DNA polymerase,
It allows a small DNA strand, called a "primer", to be extended along a "template". Fine DNA samples serve as templates. PCR regenerates the complementary sequence of deoxynucleotide triphosphate (dNTP) bases present in the template or at any part thereof. PCR can be used in conjunction with diagnostic techniques, wherein, for example, a DNA sample having a concentration below the limit of detection is amplified by PCR, and a larger sample so obtained is subsequently analyzed. In a similar manner, a DNA sample obtained from genetic material can be amplified and sequenced or studied to determine its biological effect.

Equipment for performing PCR amplification, such as the Air Thermo Cycler (Idaho Technologies) and reagents, are commercially available from a number of sources (eg, the Perkin-Elmer catalog “PCR Systems, Reagents and Consumables ”, Perkin-Elmer Applied Biosyst
ems, Foster City, California).

[0110] PCR is typically performed in a buffer of pH 5-8. The buffer is a double-stranded DNA sample to be amplified, the first primer, the second primer, magnesium chloride (MgCl _Two ), And four types of deoxy, which are units of DNA generally called "bases"
Includes trinucleotide triphosphates (dATP, dTTP, dCTP and dGTP). The reaction mixture is heated at a temperature sufficient to denature the DNA sample (typically 90 ° C).
This separates the two complementary polynucleotide strands. Or DNA
Can be denatured enzymatically at ambient temperature using a helicase enzyme. Strange
If the properties are performed by heating and using a thermostable DNA polymerase,
DNA polymerase is added before the reaction starts. Denaturation is performed by heat, and
If a thermostable DNA polymerase is used, the DNA polymerase will undergo a denaturing step.
Add after If denaturation is performed by helicase at ambient temperature, the thermostability D
NA polymerase may be included in other reagents before the reaction starts. Other denaturing conditions
Are well known to those skilled in the art, and U.S. Patent No. 5,698,400 to Cotton (1997
)It is described in. This patent specification and the references cited therein are hereby incorporated by reference in their entirety. DNA polymerases are commercially available from various sources.
For example, Perkin-Elmer Applied Biosystems (Foster City,
California) and Stratagene (La Jolla, California).

A primer is an oligonucleotide sequence, typically consisting of 7 to 25 nucleotide bases. Primers are typically chemically synthesized to a predetermined sequence that is predetermined. The primer sequence is designed to be complementary to the identified portion of the denatured DNA strand that is replicated by PCR. Primers are commercially available from various sources, for example, Synthetic Genetics (San Diego, Calif.). When the reaction is cooled to about 50 ° C., each primer anneals to a complementary base sequence in each strand of the denatured DNA sample to be replicated. By heating to about 70 ° C. in the presence of DNA polymerase, four dNTPs and Mg ⁺⁺ , replication is achieved by adding complementary dNTPs from their 3′-end along the length of the strand. Extend primer. dNTPs are available from a variety of sources, such as Pharmacia.
) (Piscataway, NJ). By repeating this process many times, a geometrical increase in the number of desired DNA strands can be achieved at an early stage of the reaction or as long as a sufficient excess of reagents is present in the reaction medium.

[0112] PCR is well known in the biotechnology art and is described in US Pat.
No., 683,202 (1987): Eckert et al., DNA used for polymerase chain reaction.
Polymerase regeneration (The Fidelity of DNA Polymerase Used In The Polymer
ase Chain Reactions), McPherson, Quirke and Taylor (Editor), “PCR: Practical Approach”, IRL Publishing, Oxford, Volume 1, Volume 22
Pages 5-244; Andre et al., GENOME RESEARCH, Cold Spring Harbor Laboratory Press, 843-852 (1977). All such references and references cited therein are hereby incorporated by reference.

[0113] The PCR method is limited by the specificity of the DNA polymerase used, as well as the ability to replicate the DNA strand due to other characteristics of the reaction. For example, a primer may bind only to a portion of the DNA strand that is partially complementary. Binding of such non-specific primers will produce a product containing the undesired sequence. In addition, the first and second primers also bind to complementary portions and form a primer dimer. The specificity of DNA polymerase varies depending on the reaction conditions used and the type of enzyme used. No enzyme can extend the primer without any mistake. Non-complementary bases are sometimes incorporated. Such an enzyme that is associated with a mistake is the original DN
A double-stranded DNA product that is not an exact copy of the A sample is generated, ie, this product contains a PCR-induced mutation. Other PCR variables that reduce accuracy or the degree of DNA replication regeneration include reaction temperature, primer annealing temperature, enzyme concentration, dNT
Including P concentration, Mg ⁺⁺ concentration, origin of enzyme and combinations thereof.

Most applications of PCR require the highest level of replication reproducibility that can be achieved. In particular, detection of mutant genes, construction of genetically engineered monoclonal antibodies,
-Analysis of cell receptor allele polymorphisms, study of HIV mutations in vivo and PCR
The cloning of individual DNA molecules from the group amplified in step 1 depends on their successful regeneration.

Prior to the present invention, PCR products and methods were monitored by gel electrophoresis or capillary electrophoresis. Although such methods separate DNA fragments by size, they do not detect PCR-induced mutations. As used herein, "PCR
The term "induced mutation" is defined as meaning the insertion of one or more bases that are not complementary to the corresponding bases in the template during the PCR method. That is, the sudden mutation induced by PCR is a deviation from the degree of replication regeneration. Such mutations have hitherto been separated from their normal pairs by gradient gel electrophoresis or gradient capillary electrophoresis. However, such techniques are difficult to perform, time consuming, require enormous skill, and are not always reproducible. Capillary electrophoresis analysis takes at least 30 minutes. Gel electrophoresis analysis takes several hours. Such an assay is readily available, easy to use, and not optimal for routine analysis of PCR methods that require high throughput, high reproducibility and quantitative results.

Thus, there is a need for an easy-to-use assay that can analyze a PCR method and minimize the bias from perfect replication and optimize the PCR method in a predictable manner. . There also exists a need for a method for easily separating and collecting pure PCR products from artifacts such as PCR-induced mutations and primer dimers.

Minimizing the bias in the PCR replication method can be achieved by changing the reaction conditions or reagents that cause the bias, if the cause of the bias can be identified. One aspect of the invention is that the product profile obtained by applying the matched ion polynucleotide chromatography (MIPC) method to PCR reaction products can be used to identify sources of bias from accurate replication. Based on the knowledge that.

“Matched-ion polynucleotide chromatography” as used herein
The term is defined as a method of separating single- and double-stranded polynucleotides using a non-polar separation medium, where the method comprises releasing a polynucleotide from the counter-ionic agent and the separation medium. Using an organic solvent. Depending on the conditions, MIPC separates double-stranded polynucleotides by size or by base-pairing sequence, and is therefore a preferred separation technique for evaluating and analyzing PCR. When the mixture of DNA fragments is added to the MIPC column, they are separated by size,
Smaller fragments elute first from the column. When performed at a temperature sufficient to partially denature the heteroduplex, MIPC is referred to as "denatured matched ion polynucleotide chromatography (DMIPC)." Typically, in DMIPC, heteroduplexes elute from the column faster than the corresponding homoduplexes.

The parameters that optimize the PCR replication reproducibility or yield are the PCR product profiles created and analyzed, and the profiles compared to standard product profiles to determine the reproducibility and yield of DNA replication. Can be predicted by predicting which of many possible parameters may need to be adjusted in order to achieve optimality. By analyzing the PCR method and identifying the undesired products in the reaction product profile, the reaction parameters responsible for the undesired products are determined, and the subsequent PCR method is used to determine such undesired products. Can be changed to eliminate or minimize things.

The term “PCR product profile” as used herein is defined to mean data generated by MIPC when applied to the products of a PCR method. This MIPC data allows the expected product and other components of the reaction mixture to be distinguished from each other. Such components comprise the desired product (s), by-products and reaction artifacts. The PCR product profile can be a visual representation, a printed representation of the data, or a status of the original data stream.

A preferred method of the invention for generating a PCR product profile is MIPC. A preferred display is a separate chromatogram output of the MIPC method as found in a video screen or printed hard copy or their corresponding data stream.
Applicants must submit the PC in the accurate and reproducible format required by MIPC to quantify the results.
It has been found that it is an efficient analytical method that can separate all possible products of the R method. Furthermore, this method is easy to implement. MIPC has never been used to analyze and optimize PCR methods.

As used herein, the term “standard profile” is defined to mean data generated by the MIPC method when the MIPC method is used to separate reference standards in connection with a PCR method. I do. The reference standard is the expected product of the PCR method, a DNA fragment of known base length, a primer, a primer dimer, and a heteroduplex of the expected PCR product that can be used to calculate the base pair length designation. And one or more such combinations. The standard profile can also include the actual PCR method separated by MIPC. The standard profile can be a visual representation of the data, a printed representation, or a state of the original data stream.

MIPC is easy to perform, provides reproducible results, and can effectively separate single and double stranded polynucleotides based on both size and base sequence. Operating MIPC at higher temperatures, as used in DMIPC, allows the separation of the desired DNA product from PCR-induced mutations that differ by only one base from the desired product. This separates the desired product from any by-products of the PCR method that represent a bias from the degree of replication regeneration. The by-products can then be evaluated and identified by comparing their product profiles with the selected standard profile as described above. Methods other than MIPC cannot separate and detect deviations from replication reproducibility and / or are inaccurate, inconvenient, time consuming, and have limited range. MIPC is generally capable of separating mixtures of single and double-stranded polynucleotides and especially DNA fragments, and has essentially no limitations of the known gel-based methods described above.
MIPC separations are typically completed in less than 10 minutes, and often in less than 5 minutes. MIPC
The system (Transgenomic, San Jose, CA) has a computer controlled oven surrounding the column and fluid inlet area. Performing a separation of the PCR mixture at the temperature required for partial denaturation (melting) of the DNA at the site of the mutation is thus automated and easily performed. The system used for MIPC separations is rugged and provides reproducible results. This can be computer controlled and the entire analysis of a large number of samples can be automated. The system provides automatic sample injection, data collection, selection of a pre-determined elution solvent based on the size of the fragments to be separated, and column temperature selection based on the base pairing sequence of the fragments to be analyzed. The separated PCR mixture components provide the reaction product profile, which can be displayed as a row of linear bands in gel format or as a row of peaks. This representation can be stored on a computer storage device. This display can be enlarged and the detection threshold can be adjusted to optimize the product profile display. The reaction profile can be displayed in real time, or later retrieved from the storage device for display. The product profile display can be viewed on a video display screen or as a hard copy printed by a printer.

Example 9 describes the effect of temperature on the separation of heteroduplexes (PCR-induced mutations) and homoduplexes by MIPC. FIG. 18 shows the results of Example 9 as product profiles of the PCR method (described in Examples 4-8) in the form of a separation chromatogram, where the separation was performed at three different temperatures by MIPC. . The product fragment contained base pairs. At 62 ° C., two poorly resolved peaks are seen. When the separation temperature is raised to 64 ° C, broad shoulders representing heteroduplexes resulting from PCR-induced mutations are seen with lower retention times than the main sharp peak. Sharp peaks represent the desired PCR product. The appearance in the 64 ° C. chromatogram indicates that the heteroduplex is at the site (s) of base pair mismatch as evidenced by a broad, low retention time that is not well separated from the product peak. This indicates that it has just begun to denature. Complete denaturation at the site of the mismatch, as evidenced by complete isolation of PCR-induced mutations from sharp product peaks that appear at higher retention times when the separation temperature is increased to 66 ° C Happened. The lower retention time peak is now partially resolved into at least two heteroduplexes. FIG. 18 also shows the primer dimer peak at very low retention times, close to the excluded volume. As can be seen from FIG. 18, the entire separation was completed in 6-8 minutes. Sample injection and temperature for each experiment were preprogrammed and performed automatically by a computer-controlled sample auto-injector and computer-controlled column oven.

A series of steps described in Example 4 represents one cycle of the PCR method. Such a cycle is repeated until the desired amount of product is obtained. After the final cycle, the mixture is usually not denatured again, since no further primers need to bind to their individual templates. However, in order to accurately analyze the final PCR mix, all possible duplexes of the complementary strand must be free of denaturation and hybridization, since all component duplexes must have the opportunity to denature and hybridize. There may be an opportunity to form a duplex combination (Example 12).

The degree of specificity of the DNA polymerase varies depending on the reaction conditions used and the type of enzyme used. No enzyme allows completely error-free primer extension. Thus, non-complementary bases may sometimes be introduced. The error associated with such an enzyme results in a double-stranded DNA product containing a PCR-induced mutation, but not an exact copy of the original DNA sample. Other PCR characteristics, such as reaction temperature, primer annealing temperature, enzyme concentration, dNTP concentration, Mg ⁺⁺ concentration and combinations thereof, can cause a decrease in the accuracy of DNA replication by PCR or the degree of regeneration Has the property.

The degree of regeneration of a DNA fragment by PCR will depend on many factors that have been long recognized in the art. Some of the factors may offset changes in the PCR product profile caused by increasing or decreasing the amount or concentration of one factor, or may be reversed by further altering a different factor. They are interrelated in the sense that they can be done. For example, increasing the enzyme concentration may reduce the reproduction of the replica, while decreasing the reaction temperature may increase the reproduction of the replica. Increasing the magnesium ion concentration or dTNP concentration increases the rate of the reaction, which may have the effect of reducing the degree of PCR regeneration. A detailed discussion about the factors that contribute to the regeneration of the PCR is, Eckert et al., [PCR: Practical Research (PCR: Practical Approach)], McPherson, Quirke and Taylor (editing), IRL Press, Okkusufu Odo, Vol. 1, 225-244 (1991); and Andre et al., Genome Research (GE
NOME RESEARCH, Cold Spring Harbor Laboratory Publishing (Cold Spring)
Harbor Laboratory Press), 843-852 (1977). All such references and references cited therein are hereby incorporated by reference. Thus, the availability of a PCR product profile allows the optimization of PCR conditions to improve results in a highly efficient manner. This work in connection with the MIPC method has never been reported before.

Thus, in another aspect of the invention, a PCR reaction product profile generated by MIPC can be analyzed and evaluated. By comparing the PCR reaction product profile to the standard profile, one can identify deviations from the standard product profile and adjust for one or more PCR variables that are known to produce the observed bias. . Multiple PCR cycles using the adjusted method variables, followed by analysis of the reaction mixture by MIPC, now represent a reduction in product profile bias from a predetermined standard.

As an example of the operation of the present invention, the following discussion relates to PCR amplification of a 500 bp DNA fragment under various conditions. Single-stranded overhangs at one or both ends of the product fragment, so-called "overhangs", are common. Due to the sensitivity and separation power of MIPC,
Such a product overhang appears as a shoulder on the product peak, and
Indistinguishable from PCR-induced mutations. To eliminate any ambiguity in the assessment of the degree of replication by MIPC, the PCR reaction products were routinely treated with Hae III endonuclease to cleave the product fragments near each end, and A 405 base pair fragment is generated as described in FIG. 6 and having blunt ends as shown in FIG. Thus, when performing MIPC separation at 66 ° C. (partial denaturing conditions), any product separated from the main peak should contain a PCR-induced mutation, and the result of an “overhang” at the end Absent. As discussed below, 405 base pair product refers to the product obtained after treating a 500 base pair PCR amplification product with Hae III endonuclease.

FIG. 19 depicts the profiles of the three PCR reaction products, which analyze and evaluate the PCR reaction product profiles and compare the PCR method variables to the predetermined standard profiles to determine the desired PCR product variables. It shows the use of MIPC to optimize the yield of the desired product generated by the PCR method by adjusting it to optimize the yield.

The product profile at the top of FIG. 19 shows the AmpliTaq ™ DNA polymerase
PCR method using Perkin-Elmer Applied Biosystems, Foster City, CA, to generate a 405 base pair DNA fragment (described in Example 4)
Represents MIPC isolation was performed at 66 ° C., a temperature that did not denature the entire 405 base pair DNA fragment, but was sufficient to cause denaturation at the site of the PCR-induced mutation (ie, base pair mismatch). Such a partially denatured fragment containing a PCR-induced mutation has a lower retention time than a fragment that does not contain base pair mismatches (and therefore is not denatured), so that AmpliTaq ™ The degree of replication regeneration of the induced PCR method can be assessed and quantified by MIPC analysis of the reaction mixture.

In addition to the large primer dimer peak near the exclusion volume, the top profile in FIG. 19 shows a 405 base pair homoduplex product peak with a retention time of just 6 minutes and a Shows a broad peak with retention time. Lower retention time peaks were obtained under known DMIPC conditions to separate PCR-induced mutations from their corresponding homoduplexes. This low retention time peak is the heteroduplex of the PCR-induced mutation. Integration of a 405 base pair peak with a retention time of just 6 minutes compared to a standard of known concentration indicated that 10 ng of the 405 base pair product was included. The only other peaks in the reaction product profile were the primer dimer peak near the exclusion volume and the peak of the PCR-induced mutation with a retention time just below 6 minutes.

Once the large PCR-induced mutations and the large primer dimer peak in the PCR reaction product profile have been identified, how can the reaction variables be adjusted to improve the yield of the desired 405 base pair product? This profile was evaluated to determine if it could be adjusted. The primer dimer artifact did not affect product yield, since the primer was in very large excess relative to the template. Thus, improved replication reproducibility should reduce the amount of heteroduplexes in the PCR-induced mutation product, while also increasing the yield of the desired 405 base pair product. Since the DNA polymerase enzyme has the greatest effect on the degree of replication regeneration, the reaction variables of the DNA polymerase were adjusted. As a result of this analysis and evaluation, the PCR cycle used AmpliTaq ™ polymerase as described in Examples 7 and 8 for Pyrococuus furiosus (Pfu) (Stratagene, La Jolla,
(California) and repeated after adjustment. All other reaction conditions were not changed. Pfu is a proofreading enzyme and is therefore expected to reduce base mismatches during PCR. D of PCR performed in the presence of Pfu
MIPC analysis gave the middle reaction product profile of FIG. The integration of the 405 base pair peak at a retention time of exactly 6 minutes was compared to a standard of known concentration, indicating a 350% increase in 405 base pair product at 35 ng. This improvement was expected as a result of analyzing and identifying the products in the PCR reaction product profile, and adjusting the reaction variables known to be responsible for the formation of undesirable products.

Further adjustment of the PCR method described in Examples 7 and 8 was performed using PFU Turbo ™ (Stratagene, La Jolla, CA), a DNA polymerase with greater proofreading capacity than PFU. Performed to optimize the PCR method. All other reaction conditions were not changed. Analysis of the PCR method containing PFUTurbo ™ by DMIPC created the reaction product profile below in FIG. The integration of the 405 base pair peak with a retention time of exactly 6 minutes was compared to a standard of known concentration and showed a further increase in yield of the 405 base pair product (265%), 93 ng. This improvement was expected as a result of analyzing and identifying the products in the PCR reaction product profile, and adjusting the reaction variables known to be responsible for the formation of undesired products.

In another example, FIG. 20 shows three PCR reaction product profiles,
Analysis and evaluation of reaction product profiles by using IPC and a PC that minimizes deviations from replication reproducibility when compared to predetermined standard profiles
It has been demonstrated that adjusting the variables of the R method provides a means to optimize the degree of PCR regeneration.

The product profile at the top of FIG. 20 shows a PCR method using AmpliTaq ™ polymerase (Perkin-Elmer Applied Biosystems, Foster City, Calif.) To generate a 405 base pair DNA fragment. (Examples 4 and 1)
0). This MIPC separation was performed at 66 ° C., a temperature that did not denature the entire 405 base pair DNA fragment, but was sufficient to cause denaturation at the site of the PCR-induced mutation (ie, base pair mismatch). Such a partially denatured fragment containing a PCR-induced mutation has a lower retention time than a fragment that does not contain base pair mismatches (and thus is not denatured at 66 ° C.), and therefore, the AmpliTaq ( The degree of replication regeneration of the TM-induced PCR method can be assessed and quantified by MIPC analysis of the reaction mixture.

In addition to the large primer dimer peak near the excluded volume, the top profile in FIG. 20 has a product peak of 405 base pairs with a retention time of just 6 minutes and a retention time of less than 6 minutes Shows a broad peak. The peak at the lower retention time is due to the known DM to separate PCR-induced mutations from their corresponding homoduplexes.
Obtained under IPC conditions. This low retention time peak is therefore a PCR-induced mutation. Integration of the product profile, which indicates the presence of a PCR-induced mutation, is of the order of 62%, indicating a very poor replication regeneration.

Having identified large PCR-induced mutations in the PCR reaction product profile,
This profile was evaluated for possible causes of the PCR-induced mutations identified.

[0139] Primer dimer artifacts are known to have no effect on replication reproducibility and can be ignored. So the obvious cause of this problem is:
It must have been a DNA polymerase, since the reaction components most affected the degree of replication regeneration. As a result of this analysis and evaluation, the PCR cycle was repeated after adjusting the enzyme to Pyrococuus furiosus (Pfu) (Stratagene, La Jolla, Calif.) As described in Example 7. All other reaction conditions were not changed. Pfu is a proofreading enzyme and is therefore expected to reduce base mismatches during PCR. Performed in the presence of Pfu
DMIPC analysis of the PCR method gave the middle reaction product profile of FIG. Quantification of the product profile indicated that the amount of undesired PCR-induced mutagenesis was greatly reduced by 25%. This improvement was expected as a result of analyzing and identifying the products in the PCR reaction product profile, and adjusting the reaction variable, AmpliTaqTM, which is known to be responsible for the formation of unwanted products .

Further adjustment of the PCR method (described in Example 7) was performed using PFU Turbo ™ (Stratagene, La Jolla, CA), a DNA polymerase with greater proofreading capacity than Pfu. By doing so, the PCR method was optimized. All other reaction conditions were not changed. Analysis by PCR of the PCR method containing PFUTurbo ™ by DMIPC produced the lower reaction product profile in FIG. Quantification of the product profile indicated that unwanted PCR-induced mutation products were further reduced by 18%. This improvement was expected as a result of analyzing and identifying the products in the PCR reaction product profile, and adjusting the reaction variables known to be responsible for the formation of undesired products.

All the 405 base pair PCR reaction products studied so far were analyzed after the last PCR cycle and after heating for post-reaction hybridization. The final hybridization procedure is described in Example 10. The last hybridization is usually not performed after the last PCR cycle. However, Applicants have found that without the last hybridization, inaccuracies and spurious low values are obtained for PCR-induced mutations. This observation is shown in FIG.
The upper trace in FIG. 21 shows a 40 ° C MIPC at 66 ° C., a temperature sufficient to cause denaturation at the site of the PCR-induced mutation before hybridization after the reaction.
1 shows a chromatogram representing the separation of a 5 base pair fragment. Heteroduplex PCR-induced mutation products appear as small peaks with retention times slightly below 6. A large, sharp 405 base pair product peak is visible with a retention time of just 6 minutes. Integration of these peaks indicates that PCR-induced mutagenesis is present at the 8% level.

The lower trace of FIG. 21 shows the same MIPC separation chromatogram except that the 405 base pair product was hybridized as described above before separation (after the reaction).
Low retention heteroduplexes representing PCR-induced mutations increased to 23.1%. Thus, the most preferred embodiment includes a hybridization step after the reaction to display the PCR-induced mutation to the true degree of accuracy.

Thus, using the method of the present invention, the PCR method was analyzed and the products were separated by MIPC to provide a PCR reaction product profile. The separated product was identified and quantified. As a result of the analysis and evaluation, it was possible to correct the problems associated with the degree of replication reproduction present in the PCR method initially investigated. Thus, it was possible to predict which reaction variables could be adjusted to improve the degree of replication regeneration. The reaction variables that seemed to be the largest cause of the observed poor replication reproducibility were adjusted, and the PCR method was repeated using the adjusted conditions. The degree of PCR replication regeneration was improved as expected. The degree of further improvement was quantified by integration of the reaction product profile using MIPC.

[0144] Once an improvement in the PCR method was expected and indicated, the method was again optimized by adjusting the reaction variables identified earlier. In essence, all those skilled in the art of mutation detection have hitherto assumed near perfect PCR replication, and the mutation observed in the sample is actually a PCR-induced mutation, Did not consider that it was not inherent in the United States. A common problem in all conventional PCR applications, especially in the field of mutation detection, was that little or no PCR-induced mutation occurrence was recognized, but it is now readily available using the methods of the present invention. Can be measured.

As shown in Example 11 and FIG. 22, in another aspect of the present invention, the desired PCR
The product was separated from reaction impurities by MIPC and isolated when eluted from the MIPC column. By analyzing the collected fractions by MIPC at 68 ° C., the heteroduplex product (s) was separated in the earlier fractions and the pure 405 bp homoduplex product was isolated. It was shown to be isolated in the last fraction. In addition to the rapid separation of heteroduplexes and homoduplexes as described herein, the isolation of the latter has not been reported previously.

The pure homoduplex fragments separated and isolated by the method of the present invention can be used for various methods. Non-limiting examples of such uses include using relatively large amounts of pure fragments as templates in PCR methods. The purity of such a template and P
Relatively large amounts in the CR method will result in large amounts of pure amplified product. Alternatively, the pure fragment can be incorporated into a plasmid and regenerated in the cell. Due to its high initial purity, few or no undesirable fragments are present and available for regeneration in cells, so large quantities of fragments are regenerated at very high purity levels from the regenerated plasmid and isolated. Will be. Highly purified PCR products are invaluable to the scientific community. Some examples where the availability of high purity PCR products is important include, but are not limited to, sequencing studies,
Includes cloning, preparation of additional amounts of the amplified polynucleotide by PCR, and preparation of polynucleotide standards.

The ability to detect mutations in double-stranded polynucleotides, and especially in DNA fragments, is of great importance in medicine and natural and social sciences. The Human Genome Project is providing a vast amount of genetic information, which is currently setting the criteria for assessing the association between mutations and human disease (Guyer et al . , Proc. Natl . Acad . Sci . USA 92: 10841 (1995)). The ultimate pathogen is described, for example, by a genetic code different from the wild type (Cotton, TIG 13:43 (1997)
). Elucidating the genetic basis of the disease is the starting point for treatment. Similarly, determining differences in the genetic code is powerful in studies of evolution and population, and can provide potentially definitive insights (Cooper et al., Human Genet. 69: 201 (1977)). Elucidating these and other issues related to the genetic code requires that D
Based on the ability to identify abnormalities (ie, mutations) in NA fragments. Thus, there is a need for techniques for detecting mutations in an accurate, reproducible, and reliable manner.

The following discussion is simply about DNA fragments. However, it is also conceivable to apply such considerations to all double-stranded polynucleotides.

As used herein, “melting temperature” is defined to mean the temperature at which 50% of base pairs in a DNA fragment separate.

"Homologous double-stranded" is defined herein to mean a double-stranded DNA fragment in which the bases in each strand are complementary to their counterpart bases in the other strand.

"Heteroduplex" is defined herein to mean a double-stranded DNA fragment in which at least one base in each strand is not complementary to at least one paired base in the other strand. I do. Since at least one base pair in the heteroduplex is not complementary,
It requires lower energy to separate bases at that site compared to its perfectly complementary base pair analog in a homoduplex. This results in a lower melting temperature at the site of the mismatched base in the heteroduplex compared to the homoduplex.

The term “hybridization” refers to the step of heating and cooling a dsDNA sample, for example, heating to 95 ° C., followed by slow cooling. Heating process is DN
Causes denaturation of the A chain. Upon cooling, the chains recombine into duplexes in a statistical manner. If the sample contains a mixture of wild type and mutant DNA, hybridization will form a mixture of hetero- and homo-duplexes.

“Heteromutant site separation temperature” T (hsst) as used herein means the temperature at which a heteroduplex DNA is selectively denatured at the site of mutation. This is the optimal temperature for performing heteroduplex and homoduplex chromatographic separations by MIPC and therefore detects mutations.

As used herein, “matched ion polynucleotide chromatography”
The term is defined as a method for separating single and double stranded polynucleotides using a non-polar separation medium, wherein the method releases the counterionic agent and the polynucleotide from the separation medium. Use an organic solvent. MIPC separation
It takes less than 10 minutes, and often less than 5 minutes. The MIPC system (WAVE ™ DNA Fragment Analysis System, Transgenomic, San Jose, CA) has a computer controlled oven surrounding the column. Therefore, the mutation can be easily detected at a temperature that requires partial denaturation (melting) of the DNA at the site of the mutation. This system used for MIPC separations is robust and provides reproducible results. It is computer controlled and can perform all analyzes of multiple samples automatically. The system provides for automated sample injection, data collection, selection of predetermined elution solvent composition based on the size of the fragments to be separated, and column temperature selection based on the base pairing sequence of the fragments to be analyzed. Separated mixture components can be displayed in the form of a linear format in a gel format, or as a sequence of peaks. This representation can be stored on a computer storage device. The display can be enlarged and the detection threshold adjusted to optimize the product profile display. The reaction profile can be displayed in real time or retrieved from a storage device for later display. A representation of the mutation segregation profile, genotype profile or other chromatographic separation profile can be viewed as a video display screen or as a hard copy printed by a printer.

Depending on the conditions, MIPC separates double-stranded polynucleotides by size or base pair sequence, and is therefore a preferred separation technique for detecting the presence of specific fragments of DNA or RNA of interest. . Convenient, automated, sensitive, and
Separation systems for mutation detection, a range of MIPC capabilities, have never been described.

When adding DNA fragments to a MIPC column, they are separated by size and smaller fragments are eluted from the column first. However, when MIPC is performed at a temperature high enough to denature portions of the domain of the DNA fragment containing the heteromutant site, the heteroduplex separates from the homoduplex. When performed at a temperature sufficient to partially denature the heteroduplex, MIPC is referred to as "denatured matched ion polynucleotide chromatography (DMIPC)."

The term “heteromutant” is defined herein to mean a DNA fragment containing a polymorphic or non-complementary base pair.

The term “mutation isolation temperature range” as used herein means the temperature range between the highest temperature at which a DNA segment is completely non-denaturing and the lowest temperature at which a DNA segment is completely denatured. Define.

The term “mutation separation temperature profile” is defined herein to mean a DMIPC separation chromatogram that shows the separation of heteroduplexes from homoduplexes. Such a separation profile is characteristic of the sample containing the mutation or polymorphism and was hybridized before separation by DMIPC. As shown in FIG.
DMIPC separation chromatograms performed at 1 ° C. illustrate the mutation separation profile as described herein.

The term “temperature titration” of DNA refers herein to the vertical axis as DM.
This is an experimental procedure in which retention-time from IPC is plotted against column temperature on the horizontal axis.

A reliable method for detecting mutations is by hybridizing the putative mutant strand in the sample with the wild-type strand (Lerman et al., Meth . Enzymol ., 155: 482). (1987)). If the mutant strand were present, two homoduplexes and two heteroduplexes as shown in FIG. 23 would be formed as a result of the hybridization step. Hence, separation of heteroduplexes from homoduplexes provides a direct way to confirm the presence or absence of a mutant DNA segment in a sample.

Current methods for detecting mutations in gel electrophoresis rely on differences in melting temperatures between heteroduplexes and homoduplexes. DNA separation methods separate DNA fragments based on the number of base pairs when the separation is performed below a temperature that denatures (melts) mismatches in the heteroduplex. However, DNA fragments with the same number of base pairs can be separated when the separation is performed at T (hsst). Such separation was achieved by denaturing gradient gel electrophoresis or denaturing gradient capillary electrophoresis. However, such techniques are difficult to operate, time consuming, require highly skilled technicians, and are not always reproducible. For example, denaturing gradient capillary electrophoresis analysis requires at least 30 minutes per run, plus a preparation time. Denaturing gradient gel electrophoresis analysis
Preparation time is required in addition to several hours. The fact that the mobility of electrophoresis decreases exponentially with the length of the denatured portion of the DNA fragment further exacerbates the long analysis time problem inherent in electrophoretic separations. Such an assay is quick to set up, easy to use, and not useful for routine analysis of PCR products that require high throughput, high reproducibility and quantitative results. An advantage of the present invention is aimed at detecting mutations
The ability to automate the determination of T (hsst) by DMIPC.

Recently, Matched Ion Polynucleotide Chromatography (MIPC) has been
Introduced as a separation method. MIPC is easy to perform, provides reproducible results, and can effectively separate single- and double-stranded polynucleotides based on both size and base sequence. It is possible to separate heteroduplexes from homoduplexes that differ by only one base. MIPC is generally capable of separating single- and double-stranded polynucleotide mixtures, and especially mixtures of DNA fragments, without the limitations of the previously known gel-based methods described above.

The temperature-dependent separation of 209 base pair homoduplexes and heteroduplexes by MIPC is shown in FIG. 24 as a series of separation chromatograms, and the separation method is described in Example 15. A sample containing a heterozygous sample of a 209 base pair homoduplex fragment (where the mutant fragment contained one base pair deviation from the wild type) was prepared as described in Example 15. Hybridized. Two homoduplexes and two heteroduplexes were generated by the hybridization method as illustrated in FIG. The mixture was separated as described in Example 16. As shown in FIG.
, One peak representing all four mixed components was seen. This result was expected because all four components had the same base pair length and the separation was performed under non-denaturing conditions (temperatures where the temperature was too low to allow denaturation). . At 53 ° C., a shoulder appeared on the retention time side below the main peak. This indicated the onset of melting, as well as the presence of heteroduplexes and the possibility of partial separation.
As the separation temperature was increased incrementally, the original single peak eventually split into four clearly defined peaks. It is shown in FIG. 24 that the two low retention time peaks represent two heteroduplexes and the two high retention time peaks represent two homoduplexes. As the AT base pair denatures at a lower temperature than the CG base pair, the two homoduplexes separate. Without wishing to be bound by any theory, this result indicates that a greater degree of denaturation in one duplex and / or one partially denatured heteroduplex compared to the other MIPC
The retention time differs on the column. Temperature titrations of homoduplex and heteroduplex species from the elution profile of FIG. 24 are shown in FIG.

As can be seen from FIG. 24, the appearance of four distinct peaks, where the temperature range of 57 ° -58 ° C. is optimal for this separation, was observed when the mutation was present in the original sample and FIG.
The results were consistent with those expected based on a hybridization summary of 3. Above this temperature, the double-stranded fragment denatures completely, rather than only at sites of base pair mismatch. This is evidenced by a single peak showing four single-stranded polynucleotides with lower retention times when the separation was performed above 59 ° C.

In some mutation analyses, two or partially separated peaks (1
Only one or more) are observed in the DMIPC analysis. Two homoduplex peaks may appear as one peak or as partially separated peaks, and two heteroduplex peaks may appear as one peak or as partially separated peaks. May appear. In some cases, only a broad peak is initially observed under partial denaturing conditions.

If a sample contains homozygous DNA fragments of the same length, hybridization and analysis by MIPC will produce only a single peak at any temperature, since heteroduplexes cannot be formed. In the procedure of the present invention, mutation determination involves homozygous samples hybridized to a known wild-type fragment and DM
Perform IPC analysis. If the sample contains only wild-type fragments, a single peak will be seen in the MIPC analysis since no heteroduplex can be formed. If the sample contains a homozygous mutant fragment, analysis by DMIPC will show homoduplex and heteroduplex separation, as seen in FIG.

The temperature at which 50% of the constant melting domain denatures can also be determined experimentally by plotting the UV absorption (UV) of the DNA sample against temperature. Absorption increases with increasing temperature and the resulting plot is called the melting profile (Breslaue
USA et al., Proc. Natl. Acad. Sci. USA 83: 3746 (1986); Breslauer, Calculating Thermodynamic Data for Transactions of any Molecularity , p. 221, Marky. Edited by J. Wley and Sons (1987)). The midpoint of the absorption axis of the melting profile represents the melting temperature (Tm), the temperature at which 50% of the double-stranded DNA strands denature. In one embodiment of the invention, the observed Tm is used as the starting temperature for performing DMIPC for mutation detection.

In another embodiment of the invention, mutation detection is performed using software such as MELT (Lerman et al., Meth . Enzymol. 155: 482 (1987)) or WinMelt ™, version 2.0. Obtain the calculated Tm to use as the starting temperature for performing DMIPC. These software programs show that, despite differences in individual base pair stability, the melting temperatures of close base pairs are closely related, ie, there is a cooperative effect.
Thus, there is a long region of 30-300 base pairs called a "domain" where the melting temperature is fairly constant. In a similar fashion, the software MELTSCAN (Brossette et al., Nucleic Acids Res. 22: 4321 (1994)) calculates the melting domains in DNA fragments and their corresponding melting temperatures. The concept of a constant temperature melting domain is important because it allows the detection of mutations in any part of the domain at one heteromutant site-selective temperature.

The use of a software package to confirm the starting temperature for performing DMIPC in connection with mutation detection has never been described before and is an important aspect of the present invention. Prior to the present invention, a time-consuming method development procedure was required to determine the starting temperature for the mutation detection assay. However, Applicants have found that the calculated melting temperature is useful for mutation detection using gel electrophoresis, but must be adjusted when applied to detect mutations by DMIPC.

Applicants have developed an equation for determining the heteromutant site separation temperature, T (hsst). Generally, this equation is of the form T (hsst) = X + mT (w), where T (hsst) is the heteromutant site separation temperature, and X is the DMIPC detector and m Is a weighting factor; both factors are used to adjust T (w) to T (hsst). X is a positive or negative value. In certain embodiments of the invention, T (w) is the melting temperature determined from the UV melting profile of a normal (ie, wild-type) DNA duplex. In another particular embodiment of the invention, T (w) is calculated by software. The value of m is preferably between 0 and 2. X depends on the sequence of the fragment to be analyzed, and this value can vary up to about 10 ° C.

In another embodiment of the present invention, T (hsst) is experimentally modified from the melting temperature calculated as described in the examples, wherein the DMIPC analysis starts with the melting temperature calculated by the software. (Example 17) or from the melting profile of UV absorption versus temperature (Example 18). Samples are subsequently injected and analyzed at incrementally reduced or increased temperatures until optimal separation is achieved. In a preferred embodiment of the invention, all aspects of the analysis are automated, and the temperature increment is selected, for example, in 2 ° C increments.

Surprisingly, Applicants have found that in most cases, T (hsst) generally exceeds the melting temperature, as determined from the UV absorption versus temperature melting curve, or from the temperature titration curve. It was found that it was only 1-2 ° C lower. After forming the temperature titration curve, T (hsst) can usually be determined in one or two experiments. Applicants have further found that the temperature range for mutation isolation of about 200-400 bp DNA fragments throughout which denaturation occurs is about 5 ° C. Thus, it has been found that even if the above procedure only reaches T (hsst), the direction of changing the temperature to achieve optimal separation is apparent. In one embodiment, the increment is about 0.3-0.
Set at .5 ° C. In a preferred embodiment, the increment is set at 0.1 ° C., allowing a very accurate determination of T (hsst).

The foregoing discussion mainly involved DNA fragments having a size of about 200-400 bp. In some cases, it may be more convenient to directly screen longer fragments of up to 1.5 kb, such as exons, for mutations. Such longer fragments generally contain multiple melting temperature domains. Double-stranded DNA fragments melt in a series of discrete steps as various domains with different thermostabilities denature with increasing temperature. These regions of different thermostability are termed "domains" and each domain is 50-300 bp in length. Each domain has its own Tm and will exhibit the thermodynamic behavior associated with that Tm. The presence of a base mismatch within a domain destabilizes it and lowers the Tm of that domain in a heteroduplex than the pair found in a fully hydrogen bonded homoduplex. As generally found by the Applicant, the presence of base mismatch reduces the Tm by 1-2 ° C. FIG. 26 shows a schematic of the theoretical melting of three domain fragments.

As mentioned above, each DNA fragment consists of one or more regions of independent thermostability or domain. The Tm of a domain serves as a thermodynamic property and determines the thermodynamic behavior of the domain. As schematically represented in FIG. 26, as the temperature gradually increases,
Domain A denatures first because Tm is lower than domains B or C. Domain B has an intermediate Tm and then melts, and domain C melts last because that domain has the highest Tm within this fragment.

Rather than "unzippering" progressively from one end to the other, base pairs within the domain open simultaneously over a very narrow temperature range. The denaturation of the domain is characterized by a sigmoid profile (FIG. 27), which indicates "cooperativity" between the base pairs comprising the domain. The midpoint of the absorption range is the Tm, and corresponds to the temperature at which the domain exists in single- and double-stranded equilibrium. When the temperature rises above Tm,
All domains will rapidly turn completely into single-stranded structures.

In the three domain molecules described in FIG. 26, putative point mutations may be present in any of the domains: A, B and C. To establish a high probability of detecting a polymorphic mutation or mutation in a previously uncharacterized DNA fragment,
One or more temperatures at which fragment analysis is performed by DMIPC must be carefully selected.

The DMIPC system can automatically profile the melting behavior of DNA fragments, possibly by incrementally increasing the temperature over the entire denaturation range (eg, 50 ° -70 ° C.) and performing a series of separations.

FIG. 27 depicts the melting of four related homo- and heteroduplex forms of a DNA fragment (homoduplexes are represented by dashed lines). Such a melting profile explains how the midpoint of the heteroduplex shifts to the left and has a lower Tm compared to the homoduplex.
And early elution from the DMIPC column. The Tm of a heteroduplex is about 1 ° to 2 ° C lower than that of a homoduplex.

FIG. 28 shows the melting profile of the 230 bp restriction fragment designated sY81. Any domains present in this fragment are now represented by a single sigmoid curve extending between about 54 ° -59 ° C. The temperature at the midpoint of this inflection is the Tm or Tm _homo of the melting profile of the homoduplex fragment. Determining the Tm _homo from the melting profile is necessary to select the appropriate temperature for performing the mutation screen. The presence of base mismatches can reduce the Tm of the corresponding heteroduplex domain being probed to about 1-2 ° C, making fairly accurate predictions for the Tm, _Tmhetero of each heteroduplex fragment. Where Tm _hetero = Tm-1 ° C. This formula is an example of the general formula for T (hsst) described above, where T (w) has a value of 1 ° C.

As noted above, the appearance of a melting profile indicates that the Tm _homo is about 56 ° C. Thus, the ideal temperature for screening for mutations in this fragment would be Tm _hetero = Tm _homo- 1 ° C, or 55 ° C. However, given the sharpness of the slope created by the inflection of both domains, and the closeness of the Tm of the two domains, it can be seen that any domain present within this fragment will be partially denatured at this temperature. When the Tm of two different domains is within 5 ° C of one, it is possible to simultaneously screen for mutations in both domains by selecting one analysis temperature. However, the temperature chosen must be below the Tm of the domain with the lower Tm. If an intermediate value is chosen, the low Tm domain in both heteroduplex and homoduplex fragments will be denatured and the ability to detect mutations in that domain will be lost. If a DNA fragment melts in a temperature range greater than 5 ° C., more than one temperature must be used to screen the fragment.

For example, if the DNA fragment contains three domains A, B and C with Tm of 55 ° C., 60 ° C. and 65 ° C., respectively, the slope of the melting profile extends over a range of 10 ° C., as shown in FIG. It will be wider than the profile shown. This indicates that more than one screening temperature must be used to extensively screen all domains in this fragment for the presence of the mutation. Domains A and B can be screened simultaneously at a temperature of 54 ° C, and domains B and C can be screened simultaneously at 59 ° C.
However, there is no single temperature at which all three domains can be screened simultaneously. FIG. 29 depicts the theoretical melting profiles of three domain fragments with Tm of 55 ° C., 60 ° C. and 65 ° C.

In a particular embodiment of the present invention, when the melting profile extends over a temperature range greater than about 5 ° to 7 ° C., the following steps are used to perform an extensive mutation screen as shown in FIG. be able to. 1. The melting curve including the inflection is divided into four parts. 2. Subtract 1 ° C from temperature at 0.25 and 0.75. 3. Perform the first analysis at 1 ° C. below the temperature corresponding to the 0.25 position. 4. Perform a second analysis at a temperature 1 ° C. below the temperature corresponding to the position of 0.75.

As indicated above, keeping all other parameters constant and increasing the temperature of the separation, melting of the DNA reduces the retention time from the liquid chromatography column.

In one aspect of the invention, samples containing mutations are examined at a range of temperatures using a heuristic optimization method. The optimal temperature obtained by this method is the temperature at which the mutant DNA fragment is most easily distinguished from the wild-type DNA by differences in peak patterns. This method is not systematic and relies on knowledge of whether heteroduplexes are present in the sample. However, prior knowledge of this mismatch,
Not always available.

A preferred embodiment of the present invention is a method for selecting T (hsst) based on temperature titration. This temperature titration can be obtained by experimental observation or by theoretical analysis of thermodynamic information. In addition, Applicants have found that the optimal temperature for mutation detection corresponds to the early stages of denaturation of the segment of the wild-type DNA fragment containing the mutation. The retention time versus temperature plot shows a parallel relationship between wild type and heteroduplexes, so that the retention time of both fragments decreases with approximately the same slope. This surprising and constant relationship found by the applicant is T (
Eliminates the need to collect data on heteroduplexes in the sample to select hsst). Alternatively, the melting properties of the wild-type fragment can be used to determine T (hsst). This relationship is illustrated by the temperature titration of Example 19, where
Both homoduplexes and heteroduplexes in the mixture resulting from the 9 bp DYS217 mutation gave a slope of about -0.9 min / ° C.

FIG. 30, which is a temperature titration for a DYS271 209 bp mutant standard mixture with a heteroduplex mismatch at position 168 bp, shows how temperature titration information can be obtained experimentally. This data shows that the two heteroduplex and two homoduplex peaks from the mismatch are well separated at 56 ° C. Increasing the temperature gives them a broad peak (60-63 ° C), and increasing the temperature shifts the peak to single-stranded DNA. Under such conditions, single-stranded DNA is separated by sequence as well as size parameters and peaks split. If the separation is optimized for a 168 bp mutation, this elution condition for fast separation moves the single-stranded (melted) peak to the first part of the gradient, thereby skipping this region.

In an example of a preferred method for determining T (hsst), FIG. 31 is the temperature titration of the slowest eluting wild type homoduplex from the data in FIG. The plot represents two inflection points. It should be noted that initially it is 56 ° C. and that this temperature separates the two heteroduplex peaks and the two homoduplex peaks well as in FIG. The retention time for the two wild type homoduplex peaks was about -0.9.
Follow the peaks of the two heteroduplexes with a slope of min / ° C. In FIG. 31, there is a second inflection point at 61.5 ° C., indicating that there is a high melting region in the fragment, but the mismatch is not in this high melting region.

In a preferred embodiment of the present invention, T (hsst) first determines the temperature range of the retention time which decreases at about 0.9 min / ° C., and secondly on the temperature titration plot within that region. The inflection point is obtained and selected from the wild-type homoduplex temperature titration graph by subtracting 1 ° C.

In another preferred embodiment of the present invention, T (hsst) is selected to correspond to the point at which the melting of the homoduplex is 25% complete. In general, the DMIPC of the actual hybridized sample at three different temperatures, for example, about 2 ° C on either side of T (hsst) and the expected T (hsst)
I do. The appearance of two inflection points, as shown in FIG. 31, or the observation of a temperature titration curve with a 0-100% melting range greater than about 5 ° C. requires two temperatures to perform mutation analysis. is there. Either of these observations indicates that the fragment has two regions within the fragment, each requiring a different temperature. An important aspect of the method of the present invention, and based on the highly constant and reproducible nature of the DMIPC method, is that once the correct T (hsst) is determined for a particular fragment, the same temperature is applied to that fragment. Can be used for all subsequent analyses. Further, a database of optimal temperatures corresponding to the analyzed sequences can be assembled for the purpose of describing the conditions required for the analysis of a particular mutation, without having to take steps to experimentally determine the optimal temperature. .

In another aspect of the invention, a thermodynamic mathematical model of the melting behavior of a known fragment can be used to predict the melting behavior of a new fragment without experimentation on the sample itself. This model predicts the optimal temperature for mutation detection and can also be used to evaluate the suitability of fragments for this technique. Indeed, temperature titrations can be determined using fragment sequence information and the behavior predicted by thermodynamic data and models, and combining these models with chromatographic behavior.

Modeling the melting behavior of DNA has been well developed in the literature. However, the reported thermodynamic melting procedure must be modified before it can be fully used for temperature prediction to detect mutations.

[0193] The hydrogen bonding energy of a nucleic acid can be measured. For example, this kind of information is Br
Eslauer et al . ( Proc. Natl. Acad. Sci. 83: 3746 (1986)). For short oligonucleotides, a simple melting model can be used, where adjacent bases (or base pairs) do not have a long-range effect beyond unit boundaries. For long fragments, the inherent helical propensity is combined with conditional probabilities such that the probability of a helical base is strongly affected next to it. Poland Del Fi
Inductive algorithms such as the xam-Freire implementation are required (Poland, Biopolymers 13: 1859 (1974) and Fixam et al. , Biopolymers 16: 2693 (1977)). The parameters used for the Fixam-Freire algorithm were optimized to predict equilibrium melting behavior in aqueous solutions. An example of the implementation of a thermodynamic method for DNA melting is shown in FIG. 32 using the DNA melting profile of the 209 bp mutation in DYS271. The program used to create this plot is BioRad Labora
tories: Program WinM, commercially available from Richmond, CA)
elt ™, version 2. This plot shows that there are two melting domains. This method is referred to as cooperative melting prediction because the melting of any particular base pair is affected next to it. This effect is widespread as long as the neighbor contains a similar GC content. The lower domain contains a heteroduplex mismatch at 168 bp. The plot correlates well with experimental data having two domains and a low melting point domain containing a mismatch.

However, WinMelt ™ or any similar program predicts the optimal temperature for performing mutation detection due to the fact that columns, buffers and solvents can affect the melting temperature of DNA. Can not be used for Since such programs use a collaborative model for melting, without choosing coefficients and cancellations that correlate with the performance of DMIPC, they do not predict well the "temperature titration" observed in DMIPC separations of DNA. In one aspect, a non-cooperative thermodynamic method for modeling DNA melting can be used. However, a preferred thermodynamic model is based on a modification of the collaborative method.

In a preferred embodiment of the invention, the calculated melting temperature is determined by the Poland model Fixam-Freire
It is derived using a first mathematical model, such as execution. The predicted temperature is then derived by adjusting the melting temperature calculated by the second mathematical model.
A preferred example of the second mathematical model is an adjustment formula developed by comparing the calculated temperature based on the first model with the empirically determined temperature observed from temperature titration. This adjustment formula can be used to predict the melting T (hsst) of DMIPC using only sequence information of wild-type or homoduplex DNA. Fi
The adjustment of the xam-Freire calculated temperature depends on the conditions used to obtain the thermodynamic data (Breslaue
USA et al., Proc. Natl. Acad. Sci. USA 83: 3746 (1986)) and the conditions used in DMIPC are necessary to account for the differences.

FIG. 33 depicts the differences between collaborative and non-cooperative studies on DNA melting. In FIG. 33, the base in the DNA sequence is a pontoon on water (horizontal gray rectangle)
And the melting temperature is represented by a ballast (black vertical bars, heavy ballasts are represented by longer bars) and low melting temperatures are represented by heavy ballasts. In a non-cooperative study (Model A), each nucleic acid base pair in the sequence has a particular stability determined by its respective specific hydrogen bonding energy. The stability or melting of each base pair is independent of any surrounding base pairs.
Model A shows that base pairs with high melting properties melt but do not affect the melting of base pairs that have a low tendency to melt. The collaborative study is shown in Model B. In this case, the entire area of the fragment is affected by the weighted cooperative effect of the specific area. In this model, fragments contain domains that have the property of melting at a particular temperature. As the temperature increases, the different domains will each melt at the appropriate temperature.

FIG. 34 represents a modified non-cooperative study on the determination of the melting profile of the 209 bp mutation standard. The plot starts at base 16 and ends at base 192 since each point was created using a 30 base moving load window and the curve was smoothed. FIG. 35 uses the Fixam-Freire cooperative method, but the loop entropy is σ
= 0.01 and represents the same plot with the fragments set to a 10% helix. Thus, it is possible to set the parameters of the cooperative method to simulate the behavior in the non-cooperative method.

An important feature of the present invention provides a method of correlating empirical temperature titration to thermodynamic parameters. Thermodynamic parameters associated with the degree of melting or retention time are used to more accurately model temperature titration. It optimizes temperature cancellation, slope, fragment size-dependent term and loop entropy, and essentially the ten nearest neighbors of free energy. For example, FIG. 36 shows the effect of loop entropy.

FIGS. 38 and 39 show how a tuning equation can be obtained to obtain the expected melting temperature. FIG. 38 is a graph of the calculated melting temperature versus the empirically determined melting temperature. In FIG. 38, the abscissa represents the melting temperature Tm _0.75 corresponding to 75% helical content, ie, Fixam-Freire using a loop entropy of 0.0001.
The point on the melting profile where the denaturation calculated by the model has just begun (75% helix equivalent to 25% melting). The ordinate represents an empirically determined melting temperature that has already been optimized for use in high-throughput screening with DMIPC, preferably for known mutations. The data plotted on the graph shows the melting temperatures determined empirically for each of the 40 experimentally determined temperature titrations of the hybridized fragments.

FIG. 39 is a graph of calculated melting temperature versus predicted melting temperature. The solid line represents the linear fit of the 40 points in FIG. As shown in FIG. 39, the straight line Tm ₀₇₅ ′ is determined by the equation Tm _0.75 ′ = 19.6 + 0.68 · Tm _0.75 .

Also, plotted in FIG. 38 are dashed lines that are one degree higher and lower than the line representing Tm ₀₇₅ ′. Such a dashed line indicates that the accuracy of the predicted Tm ₀₇₅ ′ can be expected to be within about one degree of an empirically determined value.

The predicted melting temperature is obtained by first calculating Tm ₀₇₅ according to the Fixam-Freire algorithm and then preparing Tm _0.75 according to the above equation.

Thus, in a preferred embodiment of the present invention, the above equation is used to predict the point in the melting profile where denaturation just begins (75% spiral comparable to 25% melting on an experimental titration curve). ), Where Tm ' ₀₇₅ is the predicted temperature corresponding to a helical content of 75%, and Tm ₀₇₅ is the Fixam-Freir using loop entropy 0.0001.
This is the temperature calculated from the e-algorithm. This improved the predictive power of calculating the optimal temperature for mutation detection compared to the unaltered algorithm. As the loop entropy period increases, the melting profile becomes less dominated by domains as in step melting (FIG. 36). Thus, this period can be optimized to more accurately model the experimental curve. The melting temperature of the above embodiment was selected at 75% helical content. One skilled in the art will select other points in the empirical temperature titration curve (eg, a helical content of 50% or 90%) and will recognize that the calculated temperature can be obtained and adjusted as described above. Would. The use of thermodynamic data and equations to predict temperature titrations can be accomplished in a number of different ways. The above method is preferred for the present invention. However, since this method is empirical, with the calculated temperature adapted to the actual optimal temperature for the detection of mutations, the use of the equation can be done in other ways, including various weighting factors.

The present invention provides a method for detecting a mutation in a DNA sample using MIPC. Although discussed below with respect to DNA fragments, it should be understood that the present invention can be practiced with any double-stranded polynucleotide.

In one embodiment of the invention, a mixture of homoduplexes and heteroduplexes is formed prior to MIPC analysis. A standard polynucleotide homoduplex is added to the sample, and the mixture is subjected to denaturation (eg, heating the mixture to about 90 ° C). A denatured single-stranded polynucleotide is formed in the denaturation step and is then annealed by slowly cooling the mixture to ambient temperature. If the sample contains the mutation, a new homoduplex and heteroduplex mixture is formed. If the sample does not contain the mutation, only a standard polynucleotide homoduplex will form. In a preferred embodiment, the standard polynucleotide is a "wild-type" polynucleotide.

In the DNA field, heteroduplexes are formed at temperatures lower than those required to denature the remainder of the heteroduplex, ie, the portion of the heteroduplex containing perfectly complementary base pairs. It is known to selectively denature at sites of base pair mismatches to form "bubbles". This phenomenon is generally called partial denaturation, but occurs because the hydrogen bonds between mismatched bases are weaker than the hydrogen bonds between complementary bases. Thus, since the energy required to denature the heteroduplex at the mutation site is small, the temperature required to partially denature the heteroduplex at the site of the base pair mismatch is determined by the temperature of the strand. Lower than the remaining temperature.

Although MIPC separates DNA fragments by base pair length, homoduplex and heteroduplex fragments having the same base pair length can be chromatographed under partial denaturing temperature conditions, ie, heteroduplex as described above. Separation occurs when performed at temperatures at which the main chains are partially denatured. When MIPC is performed under partially denaturing conditions to separate a mixture of heteroduplexes and homoduplexes, heteroduplexes usually elute earlier than homoduplexes.

An important aspect of the present invention is a surprising and previously unreported finding by the Applicant that states the concentration of organic solvent in the mobile phase required to elute DNA fragments from a MIPC column. There is a highly reproducible relationship between base pair length of DNA fragments. Using this relationship, the "preliminary organic solvent concentration" required to elute a DNA fragment of a known size is determined by the organic solvent concentration in the mobile phase required to elute the base pair-length fragment and the DNA. It can be obtained from a reference relating the base pair length of the fragment, eliminating the need for a method to develop elution conditions. This reference is used in the present invention to determine the pre-solvent concentration. “Preliminary solvent concentration” is defined as meaning the concentration of organic solvent obtained from the reference, which is required to elute the corresponding base pair length fragment from the MIPC column under non-denaturing conditions (about 50 ° C.). You.

References relating to the concentration of organic solvent in the mobile phase required to elute DNA fragments having various base pair lengths are represented in the graph of FIG. The relationships depicted in the graph of FIG. 40 can be represented for various ranges of base pair lengths and solvent concentrations. The data used to create the references depicted in FIG. 40 can be represented in graphs or tables. This data can be used to obtain the optimal curve equation. For example, the following equation yielded the curve shown in FIG. 38:% B _p = 19.24 + [53.6 bp / (78.5 + bp)] Reference graph FIG. Was done. Standard fragments of known base pair length were added to the MIPC column, and the concentration of organic solvent in the mobile phase sufficient to elute each fragment was determined when the chromatography was performed at 50 ° C. The organic solvent concentrations so determined were plotted against their respective base pair length fragments to produce FIG. A standard fragment of known base pair length was prepared with pUC18 DNA- Hae I
Obtained from II digestion (S6293, Sigma-Aldrich). The fragments used to create the reference of FIG. 40 were (in base pairs length) 80, 102, 174, 257, 267,
298, 434, 458 and 587. The method described herein for creating the reference of FIG. 40 is considered to be merely one of many other methods that can be used to construct such a reference. For example, other sets of standard fragments could be used.

An essential, and thus previously unrecognized, feature of the present invention on which the concept of reference is based is that under non-denaturing conditions found by the applicant, DNA fragments are separated by their size and The finding is that the separation is highly reproducible using MIPC. Therefore, there is no need to calibrate the MIPC column for each sample analysis. Daily or weekly calibration is usually not required. Once the solvent concentration is determined for a given base pair length, the retention time of the fragment at that solvent concentration is constant not only every day on the same column, but also from column to column. The finding that makes it possible to make a reference on solvent concentration for base pair length without further method development and to predict the pre-solvent concentration for eluting DNA fragments of known base pair length is surprising. is there. Good results are standard (defa
The best practice is to calibrate when a new column or eluate (buffer or mobile phase) is introduced into the instrument.

An example of a pre-selection procedure of the organic solvent concentration in the mobile phase for the detection of mutations by MIPC is described in Example 21 and is shown in FIG. In one aspect of the invention, 2
Prepare three buffers: a first buffer, a counter ionic agent (eg, 0.1M
“A” containing only TEAA) and “B” containing a second buffer, a counter ionic agent and an organic solvent (eg, 0.1 M TEAA, 25% acetonitrile). Such buffers are mixed to achieve the desired organic solvent concentration in the mobile phase during the separation.

To select the preliminary mobile phase organic solvent concentration for detecting mutations, the% B corresponding to the desired base pair length fragment was calculated from the reference graph of mobile phase concentration vs. base pair length (FIG. 40). obtain. Once the pre-solvent concentration is obtained from the reference based on the base pair length of the DNA fragment to be eluted, the "range of lumping fragments" of the organic solvent is selected. The range in which these fragments are bundled is the initial concentration and the final concentration of the organic solvent. The initial concentration includes the concentration of the organic solvent up to the amount required to first elute the DNA molecules in the mixture.
The final concentration contains a sufficient amount of organic solvent to elute the DNA fragment in the mixture last. In a preferred embodiment, the initial solvent concentration in the range that brackets the preselected fragments is% B up to about 15 percent units or less of the pre-solvent concentration. The final solvent concentration in the range that brackets the preselected fragments is a% B that is at least about 5 percent units higher than the pre-solvent concentration. When used on MIPC to detect mutations, chromatography is run on preselected fragments using a gradient based on a clustered range. Although the above technique is widely applicable in practice, the initial and final solvent concentrations can be adjusted for specific applications.

[0213] In a preferred embodiment, the chromatography system is controlled by a computer and run in an automated manner. The chromatography column is equilibrated with the initial solvent concentration. After sample injection, the solvent concentration is increased at a rate of 2% over 5-15 minutes. The gradient is preferably run for at least 10 minutes to allow the preselected fragments to reach a final concentration in the batch. Next, the solvent concentration is immediately set to 100% B, and the column is washed for 2 minutes. The solvent is then reduced to the initial solvent concentration and the column is equilibrated for 2 minutes in preparation for the next sample injection. This entire process is automated and the total time between samples, including column washing and equilibration, is less than 15 minutes.

In another embodiment, the MIPC method described above includes increased throughput in a mutation detection assay,
Alternatively, it can be optimized for other analyzes where a large number of samples need to be screened. For example, once a sample has been analyzed using the preferred automated embodiment of the present invention described above, the method may involve the use of a solvent gradient to elute the heteroduplex faster as long as homoduplex separation is maintained. Optimize by adjusting the slope. Optionally, without waiting for the appearance of the homoduplex, the solvent gradient can be programmed to increase to 100% to wash the column immediately after the retention time of the heteroduplex. After washing, the solvent concentration can be reduced to the initial concentration and the column can be equilibrated for the next analysis. In this manner, the total column chromatography time required for one sample can be reduced from about 15 to less than 10 minutes, and preferably less than about 5 to 7 minutes.

[0215] The determination of the range in which the preselected fragments are batched is performed using the formula:% B_l=% B_p-15 and% B _f =% B_p+5, where% B_lIs the initial percentage of buffer B in the mobile phase,% B_fIs the final percentage of buffer B in the mobile phase, and% B_pIs the preliminary percentage B in the mobile phase obtained from the reference.

In a preferred embodiment of the present invention, the range in which fragments are bundled is automatically selected by software installed in a computer. In this embodiment, the user enters the base pair length of the fragment to be analyzed into the user's interface screen. The software uses the reference data for base pair length versus solvent concentration and the formula shown above to
Calculate injection conditions, initial and final solvent concentrations over a range that bundles preselected fragments to achieve the desired separation, and column wash conditions.

Suitable gradients for use in MIPC mutation detection analysis are represented graphically in FIG. However, as long as the separation of homoduplex and heteroduplex fragments is maintained, 2% per minute
Both sharper linear or non-linear gradients can be used to speed up the analysis. Linear or non-linear gradients of less than 2% per minute can also be used. The latter method is useful to enhance the separation of homoduplex and heteroduplex peaks that do not resolve well.

[0219] The organic solvent in the mobile phase is selected from the group consisting of methanol, ethanol, acetonitrile, ethyl acetate and 2-propanol. A preferred organic solvent in the mobile phase is acetonitrile.

The mobile phase comprises a counter ionic agent from the group consisting of lower alkyl primary, secondary and tertiary amines, lower trialkyl ammonium salts and lower quaternary alkyl ammonium salts. Examples of counter ionic agents include, but are not limited to, octyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate, pyridinium ammonium acetate, cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, butyl ethyl ammonium acetate, methyl hexyl Ammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethyldiethylammonium acetate,
Includes triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate and tetrabutylammonium acetate. The anion in the above example is acetate, but other anions can be used, including carbonate, phosphate, sulfate, nitrate, propionate,
Includes formate, chloride and bromide, or combinations of any of the above cations and anions. Such and other reagents are described by Gjerde et al.
Ion Chromatography , 2nd edition, described by Dr. Alfred Huthig Verlag, Heidelberg (1987). A preferred counter ionic agent is triethylammonium acetate.

To analyze a polynucleotide sample, such as a DNA fragment, using a temperature at which at least partial denaturation occurs, the pH in the mobile phase is typically maintained between about 7 and 9. Preferably, the mobile phase is maintained at about 7.3.

In the use of a preferred embodiment of the present invention, where homoduplex and heteroduplex separations are used for the purpose of detecting mutations, the mixture is denatured and annealed as described above to allow the DNA sample to be wild-type. Hybridize with the type DNA fragment. The DNA sample can hybridize directly with the wild type. The DNA sample can be amplified by PCR and then hybridized to wild type. Alternatively, the wild-type fragment may be added to the sample before PCR amplification. The amplified mixture can then be hybridized after amplification. In each of these three hybridization scenarios, a mixture of homoduplexes and heteroduplexes is generated if the mutation is present in the sample. The sample so prepared is subjected to partial denaturing conditions by MIPC, preferably at 56 ° -58 ° C., using the method of the present invention for preselective pre-organic solvent concentrations and ranges for enclosing fragments as described above. And analyze for the presence of the mutation.

When the method of the invention is used to screen a large number of samples for the presence of a mutation, the throughput of the sample can be reduced by using a steeper gradient over the range of fragments to speed up the analysis of each sample. Up can increase significantly.

In all embodiments and aspects of the invention, polynucleotide fragments are detected as they are separated and eluted from the column. Any detector that can detect a polynucleotide can be used for the MIPC mutation detection method. A preferred detector is an online UV detector. If the DNA fragment is labeled with a fluorescent or radioactive tag, a fluorescence detector or a radioactivity detector can be used, respectively. After detection, the separated fragments are displayed by a video display screen or printed by a printer. The fragment so displayed is
Appears as either side-by-side peaks or bands, ie, in a virtual gel display format as described in US patent application Ser. No. 09 / 039,061, filed Mar. 13, 1998. The format can be selected by the user.

The mutation detection method of the present invention can also be used to detect a mutation in a DNA sample when the base pair length of the fragment is unknown. The base pair length in such a sample can be determined by the method of the present invention, but can only determine the presence of a mutation if the sample is of heterozygous origin. Hybridization of the heterozygous sample results in the formation of heteroduplexes and homoduplexes, which can be detected by DMIPC. However, homozygous mutants do not produce heteroduplexes after hybridization. Homozygous mutants in unknown fragments are therefore not detected. Since the sequence of a DNA fragment of unknown length is also unknown, it is impossible to generate a heteroduplex by hybridization with the wild type.

[0225] In practice, the sample is added to the MIPC column without PCR amplification. Since a primer for the unknown sequence cannot be designed, the unknown sample cannot be amplified. However, if the sample is of heterozygous origin, the sample is hybridized prior to analysis to prepare a mixture of heteroduplex and homoduplex.

Chromatography is performed under non-denaturing conditions, ie, 50 ° C., using a pre-solvent concentration selected from the lowest base pair portion of FIG. 40 of the reference or a similar reference. Since separation of polynucleotides by MIPC is dependent on base pair length under non-denaturing conditions, only fragments smaller than 80 bp will elute using a solvent concentration corresponding to 80 bp. If the peak does not elute after about 15 minutes, the sample should contain fragments longer than 80 bp. Therefore, the solvent concentration in the mobile phase is increased to a concentration corresponding to the next base pair long fragment. The method of incrementally increasing the solvent concentration is continued until a peak is observed. Adjust the solvent concentration at which the unknown sample elutes to elute within about 10 minutes. The chromatography is then repeated under denaturing conditions (56 ° -58 ° C.), using a solvent concentration range that brackets the fragments with a linear 2% gradient per minute as described above. When the sample elutes under partially denaturing conditions, the appearance of low retention time peak (s) in addition to the homoduplex (es) indicates that the unknown sample is heterozygous It is shown that. In addition, by examining the references in FIG. 40, the base pair length corresponding to the solvent concentration at which the unknown fragment was eluted is determined, thereby confirming the base pair length of the unknown fragment.

In general, mixtures of polynucleotides, and especially double-stranded DNA, are effectively separated using matched ion polynucleotide chromatography (MIPC). MIPC separation of polynucleotides at non-denaturing temperatures, typically below about 50 ° C., is based on base pair length. However, traces of multivalent cations throughout the solvent flow path can also cause significant degradation in separation resolution after multiple uses of the MIPC column. This results in increased costs and downtime resulting from the purchase of the exchange column.

Therefore, an effective means for preventing the incorporation of polyvalent metal cations into the components of the separation system, including the contact between the separation medium and the mobile phase. Although not limited to this measure, a washing protocol to remove traces of polyvalent cations from the separation medium and a guard cartridge containing the cation-encapsulated resin in-line with the mobile phase reservoir and MI
Including introducing between PC columns. Such and similar measures taken to prevent the incorporation of multivalent cations into the system have resulted in extended column life and reduced analytical downtime.

Recently, MIPC has been used to separate double-stranded DNA by separating heteroduplexes from homoduplexes.
Has been successfully applied to the detection of mutations in Such separation relies on the lower temperature required to denature the heteroduplex at the site of the base pair mismatch as compared to a perfectly complementary homoduplex DNA fragment. When MIPC is performed at a temperature sufficient to partially denature the heteroduplex, it is referred to herein as denatured matched ion polynucleotide chromatography (DMIPC). DMIPC is typically performed at a temperature between 52 ° C and 70 ° C. The optimal temperature for performing DMIPC is between 54 ° C and 59 ° C.

The precautions taken to remove polyvalent metal cations described above are sufficient to maintain the column shelf life, evidenced by good separation efficiency under non-denaturing conditions. However, Applicants have surprisingly found that the conditions for effective DMIPC separation become more stringent when performed at partial denaturation temperatures. For example, separation of a standard pUC18 Hae III digest at 50 ° C. on a MIPC column will result in all DN in the digest
Good separation of the A fragments was provided. However, homoduplex and heteroduplex standard 209b loaded on the same MIPC column and eluted under DMIPC conditions (ie, 56 ° C)
The DYS271 mutation detection mixture of p (Transgenomic, San Jose, CA) had poor separation of mixture components. Effective separation of homoduplex from heteroduplex at partial denaturation temperatures as required for mutation detection to optimize column shelf life and specific column washing and storage procedures Used in the embodiments of the present invention to be described hereinafter.

Therefore, in one aspect of the present invention, the separation efficiency is maintained by flowing an aqueous solution of a polyvalent cation binder through a column. To maintain the separation efficiency of the MIPC column at the partial denaturation temperature, the column is preferably washed with a polyvalent cation binder solution after about 500 uses or when performance begins to degrade.

Non-limiting examples of polyvalent cation binding reagents that can be used in the present invention include acetylacetone, alizarin, aluminone, chloranilic acid, kojic acid, morin, rhodizonic acid, thionalide, thiourea, α-furyldioxime, nioxime, salicyl Aldoxime, dimethylglyoxime, α-furildioxime, cuperon, α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN, SPADNS, glyoxal-bis (2-hydroxanyl), murexide, α-benzoin oxime, mandelic acid, anthranilic acid,
Ethylenediamine, glycine, triaminotriethylamine, thionalide, triethylenetetraamine, EDTA, metalphthalein, arsonic acid, α, α'-bipyridine, 4-hydroxybenzothiazole, β-hydroxyquinaldine, β-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldic acid, α, α ', α''
-Terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol, rhodizonic acid, salicylaldoxime, salicylic acid, thyrone, 4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzo It is selected from the group consisting of thiazole, rubeanic acid, oxalic acid, sodium diethyldithiocarbamate and zinc dibenzyldithiocarbamate. These and other examples are described by Perrin in Organic Complexing Reagents : Structure, Behavior, and Application to Inorganic Analysis , Robert E.
.Krieger Publisher (1964).

In a preferred embodiment, the polyvalent cation binder is water-soluble. Solubility in water can be enhanced by attaching covalently attached ionic functional groups such as sulfate, carboxylate or hydroxy. The cationic binder must be easily removed from the column by washing with water, an organic solvent or a mobile phase. The cationic binder should not interfere with the use of the column. A preferred multivalent cation binder is EDTA.

The concentration of the cation binding agent can be between 0.01M and 1M. In a preferred embodiment, the column wash solution contains EDTA at a concentration of about 0.03-0.1M.

In another embodiment, the solution comprises an organic solvent selected from the group consisting of acetonitrile, ethanol, methanol, 2-propanol and ethyl acetate. Suitable solutions contain at least 2% of an organic solvent to prevent the growth of microorganisms. In a most preferred embodiment, a solution containing 25% acetonitrile is used for washing the MIPC column.

The multivalent cation binding solution can optionally include a counter ionic agent. The counter ionic agent is selected from the group consisting of lower primary, secondary and tertiary amines, and lower trialkyl ammonium and quaternary ammonium salts. Examples of counter ionic agents include, but are not limited to, octyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate, pyridinium ammonium acetate, cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, butyl ethyl ammonium acetate, methyl hexyl. Ammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethyldiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethyl Comprises ammonium acetate, tetrapropylammonium acetate and tetrabutylammonium acetate. The anion in the above example is acetate, but other anions, including carbonates, phosphates, sulfates, nitrates, propionates, formates, chlorides and bromides, or any combination of cations and anions can be used. These and other reagents are described by Gjerde et al. In " Ion Chromatography , 2nd Edition, Dr. Alfred Heutschig, Heidelberg (1987)."

In one embodiment, the MIPC separation column is washed with a multivalent cation binding solution at a heating range of 50 ° C. to 80 ° C. In a preferred embodiment, the column is washed with a solution containing EDTA, TEAA and acetonitrile in a temperature range from 70 ° to 80 ° C. In a specific embodiment, the solution contains 0.032M EDTA, 0.1M TEAA and 25% acetonitrile.

[0238] Column washing can range from 30 seconds to 1 hour. For example, in a high throughput DMIPC assay, the column can be washed for 30 seconds after each sample and subsequently equilibrated with the mobile phase. Since DMIPC can be automated by computer, the column washing procedure can be incorporated into the mobile phase selection program without further operator involvement. In a preferred procedure, the column is washed with the polyvalent cation binding agent for 30-60 minutes, preferably at a flow rate in the range of about 0.05-1.0 mL / min.

In one embodiment, the DMIPC column is tested using a homoduplex and heteroduplex standard mutation detection mixture after approximately 1000 sample analyzes. If the separation of the standard mixture is poor compared to the freshly washed column, the column is then washed with the polyvalent cation binding solution for 30-60 minutes at a temperature above about 50 ° C to restore the resolution.

In another aspect, Applicants have found that column separation efficiency can be protected by storing the column separation medium in a column containing a solution containing a polyvalent cation binder. The binder solution may also include a counter ionic agent.
Any of the above multivalent cation binders, counter ionic agents and solvents are suitable for the purpose of storing MIPC columns. In a preferred embodiment, the column packed with the MIPC separation medium is stored in an organic solvent containing a polyvalent cation binder and a counter ionic agent. An example of this preferred embodiment is 0.032 M EDTA and 0.1 M tetraethylammonium acetate in 25% aqueous acetonitrile. In preparation for storage, a solution of the above polyvalent cation binder is passed through the column for about 30 minutes. Next, change the column to HP
Disconnect from the LC instrument and close the column end with a commercially available threaded end cap made of a material that does not release polyvalent cations. Such end caps can be made from coated stainless steel, titanium, organic polymers, or any combination thereof.

Applicant states that washing the MIPC column with a multivalent column binder restores the column's ability to separate heteroduplexes and homoduplexes in a mutation detection protocol under DMIPC conditions. The effect of the surprising findings by is described in Example 24 and is shown in FIGS.

As described in Example 24, Applicants have used MIPC columns in mutation detection.
During use, a decrease in the resolution of homoduplexes and heteroduplexes was noticed. But pUC18 Hae When a DNA standard containing the III digest (Sigma / Aldrich Chemical Co.) was added at 50 ° C., no apparent decrease in resolution was observed.
(Not shown). To further test column performance, a mixture of homoduplex and heteroduplex in a 209 bp DNA standard was added to the column under DMIPC conditions at 56 ° C (Kuklin et al.Genetic Testing 1: 201 (1988). Surprisingly, the peaks representing the homoduplex and heteroduplex of the mutation detection standard are not well separated.
Was observed (FIG. 42).

In FIG. 43, when the guard cartridge containing the cation capture resin is placed in-line between the solvent reservoir and the MIPC system, some amount of homoduplex and heteroduplex separation of the standard mutation detection mixture was observed. The improvement is shown. The chromatography shown in FIG. 43 was performed at 56 ° C. The column used in FIG.
The same column was used for the separation shown in Figure 2 and for the separation of the standard pUC18 Hae III digest.

FIG. 44 represents the separation of the homoduplex and heteroduplex of the standard mutation detection mixture at 56 ° C. on the same column used to generate the chromatograms of FIGS. 42 and 43. . However, in FIG. 44, the column was washed at 75 ° C. with a solution comprising 32 mM EDTA and 0.1 M triethylammonium acetate in 25% acetonitrile for 45 minutes before adding the sample. FIG. 44 shows four distinct peaks representing two homoduplexes and two heteroduplexes of the standard 209 bp mutation detection mixture. Restoration of the separation capacity of the MIPC column under DMIPC conditions after washing with a solution containing a cation binder compared to the chromatograms in FIGS. 42 and 43 clearly demonstrates the efficacy and utility of the present invention. ing.

In an important aspect of the present invention, Applicants have developed a standardized standard for evaluating the performance of DMIPC separation media. DMIPC, as used herein, is defined as a method for separating heteroduplexes and homoduplexes using non-polar beads in a column, where the method removes nucleic acids from the counterion agent and beads. Using an organic solvent to separate the beads, where the beads have at least 0.1
utation Separation Factor (MSF). In an operational embodiment, the beads have a mutational segregation factor of at least 0.2. In a preferred embodiment, the beads have a mutation segregation factor of at least 0.5. In an optimal embodiment, the beads have a mutation segregation factor of at least 1.0.

The performance of the column is demonstrated by the highly efficient separation of heteroduplex and homoduplex by DMIPC. The highest criterion for measuring performance was found to be the mutation segregating factor as described in Example 23. It is measured as the difference between the peak areas of the separated heteroduplex and homoduplex. A correction factor can be applied to the region generated below the peak. The following factors can affect the calculated peak area and its reproducibility: baseline drawn, peak normalization, variable temperature control, variable elution conditions, detector instability, flow rate Instability, variable PCR conditions and degradation of standards and samples.

Mutation segregation factor (MSF) is determined by the following formula: MSF = (area peak 2−area peak 1) / area peak 1 where area peak 1 was measured after wild type DMIPC analysis Peak area, and area peak 2 is the DMIP of the hybridized mixture containing the putative mutation.
C The total area of the peak (s) measured after the analysis, taking into account the correction factors described above, where the peak height has been normalized to the wild type peak height.
The separating particles are loaded onto an HPLC column and they separate a standard hybridized mixture containing the wild-type 100 bp lambda DNA fragment fragment and the corresponding 100 bp fragment containing the A to C mutation at position 51. Test your ability.

[0248] Other features of the present invention will become apparent from the following description of embodiments, which provide, but do not limit, the invention in detail.

All references cited herein are hereby incorporated by reference.

The procedures described in the past in the examples below were performed in the laboratory. The procedure described in the present tense has not yet been performed in the laboratory and has been reduced constructively for the purpose of this application.

[0251]

【Example】

Example 1 Sample Concentration by Adding Multiple Sample Aliquots 0.2 μg of a standard sample containing 80, 102, 174, 257, 167, 298, 434, 458, 587 base pair DNA fragment 0.2 μg pUC18 Hae III restriction The digest (D6293, Sigma / Aldrich Chemical Co.) was injected onto the MIPC column and the column was eluted under gradient conditions to generate the reference chromatogram shown in FIG. The pH 7 mobile phase consisted of component A, 0.1 M triethylammonium acetate (TEAA) and component B, 0.1 M TEAA, 25% acetonitrile. The gradient conditions used for the gradient shown in FIG.
The column was then equilibrated with 55-65% B, 65% B for 2.5 minutes, 100% B (column wash) for 1.5 minutes, and 35% B for 2 minutes for the next 7 minutes. Back pressure 2100
psi, temperature 50 ° C., UV detection 260 nm, flow rate 0.75 mL / min. The column was DNASep ™ (621-0546, Transgenomic, San Jose, Calif.) 50 × 4.6 mm id.

In another experiment, another 5 μL of the sample pUC18 DNA- Hae III digest was added to the MIPC column and washed in a single mode of 35% B for 10 minutes. FIG. 2 shows that no DNA fragments were eluted, as represented by the flat baseline of the chromatogram. A second 5 μL of the pUC18 DNA- Hae III digest was added to the same MIPC column, and the column was eluted with 35% B followed by the gradient shown above for FIG. 1 (FIG. 3).

Example 2 Melting Profile of a 200 bp Fragment Based on Primer Design and Mutation Detection PCR of 6 genomic DNAs of the p53 exon with a C to T mutation at position 13346 using three different sets of primers Amplified by Each primer set was designed to generate DNA fragments (amplicons) with mutations located in different melting domains. The melting profiles calculated by WinMelt ™ showing the melting domains of the three fragments and their relative positions in the fragments are shown in FIG. 200b
The primers that generated pFragment 1 were designed to place a mutation at position 159 near the 3 'end of the fragment. The primers that generated the 201 bp fragment 2 had 5 primers near the 5 'end of the fragment.
It was designed to place the mutation at position 9. The primer that generated the 200 bp fragment 3 was designed to place a mutation at position 91 near the center of the fragment. The melting profiles of the three fragments in FIG. 4 are represented in terms of mutations. The temperature and sequence position do not specifically indicate the temperature and base pair length, but are shown in relative temperature and sequence position.

The set of primers used is shown in the following table:

[0255]

[Table 1]

The PCR conditions used with each of the three sets of primers are described below. For each set of primers, the components shown in the following table were mixed, vortexed to ensure mixing, and centrifuged:

[0257]

[Table 2]

Next, aliquots were dispensed into PCR tubes. The PCR tube was placed in the thermocycler and the temperature cycling program was started.

The cycling program parameters are shown in the following table:

[0260]

[Table 3]

The PCR products generated by the above procedure were analyzed by DMIPC using the Transgenomic WAVE ™ DNA Fragment Analysis System (Transgenomic, San Jose, CA). Following the initial DMIPC analysis, the samples were hybridized by heating at 95 ° C for 4 minutes and then slowly cooled to 25 ° C. The sample was then analyzed again by DMIPC.

The DM used for the separation of mutation detection shown in the chromatograms of FIGS. 5, 6 and 7
The IPC conditions are shown below:

[0263]

[Table 4]

The mobile phases were solvent A: 0.1 M TEAA and solvent B: 0.1 M TEAA in 25% acetonitrile
At a flow rate of 0.9 mL / min. The column temperature in DMIPC was 61 ° C for Fragment 1, 61 ° C for Fragment 2 and 62 ° C for Fragment 3.

Example 3 Melting Profile of 100-400 bp Fragment Based on Primer Design and Detection of Mutations The template was a bacteriophage lambda with a mutation at position 32061 (31500-32500
(Available from FMC, Bioproducts, Rockland, ME) and amplified by PCR using four different sets of primers. Lambda array is O'Conne
J et al., Biophys . J 74: A285 (1988), and Garner et al., Mutation Detection 97 4th International Workshop, Mutation Detection 974 ^th Interna.
National Workshop, Human Genome Organization), published May 29-June 2, 1997, in poster No. 29, Brno, Czech Republic. Each set of primers was designed to generate amplicon fragments such that each had a mutation located in a different melting domain. The melting profiles, calculated using WinMelt ™, indicating the melting domains of the four amplicon fragments and their relative positions in the fragments are shown in FIG. The primer that generated the 248 bp fragment 1 was designed to place a mutation at position 198 near the 3 'end of the fragment. The primer that generated the 253 bp fragment 2 was designed to place the mutation at position 50 near the 5 'end of the fragment. The primer that generated the 400 bp fragment 3 was designed to place the mutation at position 199 near the center of the longest fragment. The primers that generated 100 bp fragment 4 were designed to place the mutation at position 51 near the center of the shortest fragment. The melting profiles of the four fragments in FIG. 8 are expressed in terms of mutations. Temperatures and sequence positions do not show specific temperatures and base pair lengths, but are shown as relative temperatures and sequence positions.

The set of primers used is shown in the table below:

[0267]

[Table 5]

The PCR conditions used with each of the three sets of primers are described in the table below. All components were mixed, vortexed to mix well, and centrifuged. Aliquots were then dispensed into PCR tubes as shown in the table below:

[0269]

[Table 6]

The PCR tubes were placed in the thermocycler and the temperature cycling program was started. The cycling program parameters are shown in the following table:

[0271]

[Table 7]

The PCR products generated by the above procedure were analyzed by DMIPC using the Transgenomic WAVE ™ DNA Fragment Analysis System (Transgenomic, San Jose, CA). Following the initial DMIPC analysis, the samples were hybridized by heating at 95 ° C for 4 minutes and then slowly cooled to 25 ° C. The sample was then analyzed again by DMIPC.

The DMIP used to isolate the mutation detection shown in the chromatograms in FIGS. 9 and 10.
The C conditions are shown below:

[0274]

[Table 8]

The DMIPC conditions used for the isolation of mutation detection shown in the chromatogram in FIG. 11 are shown below:

[0276]

[Table 9]

The DMIPC conditions used for the separation of mutation detection shown in the chromatogram in FIG. 12 are shown below:

[0278]

[Table 10]

For FIGS. 9-12, the mobile phase contained solvent A: 0.1 M TEAA and solvent B: 0.1 M TEAA in 25% acetonitrile at a flow rate of 0.9 mL / min. The column temperature in DMIPC was 62 ° C. for fragment 1, 62 ° C. for fragment 2, and 63 ° C. for fragment 3, and 60 ° C. for fragment 4.

Example 4 Polymerase Chain Reaction (PCR) Materials and Procedures Samples for PCR amplification were obtained from Perkin-Elmer Applied Biosystems (Foster, CA) using GeneAmp (PCR reagent kit (Part No. N801-0055).
) And included AmpliTaq ™ (DNA polymerase, GeneAmp (10x PCR buffer, dNTP 'and DNA templates and primers. To compare the effect of DNA polymerase on the degree of PCR regeneration, A further sample is AmpliTaq (
® DNA polymerase cloned Pfu DNA polymerase (Cat. No. 60015)
3, Stratagene, La Jolla, California, USA) and PFUTubo (
(Trademark) DNA polymerase (Cat. No. 600250, Stratagene). In these samples, the GeneAmp 10 × PCR buffer was replaced with a 10 × cloned Pfu DNA polymerase reaction buffer (Cat. No. 600153-82, Stratagene). DNA template was 10 mM Tris-HCl, pH 8.0 (Cat. No. 0291, technova: Teknova, Half Moon Bay, CA, USA), 1 mM EDTA, pH 8.0 (Cat. No. 0306, technova), 10 mM NaCl (Cat. No. S7653, Sigma, St. Louis, MT, USA) diluted to 100 ng / mL. A 500-bp product was amplified from a DNA control template (bacteriophage lambda DNA) from control primer # 1 (5'-GATGAGTT CGTGTCCCTACACTACTGG-3 ') and control primer # 2 (5'-GGTTATCGAATCAGCCACAGCGCC-3'). .

The components were added to PCR tubes (Part No. TFI-0201, MJ Research, Watertown, Mass., USA) in the following order: 53 μL ddH20, 10 μL 10 × PCR buffer, 200 μM each dNTP, 2.5 U / 100 μL AmpliTaq ™, cloned Pfu or PFUT
urbo ™ DNA polymerase, 1 μM control primer # 1, 1 μM control primer # 2 and 1 ng DNA control template for a total of 100 μL. Amplification was performed with MJ Research (Watertown, Mass., USA) PTC-100 thermocycler or 15
Performed using 35 PCR cycles.

Example 5 Cleavage of a 500 bp PCR Product to Create Blunt Ends A 500 bp PCR product was prepared at bases 37, 47, 452 and 457 using Hae III endonuclease (R5628, Sigma-Aldrich, St. Louis). (Mt., Montana, USA), yielding a 405 bp blunt-ended product. Hae III with ddH20
Diluted at 1:30. Hae III diluted in PCR tubes was added to the PCR product (1 part diluted Hae III: 2 parts PCR sample), vortexed and then incubated at room temperature. PCR samples were analyzed before, during and after digestion with Hae III to ensure complete cleavage.

Example 6 MIPC of a 500 bp PCR product and a 405 bp blunt-ended product As shown in FIG. 17, a 405 bp blunt-ended product was replaced by a 500 bp PCR product by a Hae III
Prepared after cleavage with endonuclease. At 0 minutes, a complete 500 bp product peak was seen before cleavage by Hae III. After 15 minutes, the 500 bp product was partially cleaved showing three species: uncleaved 500 bp, partially cleaved intermediate species with truncated ends, and the cleaved 405 bp product. After a final 30 minutes, cleavage of all four final fragments was completed. MIPC analysis conditions using the WAVE ™ DNA Fragment Analysis System are as follows: solvent A: 0.1 M TEAA, solvent B: 0.1 M TEAA, 25% acetonitrile, 31-53% solvent B for 0.1 min, 12 min With a linear gradient of 53-77% B; flow rate: 0.9 mL / min; temperature: 50 ° C .; detection: UV, 254 nM.

Example 7 Analysis of PCR-Induced Mutations by MIPC and Corresponding Reaction Product Profiles At baseline, samples intentionally enhance the mutation rate, but are found in a typical PCR lab. Shed under the conditions to get. This control sample, which contains a PCR mutation at 61.7% in the fragment, was amplified in 35 PCR cycles using AmpliTaq ™ DNA polymerase. Further samples were run to study the factors that had an effect on the degree of PCR regeneration. Such samples were identical to the control but differed in the number of cycles or the type of DNA polymerase. PCR samples amplified using AmpliTaq ™ for 15 cycles had a mutation rate of 51.6%. The 10.1% reduction from 35 amplification cycles is expected because fewer cycles have less opportunity for mutations to occur. Replacing AmpliTaq ™ DNA polymerase with Pfu or PFU Turbo ™ had a positive effect on the regeneration of PCR products. PFU Turbo ™ had a rate of error equal to Pfu , but resulted in higher yields due to a new thermostability factor (Stratagene, PFU Turbo ™ DNA Polymerase Instruction Manual, Rev. # 107001) . Mutation rates were reduced to 24.8% and 18.4% for Pfu and PFU Turbo ™, respectively (FIG. 20).

Further experiments were performed to further optimize the PCR conditions using Pfu and PFU Turbo ™. The dNTP concentration was reduced from 200 μM to 100 μM for each dNTP, the primer concentration was reduced from 1 μM to 0.2 μM, and the sample was changed to “touchdown”
After amplified using down) "PCR protocols, mutation rate was reduced to 23.1% and 17.8% respectively for Pfu and PFUTu rbo (TM).

Example 8 Analysis of PCR yield by MIPC and corresponding reaction product profile A 405 bp PCR product was amplified using AmpliTaq ™, Pfu and PFU Turbo ™. Replacing AmpliTaq ™ DNA polymerase with Pfu or PFU Turbo ™ had a positive effect on PCR product yield. The yield of homoduplex product was determined from the integral of the peak area and the yield of a standard of known amount. AmpliTaq ™ gave the lowest yield (10 ng) and a large amount of primer dimer (2 min peak). Pfu and PFU Turbo ™ gave yields of 35 ng and 93 ng, respectively, and low amounts of primer dimer (FIG. 19). MIPC analysis conditions using the WAVE ™ DNA fragment analysis system are as follows: solvent A: 0.1 M TEAA, solvent B: 0
.1M TEAA, 25% acetonitrile; linear gradient from 31-53% solvent B for 0.1 min, 53-71% B for 9 min; flow rate: 0.9 mL / min; temperature: 66 ° C; detection: UV, 254 nM.

Example 9 Effect of Temperature on Separation of Homoduplexes and Heteroduplexes by MIPC A 405 bp PCR product amplified using AmpliTaq ™ was chromatographed under partially denaturing conditions. (FIG. 18). The samples were run at 62 ° C, 64 ° C and 66 ° C. The main peak corresponded to pure homoduplex DNA of 405 bp in length. It was concluded that increasing the temperature decreased the retention time due to partial denaturation. At 66 ° C, the sample showed the greatest separation between homoduplex and heteroduplex. MIPC analysis conditions using the WAVE ™ DNA fragment analysis system are as follows: solvent A: 0.1 M TEAA, solvent B: 0.1 M TEAA, 25% acetonitrile; 31-53% solvent B in 0.1 min, 9 53 ~ in minutes
Flow rate: 0.9 mL / min; temperature: 66 ° C .; detection: UV, 254 nM.

Example 10 Effect of Hybridization on the Analysis of PCR Replication Reproduction by MIPC The final hybridization cycle is required to melt and reanneal the strands to form homoduplexes and heteroduplexes It is. To inhibit further polymerization activity, EDTA (Cat. No. 0306, Technonova, Half Moon Bay, USA) was added to a final concentration of 20 mM to chelate any free magnesium remaining in the PCR samples. The sample was then placed in a thermocycler (model PTC-100, MJ Research), heated at 95 ° C for 4 minutes, and then slowly cooled to 25 ° C. After hybridization, the heteroduplex peak (at 5.6 minutes) increased from 8.2% to 23.1% mutation, indicating that an artificially low mutation rate was achieved by eliminating this step.
The MIPC analysis conditions using the WAVE ™ DNA fragment analysis system are as follows: solvent A: 0.1 M TEAA, solvent B: 0.1 M TEAA, 25% acetonitrile; 31-53% solvent B in 9 minutes, 9 Linear gradient of 53-71% B in min; flow rate: 0.9 mL / min; temperature: 66 ° C
Detection: UV, 254 nM.

Example 11 Separation and Isolation of Pure PCR Product by MIPC 138 ng of a 209 bp DNA fragment (DYS271 from human Y chromosome) was injected onto a MIPC column and separated under non-denaturing conditions (52 ° C). The peak corresponding to this fragment is
Collected in 300 μL vials. Aliquots of the collected liquid (5 μL and 20 μL in separation experiments) were subsequently amplified by direct PCR and quantified (in nanograms) by re-analysis on the WAVE ™ system. A control was made by diluting a portion of the original so that the control and the collected fractions gave the same area when reinjected into the WAVE ™ system. The initial copy number of the DNA in the control then matched the copy number of the collected fraction. Differences in PCR yield reflected only the effect of the elution buffer from which the fraction was collected. Using the WAVE ™ DNA Fragment Analysis System
MIPC analysis conditions are as follows: solvent A: 0.1 M TEAA, solvent B: 0.1 M TEAA, 25%
Acetonitrile; linear gradient 43-57% solvent B in 0.5 min, 29-71% B in 7.5 min; flow rate: 0.9 mL / min; temperature: 52 ° C; detection: UV, 254 nM. Using the following reagents: 10 mM
Tris buffer pH 8, 100 μM each dNTP, 0.2 μM primer, 2.5 mM MgCl ₂ , 2
.5U Perkin-Elmer AmpliTaq ™.

Example 12 Preparation of PCR Primers Based on MIPC Detection of Excessive Primer Dimer Formation A long range primer design requires a highly sensitive and reproducible method for detecting unknown mutations. is necessary. To achieve such a level of accuracy, it is necessary to fragment exons into 150-450 bp segments, despite the fact that a single base mutation was detected in the 1.5-kb fragment. Good. If the sequence is known, the melting profile is constructed using the appropriate software. Areas different than 15 ° C in the same fragment should be avoided. If this is not possible, replace dGTP with N ⁷ -dGTP (7-deaza-2′-deoxyguanosine 5′-triphosphate) to lower the melting temperature of the GC-rich region, or use a short G or G base of 3 or 4 bases. -Include a C clamp.

For the design of partial primers, universal sequencing primers or primers with non-template tails such as the T7 promoter should be avoided. The difference in Tm between primers 1 and 2 is less than 1 ° C. The difference between the primer and the template is about 25 ° C. 3'-Hentomer of each primer is G, -6kcal
/ Mole more stable. Any possible primer dimer is at least 5 kcal / mole less stable than the 3'-pentomer. To avoid degradation, Tris-HCl (pH 8.0
) Storage in a buffer is preferred over pure water.

Example 13 Adjustment of PCR Variables to Reduce PCR-Induced Mutations Detected by MIPC Despite Using Proofreading Enzymes For small fragments up to 250 bp, Taq or Taq Gold (trademark) ) (Perkin-Elmer Applied Biosystems, Foster City, Calif.) Usually gives satisfactory results, but Pfu still gives the best results, especially at 25 cycles of amplification (Taq is one in 15% of dsDNA fragments). These mutations can easily occur). As a guideline, use Taq only when the number of cycles multiplied by base pairs is less than 10,000. PCR reactions using the buffer recommended by manufacturer, and should be carried out under conditions that the following recommendation: 2.0~2.5mM MgCl _2, each dNTP 100 [mu] M, each primer 0.2 [mu] M, Pfu of 2.5U per 100 [mu] L, Taq (Or Taq Gold ™); about 50 ng mold.

Example 14 Reduction of Excessive By-products Detected by MIPC Resulting from Excessive PCR Cycles The probability of DNA sequence change is the number of cycles (n) and / or polymerase error per nucleotide (p) : F = np / 2 (where f is the frequency of errors, n is
It can be manipulated experimentally by varying the number of pcr cycles, and p is the percentage of errors. For example, the expected error frequency is 1 × 10 ⁻³ after 20 cycles, ie, one error at 1000 nucleotides, when p = 1 / 10,000. The number of cycles should be kept to the minimum necessary to prepare a workable amount of product DNA.

Example 15 Hybridization Procedure for Analyte The PCR method is stopped by adding 5 mM EDTA, 60 mM NaCl, 10 mM Tris-HCl pH 8.0 to the reaction mixture. The reaction mixture is heated to 95 ° C. for 3 minutes and
Cooled to 25 ° C. Homozygous mutants must be combined with wild type in an approximately 1: 1 ratio prior to hybridization.

Example 16 Description of Temperature-Dependent DMIPC Analysis Method The following example relates to FIG. 24 (heteroduplex separation over a temperature range of 51 ° C. to 61 ° C.).

A 209 base pair fragment from the human Y chromosome DYS271 locus with an A to G mutation at position 168 was hybridized with the wild type as described in Example 15 above, and the sample was subjected to MIPC The column (50 mm × 4.6 mm id) was injected at 51 ° C. The column is 0.9mL /
Eluted with a gradient of acetonitrile in 0.1 M TEAA over 7 minutes. Chromatography was monitored at 260 nm using a UV detector. Heteroduplexes present in the mixture did not denature at 51 ° C .; thus, a single peak was observed.

As can be seen in FIG. 24, sample injection and chromatography was performed at a temperature of 1 ° C.
Was repeated with an incremental rise. A shoulder was observed at 53 ° C. on the lower retention time side of the main peak, indicating the possibility of the presence of a heteroduplex. At 54 ° C., three peaks were observed. And four peaks were observed at 55 ° -58 ° C., indicating a clear presence of the mutation. The two lower retention time peaks were two heteroduplexes, and the higher retention time peak was homoduplex.

Example 17 Determination of T (hsst) by Initiating DMIPC Analysis at the Melting Temperature Calculated by Software Site Separation Temperature T ((209) of the 209 base pair heteroduplex heteromutant of Example 16 above. hsst) is determined by applying the formula T (hsst) = X + mT (w) using a software package such as MELT. The melting temperature T (w) determined by MELT was 52 ° C. The sample was applied to the MIPC column at 52 ° C and eluted as described in Example 16 above. A single peak is seen. This result indicates that the sample contains no mutations (heteroduplex) or that the temperature at which the chromatography was performed was T (
hsst). Therefore the temperature is raised by 2 ° C. to 54 ° C. and the chromatography is repeated. Three peaks appear, indicating the presence of the mutation. The temperature is raised an additional 2 ° C. to 56 ° C. and the chromatography is repeated. Separation is optimized as evidenced by the appearance of two distinct heteroduplex peaks at low retention times, as well as two distinct homoduplex peaks at high retention times. Using T (w) = 52, m = + 1 and X = + 4 in the above equation, T (hsst) is determined to be 56 ° C.

Example 18 Determination of T (hsst) by Initiating DMIPC Analysis at the Melting Temperature Determined from the UV Melting Profile T (hsst) follows the format of Example 17, but S.Lim [Varian Technical Note (Varian Technical Note) “DNA denaturation using the Cary 1/3 Thermal Analysis System”, No.
UV-51, pages 1-5, June 1991, Varian Associates,
Palo Alto, Calif.)] And used a pH 7.3 buffer comprising 0.1 M TEAA and acetonitrile at the concentration obtained from FIG. 38 for the 209 bp fragment, based on the UV melting profile obtained. To decide.

Example 19 Temperature Effect on Retention Time and Resolution of Homoduplex / Heteroduplex Mixtures A 209 bp fragment from the DYS271 locus on the human Y chromosome with an A to G mutation at position 168 Was hybridized with the wild type as described in Example 15 above, and the sample was injected at 51 ° C. onto a MIPC column (50 mm × 4.6 mm id). Chromatography was monitored at 260 nm using a UV detector. Heteroduplexes present in the mixture did not denature at 51 ° C .; thus, a single peak was observed. Column is 0.9m
At L / min, eluting with solvent A: 0.1 M TEAA and solvent B; 0.1 M TEAA, 25% acetonitrile using the following gradient;

[0301]

[Table 11]

DYS271 209 bp mutation standard mixture of heteroduplex and homoduplex species (available as a mutation standard from Transgenomic, San Jose, Calif .; mutations were performed by Seielstad et al., Hum . Mol. Genet. .3: 2159 (described by 1994) was measured as a function of oven temperature starting at 50 ° C., and 0.5 and 0.3 degree increments at 57.5 ° C. (FIG. 37) for temperature titration. )
Continued until. The HPLC instrument was controlled from the customized system software via the RS232 interface. Software control was from a custom prototype front-end software package (a thoroughly modified version of WAVEMaker ™) from Transgenomic, Inc. (San Jose, CA). The oven was manufactured from a model PTC200 MJ Research Thermocycler modified to include a preheat line (150 cm x 0.007 "id) made of DNASep ™ columns and PEEK tubes.The preheat tube was a PCR. Interwound between the wells of the tube (ie physically located around the well itself and in contact with the 96-well heating block) and then placed in the machined cavity of the thermocycler The reaction in the oven was high, requiring about 10 seconds to reach the set point temperature and about 2 minutes to bring the fluid to the set point temperature. It was much faster than a regular oven, because the oven is Peltier cooled,
The temperature rise and fall occurred quickly.

The mobile phase used for the separation comprised 0.1 M TEAA (solvent A) and 0.1 M TEAA in 25% acetonitrile (solvent B). MIPC columns were run at 0 using the gradient shown below.
Eluted at a flow rate of .9 mL / min.

[0304]

[Table 12]

FIG. 37 illustrates an important dependence on separation oven temperature, and more importantly on mobile phase fluid temperature. Even a temperature change of only 0.1 ° will be reflected in the change in retention time and peak pattern. Therefore, to determine the genotype of a mutation,
It is important that the temperature be reproducible between experiments and between instruments, preferably at least 0.1 ° C and most preferably at least 0.05 ° C. This data indicates that temperature control is important within a range better than 0.1 ° C based on the resolution of homoduplexes and heteroduplexes. Actual slope d from the plot (retention time in minutes)
/ D (temperature (° C)) was measured to be -0.875 min / ° C.

Example 20 Generation of a reference graph of mobile phase versus nucleotide base length Standard pUC18 Hae III restriction enzyme containing DNA fragments having base lengths of 80, 102, 174, 257, 267, 298, 434, 458 and 587 The digest is applied to the MIPC column at non-denaturing temperature
At ℃. The column was eluted with a mobile phase linear gradient comprising solvent A (0.1 M TEAA, pH 7) and solvent B (0.1 M TEAA in 25% acetonitrile). The flow rate was 0.75 mL / min and detection was UV at 260 nm. The gradient is shown below:

[0307]

[Table 13]

The reference curve (FIG. 38) takes the retention time of each fragment and calculates the corresponding% from the slope.
It was constructed by finding B. Next, as shown in FIG. 38,% B was plotted against base pairs.

Example 21 Selection of Mobile Phase for Mutation Detection The next example is a 500 base pair DNA fragment. However, the same method of solvent selection is used for DNA fragments of any base pair length.

To select the preliminary mobile phase organic solvent concentration for mutation detection, the% B corresponding to the desired base pair length fragment is obtained from the reference graph (FIG. 40). Using this reference, a pre-solvent concentration of 65% B is required to elute the 500 bp fragment from the MIPC column under non-denaturing conditions at 50 ° C. In this example, the solvent concentration of the mobile phase was increased by 5 percent to 70% B to ensure complete removal of the fragments from the column during the mutation detection analysis. This is the final mobile phase organic solvent concentration.
The initial mobile phase solvent concentration is set by subtracting 15 percent units from the preliminary concentration B determined from the reference. In this example, the initial mobile phase solvent concentration is set to 50% B (65% minus 15%). The mobile phase concentration for the range that brackets the fragments used for mutation detection by MIPC increases the percentage of B in the mobile phase at 2% per minute over 10 minutes. This is followed by about 10 minutes to wash the column.
Immediately increase to 0% B. After this wash, the mobile phase concentration is returned to the initial 50% B for 2 minutes to equilibrate the column for the next sample injection. The graphical representation of the above gradient is
This is shown in FIG.

The above procedure is initially used under non-denaturing conditions, eg, at 50 ° C., to establish the quality of a DNA sample amplified by PCR. To detect mutations in the amplified sample, the above procedure is repeated under partially denaturing conditions, eg, 57 ° C. The appearance of one or more low retention time peaks in the chromatogram under partially denaturing conditions indicates the presence of one or more mutations (heteroduplex).

Example 22 Use of PEEK and Titanium Frit in Mutation Detection The resin lot used in this example passed the Mutant Separation Factor test with a value> 0.1 as shown in Example 23 and It was shown to be suitable for detection.

The type of titanium frit used for the column has a significant effect on the ability to separate heteroduplexes and homoduplexes. The experiments were performed using titanium frit from two separate lots. All frit procurement used in this example was obtained from Isolation Technologies (Hopedale, Mass.). The elution conditions used in this example were the same as those used in FIG.

FIG. 45 shows the separation of the 209 bp mutagenic standard of DYS271 from lot A using titanium frit. Four peaks appear and the two heteroduplex peaks clearly separate from the two homoduplex peaks, with a retention time of 3.07 for the heteroduplex.
And 3.24 minutes, and 3.65 and 3.82 minutes for the homoduplex. However, in FIG. 46, the same 209 mutation standard using the titanium frit from lot B gave different results: only three peaks appeared and the second homoduplex peak disappeared altogether. The type of titanium frit used in this way can affect such separation of heteroduplexes from homoduplexes. Treatment with 0.5 M EDTA tetrasalt (sonication for 10 minutes and soaking for several days) improved Lot B performance.

In general, columns containing PEEK frit cannot perform satisfactory separation of heteroduplexes from homoduplexes without very rigorous washing. FIG. 47 shows that the optimal temperature at which heteroduplexes are observed to separate from homoduplexes is 56 ° C. The only peak appears at a retention time of 3.45 minutes.

Example 23 Determination of Mutation Isolation Factor Mutation Isolation Factor (MSF) is determined according to the following formula: MSF = (area peak 2−area peak 1) / area peak 1 where peak 1 is wild Area is the area of the peak measured after DMIPC analysis of the form, and area peak 2 is the area of the hybridized mixture containing the putative mutation.
The total area of the peak (s) measured after DMIPC analysis, where correction factors were considered above, where the peak heights were normalized to the wild type peak height. The separating particles are packed on an HPLC column and their ability to separate a standard hybridized mixture containing a wild-type 100 bp lambda DNA fragment and a corresponding 100 bp fragment with an A to C mutation at position 51. Test for

Depending on the filling volume and the polarity of the filling, this procedure requires the choice of propellant concentration, pH and temperature. Any solvent can be used: acetonitrile, tetrahydrofuran, methanol, ethanol or propanol. The counter ionic agent is selected from trialkylamine acetate, trialkylamine carbonate, trialkylamine phosphate or any type of cation capable of forming a matched ion with the polynucleotide anion.

As an example of determining the mutation segregation factor, FIG. 49 shows the resolution of the separation of the hybridized DNA mixture into heteroduplexes and homoduplexes.

The PCR conditions used for each primer are described in the table below. All components were combined and vortexed to mix well and centrifuged. Aliquots were then dispensed into PCR tubes as shown in the table below.

[0320]

[Table 14]

The PCR tubes were placed in a thermocycler and a temperature cycling program was started. The cycling program parameters are shown in the following table:

[0322]

[Table 15]

The conditions used for the mutation detection separation are as follows: Eluate A: 0.1 M TEAA; Eluate B: 0.1 M TEAA, 25% acetonitrile;
900 mL / min; gradient:

[0324]

[Table 16]

The lambda sequence was analyzed by O'Conner et al. In Biophys . J. 74: A285 (1988) and by FMC Inc. for mutation detection 97 4th International Workshop, Human Genome Association (Mutation De
^{tection 97 4 th International Workshop, Human} Genome Organization), 5 May 29 - June 2, 1997, Brno, Czech Republic, has been published in Hosuta number 29. The 100 bp lambda fragment sequence (bases 32011-32110) (available from FMC) was used as a standard and the mutation was 32061. The following chart lists the primers used:

[0326]

[Table 17]

FIG. 48 is a chromatogram of a wild-type chain analyzed under the above conditions. The peak that appears has a retention time of 4.78 minutes and an area of 98621.

FIG. 49 shows the lambda mutation analyzed under the same conditions as in FIG. 48 above. Two peaks are evident in this chromatogram, with retention times of 4.32 and 4.68 and a total area of 151246.

The mutation segregating factor can be calculated by applying such various peak areas to the above MSF equation. That is, using the above definition, MSF = (area peak 2−area peak 1) / area peak 1, the MSF would be (151246-98621) / 98621, or 0.533.

Example 24 Effect of Multivalent Cation Decontamination Means on Sample Resolution by DMIPC The separation shown in FIG. 42 was performed using the WAVE ™ DNA fragment analysis system (transgenomic
Co., San Jose, Calif.) Under the following conditions: Column: 50 × 4.6 containing alkylated poly (styrene-divinylbenzene) beads (DNASep ™, Transgenomic). mm id; mobile phase 0.1 M TEAA (1 M concentration, available from Transgenomic) (eluent A), pH 7.3; gradient: 50-53%
1M TEAA and 25.0% acetonitrile (eluent B) for 0.5 minutes; 53-60% B for 7 minutes; 60-100% B for 1.5 minutes; 100-50% B for 1 minute; 50% B for 2 minutes. Flow velocity is 0.9
mL / min, detection at 254 nm UV, and column temperature at 56 ° C. 2 μL of sample (= 0.2 μg DN
A, DYS271 209 bp mutation standard with A to G mutation at position 168).

FIG. 43 is the same as the separation performed in FIG. 42, but after replacing the guard cartridge (20 × 40 mm, complexing cartridge, transgenomic part no. 530012) and replacing the pump valve filter. (Part no. 638-1423, Transgenomic). The guard cartridge had dimensions of 10 × 3.2 mm, contained 2.5 meq / vol and a particle size of 10 μm iminodiacetate chelate resin and was placed directly in front of the injection valve.

FIG. 44 is the same as the separation performed in FIG. 43, but with the column loaded with 0.1 M TEAA, 2M for 45 minutes.
The test was performed after flowing 5% acetonitrile and 32 mM EDTA at 75 ° C.

Although specific embodiments of the present invention have been given above, it should be understood that such embodiments have been presented by way of example only. Others will recognize and make modifications to the described embodiments that do not depart from the spirit and scope described and claimed herein.

[Brief description of the drawings]

FIG. 1: MIPC separation of pUC18 DNA- Hae III digest fragment on a column containing alkylated poly (styrene-divinylbenzene) beads.

FIG. 2 is a chromatogram obtained after 5 μL of a sample was added as in FIG. 1 and a fixed concentration of 35% B was applied to the column under a single condition.

FIG. 3 is a chromatogram obtained after a second 5 μL aliquot of a standard pUC18 DNA- Hae III digest was added to the column of FIG. 2 and eluted with the gradient described in FIG.

FIG. 4 shows melting profiles of three DNA fragments having mutation sites indicated by arrows.

FIG. 5 shows the DMIPC elution profile of Fragment 1 of FIG.

FIG. 6 shows the DMIPC elution profile of fragment 2 of FIG.

FIG. 7 shows the DMIPC elution profile of fragment 3 of FIG.

FIG. 8 shows melting profiles of four DNA fragments having mutation sites indicated by arrows.

FIG. 9 shows the DMIPC elution profile of fragment 1 of FIG.

FIG. 10 shows the DMIPC elution profile of fragment 2 of FIG.

FIG. 11 shows the DMIPC elution profile of fragment 3 of FIG.

FIG. 12 shows the DMIPC elution profile of fragment 4 of FIG.

FIG. 13 is a schematic diagram showing the stepwise melting of a theoretical three domain DNA molecule.

FIG. 14 depicts the temperature titration curves of two homoduplexes (top two curves) and two heteroduplexes (bottom two curves).

FIG. 15 shows a melting profile for DNA fragment sY81.

FIG. 16 depicts the theoretical melting profiles of three domain DNA fragments whose domains have melting temperatures of 55 ° C., 60 ° C., and 65 ° C., respectively.

FIG. 17 shows a MIPC chromatogram of a 500 bp PCR product and a 405 bp blunt-ended fragment.

FIG. 18 shows the effect of temperature on the separation of homoduplexes and heteroduplexes by MIPC.

FIG. 19 is a comparison of MIPC chromatograms showing the yields obtained after PCR using various DNA polymerase enzymes.

FIG. 20 shows the degree of regeneration of PCR products obtained using various DNA polymerase enzymes.
This is a comparison of MIPC chromatograms.

FIG. 21 shows the effect of post-PCR hybridization on the results of a PCR reaction when analyzed using MIPC.

FIG. 22 depicts the use of MIPC to collect pure PCR products.

FIG. 23 shows a schematic representation of hybridization to form homoduplexes and heteroduplexes.

FIG. 24 depicts temperature-dependent separation of homo- and heteroduplexes.

FIG. 25 shows the change in retention time according to the temperature of the homo- and heteroduplex peaks in FIG. 24.

FIG. 26 is an illustration of stepwise melting of a theoretical three domain DNA molecule.

FIG. 27 depicts the temperature titration of two homoduplexes (top two curves) and two heteroduplexes (bottom two curves).

FIG. 28 shows temperature titration for DNA fragment sY81.

FIG. 29 represents the theoretical temperature titration of three domain DNA fragments whose domains have melting temperatures of 55 ° C., 60 ° C., and 65 ° C., respectively.

FIG. 30 depicts the effect of column temperature on the separation of homoduplex and heteroduplex DNA for a 209 bp mutation mixture of DYS271 containing a heteroduplex mismatch at position 168.

FIG. 31 shows the temperature titration of the wild type homoduplex from FIG. 30 with the inflection point indicated by the arrow.

FIG. 32 is a DNA melting profile of the DYS271 209 bp mutation mixture.

FIG. 33 is a schematic representation of a cooperative method (Model A) and a non-cooperative method (Model B) for modeling DNA melting within fragments.

FIG. 34 depicts the melting profile of the 209 bp mutation mixture of DYS271 using a non-cooperative weighting model.

FIG. 35 depicts the melting profile of the 209 bp mutation mixture of DYS271 using a cooperative loading model, where loop entropy was varied to mimic a non-cooperative model.

FIG. 36 depicts the melting profile of the 209 bp mutation mixture of DYS271 using a cooperative model using various loop entropies, using temperature cancellation, and including slope and fragment size-dependent duration.

FIG. 37 shows retention time changes with temperature for heteroduplex and homoduplex species in the 209 bp mutation mixture of DYS271.

FIG. 38 is a graph of calculated melting temperature versus empirically determined melting temperature.

FIG. 39 is a graph of calculated melting temperature versus predicted melting temperature.

FIG. 40 is a reference chart used to select a mobile phase composition for eluting a double-stranded polynucleotide.

FIG. 41 depicts a mobile phase gradient embodiment for mutations detected by DMIPC.

FIG. 42 is an elution profile representing the separation of 209 base pair homoduplex / heteroduplex mutation detection performed by DMIPC at 56 ° C.

FIG. 43. Elution profile after separately injecting the same 209 bp mixture and using the same column as in FIG. 42, but replacing the guard cartridge and replacing the pump valve filter.

FIG. 44. Elution profile after separately injecting the same 209 bp mixture and using the same column as in FIG. 43, but washing the column with 0.1 M TEAA, 25% acetonitrile and 0.32 M EDTA at 75 ° C. for 45 minutes. It is.

FIG. 45 is an elution profile of the 209 bp mutation standard for DYS271 using the lot A titanium frit.

FIG. 46 is an elution profile of a 209 bp mutation standard of DYS271 using lot B titanium frit.

FIG. 47 is an elution profile of the 209 bp mutation standard for DYS271 using PEEK frit.

FIG. 48 is a DMIPC elution profile of a 100 bp PCR product derived from the wild-type strand of lambda DNA.

FIG. 49 is a DMIPC elution profile of a hybridized mixture containing lambda DNA containing a mutation and wild-type strand.

[Procedure amendment]

[Submission date] March 15, 2000 (2000.3.15)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

3. A method for preparing a double-stranded DNA fragment for detecting a mutation by denaturing matched-ion polynucleotide chromatography, comprising the steps of:
And also to the A fragment corresponds to the wild-type double stranded DNA fragments with known nucleotide sequence, the following steps (a) ~ (c): a (a) (i) sequence of wild-type double-stranded DNA fragment Analysis shows that the double-stranded DNA fragment
Dividing into sample sequences of nucleotides having a melting point range of less than degree C, each sample sequence having a first end and a second end opposite thereto; and (ii) one of the sample sequences
One is amplified by PCR using a set of primers adjacent to the one of the first and second ends of the sample sequence; (b) (i) analyzing the sequence of the wild-type double-stranded DNA fragment Te, one of the divided the sample sequence of nucleotides, and (ii) said sample sequence having a low melting domain high have melting domain and the mutation site double-stranded DNA fragment is positioned, first of the sample sequences was amplified by PCR using a set of primers flanking the first and second end; (c) (i) analyzing the sequence of the wild-type double-stranded DNA fragments, mutation of double-stranded DNA fragment Splitting the sample sequence into nucleotides whose sites are within 25 percent of the total base pairs from the end of the fragment , and (ii) dividing one of the sample sequences into a contiguous set of first and second ends of the sample sequence using primers to amplify by PCR, of 1 The above method of preparation, comprising the steps of

4. A method for detecting a mutation in a double-stranded DNA fragment by denaturing matched-ion polynucleotide chromatography, comprising the steps of: (a) analyzing the sequence of a wild-type double-stranded DNA fragment; Splitting the DNA fragment into sample sequences of nucleotides having a melting point range of less than 15 degrees C, wherein each sample sequence has a first end and a second end opposite thereto; One is amplified by PCR using a set of primers adjacent to the first and second ends of the one of the sample sequences; (c) analyzing the amplified sample by denaturing matched-ion polynucleotide chromatography The above method, comprising:

6. A PCR method is an evaluation method of PCR methods to determine whether to induce mutations, following (a) and (b): (a) ( i) a polynucleotide a plurality of PCR PCR amplification product to obtain a PCR amplification product , and (ii) analyzing the PCR amplification product by matched ion polynucleotide chromatography to identify any mutations generated by the PCR-induced mutation. the resulting PCR amplification products profiles including profiles, the (b) as compared to the reference profile of the PCR amplification product profile, PCR determines the presence of a mutation induced in the PCR amplification production was Kotono 1 or more The above method comprising:

7. A method for identifying a deviation of a PCR method from a previously determined reference profile, the following (a) and (b), (a) ( i) a polynucleotide by making a plurality of PCR method cycle and amplified by the PCR amplification product obtained; and the (ii) PCR amplification products were analyzed by matched ion poly quinuclidine octreotide chromatography, the PCR amplification product profile including the profile of mutations any PCR-induced obtained, the (b) PCR amplification product profile, as compared to the reference profile, PCR is to identify the deviation of the PCR reaction products from the reference profile previously determined, including mutations and induction, one or more The above method comprising:

8. A method for reducing mutations induced by PCR in a PCR amplification method, comprising: (a) amplifying a polynucleotide by performing a plurality of PCR amplification cycles to obtain a first PCR amplification product; (B) analyzing the first PCR amplification product by matched ion polynucleotide chromatography to obtain a PCR amplification product profile; (c) comparing the PCR amplification product profile to a reference profile to determine the presence of PCR-induced mutations; (D) amplifying the polynucleotide by performing multiple PCR amplification cycles with one or more method-adjusted variables to produce a second PCR amplification product having reduced PCR-induced mutations; Forming above.

Claims9] Furthermore,(E) Analyzing the PCR reaction product obtained in step (d) by matched ion polynucleotide chromatography to obtain a second reaction product profile;(F) The second reaction product profile is compared to a set of standard profiles and
Determine the bias of the PCR method from established standards;(G) Performing a plurality of PCR cycles by adjusting one or more method variables to form a third PCR reaction product with reduced PCR bias from the predetermined standard. Claims for further reducing the bias of the PCR method from 8 The method described in.

10. The following (i) ~ (vii): (i) PCR method is rope lines using DNA template, and PCR method PCR method in step (a) I-line using a first primer We are predefined generated bias of the 1PCR reaction products from standard primer dimers, and the first primer in step (d), it has a greater affinity for the DNA template than the first primer cash example the second primer, cycle PCR method non of step (ii) (a) - in the presence of a performed using the proofreading enzyme, bias polymorphism of the 1PCR reaction product from a predetermined standard There, and instead the enzyme used in step (d) the proofreading enzyme, (iii) step (a) cycles of PCR methods line cracks with a proofreading enzyme, the 1PCR reaction from predetermined standards Product bias is the existence of polymorphism A stationary, its to multiple PCR method cycle one or more nucleotides of step (d), to reduce the magnesium ion or the enzyme concentration, or raising the temperature, or carried out by combining them in advance (iv) deviation of the 1PCR reaction product from prescribed standards are low product yield, and a plurality of PCR method cycle one or more nucleotides of step (d), increasing the magnesium-ion or enzyme concentration, or reducing the temperature It is, or subjected to a combination thereof, (v) a predetermined byproduct bias of excessive levels of the 1PCR reaction product from the standard, wherein reducing the number of cycles step (d), (vi) step analysis of the PCR reaction product by matched ion polynucleotide chromatography (b) is conducted at a temperature which causes the polymorphic partial denaturation of該分 analysis is most Are rope lines after hybridization in the PCR reaction products PCR cycles, (vii) PCR reaction product purified is separated from impurities during the analysis of the by that PCR reaction product matched ion port chromatography pure product is formed Te, or their to where step (d) is performed using a pure product, or where pure product is further amplified by cloning in a host system in, for 10. A method according to claim 8 or claim 9 comprising one or more .

11. A detection method for DNA gene mutation, and heated to a temperature at which a mixture of wild-type double stranded DNA segment, strands are completely denatured double-stranded DNA segment and corresponding (a) Sample (B) cooling the product of step (a) until the strands have completely annealed, thereby containing two homoduplexes and two heteroduplexes if the sample segment contains a mutation; (C) determining the heteromutant site separation temperature; (d) using the denatured matched ion polynucleotide chromatography at the heteromutant site separation temperature to determine the product of step (b). Analyzing to identify the presence of a component in the product wherein the site of the heteromutant has been separated.

12. The following (i) to (iv): (i) the sequence of the normal double-stranded DNA is known, and the heteromutation site separation temperature has the formula: T (hsst) = X + m · T (w) [where T (hsst) is the heteromutant site separation temperature and T (w) is the temperature calculated by software or determined experimentally, at which There is a selected equilibrium between the denatured and non-denatured state of the single-stranded DNA, X is the detector of denaturing matched ion polynucleotide chromatography, and m is the weighting factor selected between 0 and 2. in a] is determined by, (ii) hetero mutant site separation temperature is mutated separation temperature range product of step (b), modified matched in a series of incremental modified matched ion polynucleotide chromatography separation Ion polynucleotide chromatography It determined by Rukoto be analyzed by Rafi, or each successive separation mutation separation profile is observed, or mutation separation temperature range, until the absence of the mutation separation profile is observed, than the immediately preceding separation Has a high temperature, wherein the mutation segregation profile identifies the presence of the mutation, and the absence of the mutation segregation profile indicates the absence of the mutation in the sample ; , mutated separation temperature range product of step (b), is determined by analyzing the modified matched ion polynucleotide chromatography in a series of incremental modified matched ion polynucleotide chromatography I over separate, each successive separation mutation separation profile is observed Luke or mutations separated flop mutated separation temperature range, Has a temperature lower than the separation of the immediately preceding up feel of the absence is observed, wherein a mutation separation profile identifies the presence of a mutation and the mutation separation absence profiles absence of mutation in the sample are shown, (iv) determination by modified matched ion polynucleotide chromatography of the T (hsst) is computer controlled, and the method of claim 11 including is automated, one or more.

13. A detection method for DNA genetic mutations, calculation step for obtaining a hetero mutant site separation temperature calculated (a); a (b) the expected heterodimer mutants site separation temperature (C) heating the mixture of the sample double-stranded DNA segment and the corresponding wild-type double-stranded DNA segment to the expected heteromutant site separation temperature; (d) step (c ). A) analyzing the product using denatured matched-ion polynucleotide chromatography at the expected heteromutant site separation temperature to identify the presence of the heteromutated site-separated component in the product. The above method comprising:

14. The following (i) ~ (iii): (i) calculation process, Ri comprises calculating a hetero mutant site separation temperature calculated in accordance with a first mathematical model, (ii) prediction step, comprises adjusting the hetero mutant site separation temperature calculated in accordance with a second mathematical model, and wherein the second mathematical model is empirically determine heteroaromatic mutant sites separated based on the comparison between the temperature and the calculated hetero mutant site separation temperature, including (iii) hetero mutant site separation temperature calculated is calculated using the first mathematical model, one or more The method according to claim 13 .

The 15. The mixture of first heteroduplex comprising a DNA molecule to elute DNA molecules and finally to elute and homoduplex DNA molecules, heteroduplex D
A chromatographic method for separating mutation sites present in NA molecules under conditions that selectively denature, comprising: (a) adding the mixture to a matched ion polynucleotide chromatography column; Eluting the molecules of the mixture using a mobile phase comprising an organic solvent concentration range that brackets the agent and the preselected fragment, wherein the range comprises the initial and final concentration of the organic solvent; Is D which elutes first in the mixture
Containing an organic solvent concentration up to the amount required to elute the NA molecule, and wherein the final concentration contains an organic solvent concentration sufficient to elute the last eluting DNA molecule in the mixture. The above method comprising:

16. The following (i) ~ (iv): (i) the range to collectively preselected fragment above reporting, required to elute the DNA molecules of a variety of base pairs in length and base pairs in length resulting et is from a reference related to the organic solvent concentration, (ii) preliminary organic solvent concentration that can elute the DNA molecules of the special base pairs long, in order to elute the DNA molecules of a variety of base pairs in length and base pairs in length Obtained from a reference relating to the required organic solvent concentration , and wherein said preliminary organic solvent concentration is used to select a range for bundling said fragments ; (iii) said heteroduplex molecule and said homoduplex chain molecule, have a same base pairs in length, (iv) the heteroduplex molecule Ri comprises at least two different heteroduplexes, and wherein said homoduplex molecule at least two different comprising homoduplex molecule, one or more The method according to no claim 15.

17. Prior to step (a), (A) the relationship between the organic solvent concentration in the mobile phase required to elute DNA molecules of various base pair lengths from the column is a function of base pair length. 17. The method of claim 15 or claim 16 , comprising: (B) determining from the resulting relationship an organic solvent range that brackets the preselected fragment.

18. The method of claim 19 , further comprising flowing a solution containing a polyvalent cation binding agent through the column to improve the resolution of the double-stranded DNA fragments separated on the column, wherein the solution comprises about 50%. A method for treating a matched ion polynucleotide chromatography column having a temperature of ~ 90 ° C.

19. The following (a) ~ (c): (a) the polyvalent cation binding agent Ri comprises a coordination compound, wherein the coordination compound is a water-soluble chelating agent, crown ether, acetylacetone , alizarin, Al Minon, chloranilic acid, kojic acid, morin, rhodizonic acid, Chionarido, Chioure A, alpha-furyl dioxime, Niokishimu, salicylaldoxime, dimethylglyoxime, alpha-furyl dioxime, cupferron, alpha-nitroso - β- naphthol, nitro nitroso -R- salt, diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN, SPADNS, glyoxal - bis (2-Hidorokisaniru), murexide, alpha-benzoin oxime, mandelic acid, anthranilic acid, ethylenediamine, glycine, triamino triethylamine, Chionarido, triethylene Tet Amin, EDTA, metal phthalein, Arusonin acid, alpha,. Alpha .'- bipyridine, 4-hydroxy sheet benzothiazole, beta-hydroxyquinaldine, beta-hydroxyquinoline, 1,10 - phenanthroline, picolinic acid, Kinaruji acid, alpha , α ', α''- terpyridyl, 9-methyl -2,3,7- trihydroxy-6-fluorone, pyrocatechol, rhodizonate, salicylic aldoxime, salicylic acid, tiron, 4-chloro-1,2 dimercapto benzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid, a member selected from the group consisting of diethyl Le dithiocarbamate sodium and dibenzyl dithiocarbamate, zinc, (b) wherein said solution further organic solvents comprises, where the organic solvent, a alcohol, nitrile, dimethylformamide, tetrahydrofuran, Contact ester Is selected from the group consisting of finely ether, (c) wherein it comprises the solution further counter-ionic agent, wherein the counters ion agent, lower primary, secondary and tertiary amines, as well as lower trialkylammonium salts, quaternary ammonium, octyl ammonium acetate tape DOO, decyl ammonium acetate, ammonium acetate, Pi lysine bromide ammonium acetate, cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, blanking methyl ethyl ammonium acetate, Methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium Iodonium acetate, dimethyl diethyl ammonium acetate, triethylammonium acetate tape, tri-propyl ammonium acetate, tributyl ammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate tape DOO, tetrabutyl ammonium acetate, carbonate, phosphate, sulfates Feto, nitrate, propionate, formate, chloride the method of claim 18, bromides Contact and selected from the group consisting of any one or more of the above mixture, including one or more.

20. A method for improving the resolution of a double-stranded DNA fragment separated on a column, comprising passing a solution containing a polyvalent cation binder through the column before storing the column. How to store nucleotide chromatography columns.

21. The following (a) ~ (c): (a) the polyvalent cation binding agent Ri comprises a coordination compound, wherein the coordination compound, water soluble chelating agent, crown ether , acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid, morin, rhodizonic acid, Chionarido, thiourea, alpha-furyl dioxime, Niokishimu, salicylaldoxime, dimethyl glyoxime, alpha-furyl dioxime, cupferron, alpha-nitroso - β- naphthol, nitroso -R- salt, diphenylthiocarbazone, diphenylcarbazone, Erioku chromium black T, PAN, SPADNS, glyoxal - bis (2-Hidorokisaniru), stuffiness Kishido, alpha-benzoin oxime, mandelic acid, anthranilic acid , ethylenediamine Min, glycine, triamino triethylamine, Chionarido, triethylene DOO Raamin, EDTA, metal phthalein, Arusonin acid, alpha,. Alpha .'- bipyridine, 4-hydroxycarboxylic benzothiazole, beta-hydroxyquinaldine, beta-hydroxy quinoline, 1,10-phenanthroline, picolinic acid, Kinaruji acid, α, α ', α'' - Terupiriji le, 9-methyl -2,3,7- trihydroxy-6-fluorone, pyrocatechol, rhodizonate, salicylaldoxime, salicylic acid, tiron, 4-chloro-1,2 - dimercapto benzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid, a member selected from the group consisting of sodium diethyldithiocarbamate and dibenzyl dithiocarbamate zinc, comprise further organic solvent (b) the solution made, wherein the organic solvent, alcohol, nitrile, dimethylformamide, tetrahydrofuran, esters and Is selected from the group consisting of d ether, (c) said solution further comprises a counter-ionic agent, wherein the counter ion-agent is a lower primary, secondary and tertiary amines and lower Toriarukirua, It is selected from the group consisting of ammonium salts and quaternary ammonium salts, wherein the counters ion agent, octyl ammonium acetate, decyl ammonium acetate lactate, ammonium acetate, pyridinium ammonium acetate lactate, cyclohexyl ammonium acetate, diethylammonium a cetearyl over preparative , propyl ethyl ammonium acetate, butyl ethyl ammonium acetate lactate, methyl hexyl ammonium acetate, tetramethylammonium A Seteto, tetraethylammonium acetate, Tetorapuropi Le ammonium acetate, tetrabutyl ammonium acetate, dimethyl diethyl Ann monitor um acetate, triethylammonium acetate, tripropyl ammonium Niu beam acetate, tributyl ammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium Niu arm acetates, carbonates, phosphates, sulfates, nitrates, pro Pioneto, formate, chloride, the method of claim 20 which is selected from the group consisting of any one or more of a mixture of bromide and above containing one or more.

The method according to claim 22 mixture of polynucleotides having the highest 1500 base pairs, the separation column beads containing polymer beads having an average diameter of 0.5 to 100 microns and having a mutation separation factor of at least 0.01 Separating the mixture of double-stranded polynucleotides, comprising flowing and separating the mixture of polynucleotides.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/70 C07H 21/04 A // C07H 1/06 C12N 15/00 A 21/04 E (31) Priority claim number 60 / 056,500 (32) Priority date August 20, 1997 (August 20, 1997) (33) Priority claim country United States (US) (31) Priority claim number 60/061, 445 (32) Priority Date October 9, 1997 (Oct. 9, 1997) (33) Priority Claimed Country United States (US) (31) Priority Claim Number 60 / 062,690 (32) Priority Date 1997 October 22, 1997 (Oct. 22, 1997) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 067,269 (32) Priority date December 3, 1997 (1997. 12.3) (33) Priority claim country United States (US) (31) Priority claim number 60/07 0,572 (32) Priority date Jan. 6, 1998 (1998.1.6) (33) Priority country United States (US) (31) Priority number 60 / 070,585 (32) Priority date January 6, 1998 (1998.1.6) (33) Priority claim country United States (US) (31) Priority claim number 60 / 093,844 (32) Priority date July 22, 1998 ( (22 Jul. 1998) (33) Priority country United States (US) (31) Priority claim number 09/129, 105 (32) Priority date August 4, 1998 (1998.8.4) (33) ) Priority country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, D K, EE, ES, FI, GB, GE, GH, GM, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US , UZ, VN, YU, ZW (72) Inventor Hefuel, Robert M. 94301 Palo Alto Hawthorne, Avenille, Calif., United States 248 F term (reference) QA17 QQ12 QQ42 QR08 QR32 QR42 QS17 QX04 4B064 AF27 CA21 CB24 CD04 CD12 CD16 CE10 CE14 DA13 4C057 AA10 BB02 BB05 MM01 MM04 MM09

Claims (79)

[Claims] 1. An improved method for separating a sample mixture of polynucleotides having a polynucleotide concentration in a sample mixture that is less than a predetermined threshold concentration by matched ion polynucleotide chromatography, the improvement comprising: loading the sample mixture onto a column. Such an improved method comprising accumulating a sample mixture of polynucleotides by adding. 2. A sample mixture added to the column as an aliquot greater than 10 μL is added to the column, and the solvent mixture comprises a counter ionic reagent.
The method of claim 1. 3. The method of claim 1, wherein the improvement comprises adding the sample in a mobile phase having an organic solvent concentration less than that required to elute the polynucleotide. 4. The method of claim 1, wherein the defined threshold concentration is a lower limit of detection of the polynucleotide. 5. The method of claim 1, wherein said mobile phase comprises a counter ionic agent and an organic solvent. 6. The method of claim 1, further comprising adding the mixture to a matched ion polynucleotide chromatography column and flowing an aqueous mobile phase through the column under isocratic conditions, wherein impurities are removed from the mixture. Item 1
The method described in. 7. A method for preparing a double-stranded DNA fragment for detecting a mutation by denaturing matched ion polynucleotide chromatography, comprising the steps of:
A fragment corresponds to a wild-type double-stranded DNA fragment having a known nucleotide sequence, and (a) analyzing the sequence of the wild-type double-stranded DNA fragment, Splitting into sample sequences of nucleotides having a melting point range of, each sample sequence having a first end and a second end at an opposite position; and (b) one of the sample sequences Amplifying by PCR using a set of primers adjacent to said one of the first and second ends. 8. A method for detecting a mutation in a double-stranded DNA fragment by denaturing matched-ion polynucleotide chromatography, comprising the steps of: (a) analyzing the sequence of a wild-type double-stranded DNA fragment; Dividing the DNA fragment into sample sequences of nucleotides having a melting point range of less than 15 degrees C, wherein each sample sequence has a first end and a second end at a position opposite thereto; (b) the sample sequence Is amplified by PCR using a set of primers flanking the first and second ends of the sample sequence; (c) analyzing the amplified sample by denaturing matched-ion polynucleotide chromatography The above method comprising the steps of: 9. A step (c) comprising: (a) heating the mixture of the one of the sample sequences and the corresponding wild-type double-stranded DNA segment to a temperature at which the strands are completely denatured; The product of step (a) is cooled until the strands have completely annealed, so that if said one of said sample sequences contains a mutation, it comprises two homoduplexes and two heteroduplexes (C) transforming the product of step (b) without denaturing the one other portion of the sample sequence;
9. The method of claim 8, comprising analyzing by matched ion polynucleotide chromatography at a temperature that causes denaturation at any base pair mismatch sites in the heteroduplex. 10. The method of claim 7, wherein step (b) comprises using a dGTP analog instead of dGTP. 11. The method of claim 10, wherein said analog is 2,6-aminopurine. 12. The primer comprises a GC clamp of up to 40 bases,
The method according to claim 7. 13. A method for evaluating a PCR method for determining whether PCR induces a mutation, comprising: (a) amplifying a polynucleotide by performing a plurality of PCR cycles; (B) analyzing the PCR amplification product by matched ion polynucleotide chromatography to obtain a PCR amplification product profile, including the profile of any mutations generated by the PCR-induced mutation. The above method comprising: 14. The method of claim 13, further comprising comparing the PCR amplification product profile to a reference profile to determine the presence of a PCR-induced mutation in the PCR amplification product. 15. A method for identifying PCR bias from a predetermined reference profile, comprising: (a) amplifying a polynucleotide by performing a plurality of PCR cycles to obtain a PCR amplification product; b) analyzing the PCR amplification product by matched ion polynucleotide chromatography to obtain a PCR amplification product profile, including the profile of any PCR-induced mutations. 16. The method of claim 15, further comprising comparing the PCR amplification product profile to a reference profile to identify biases of the PCR reaction product from the predetermined reference profile, including PCR-induced mutations. The method described in. 17. A method for reducing PCR-induced mutations in a PCR amplification method, comprising: a) amplifying a polynucleotide by performing a plurality of PCR amplification cycles to obtain a first PCR amplification product; b) analyzing the first PCR amplification product by matched ion polynucleotide chromatography to obtain a PCR amplification product profile; c) comparing the PCR amplification product profile to a reference profile to determine the presence of PCR-induced mutations; d. A) amplifying the polynucleotide by performing multiple PCR amplification cycles with the adjustment of one or more method variables to form a second PCR amplification product with reduced PCR-induced mutations; The above method comprising: 18. The PCR method is performed using a DNA template, and the PCR method is performed using a step (
Performed in a) using the first primer, the bias of the first PCR reaction product from a predetermined standard is the formation of a primer dimer, and in step (b) the first primer is 18. The method according to claim 17, wherein also replacing the second primer with a large affinity for the DNA template. 19. The PCR cycle of step (a) is performed using a non-proofreading enzyme, wherein the bias of the first PCR reaction product from a predetermined standard is the presence of a polymorphism, and 18. The method according to claim 17, wherein the enzyme used in d) is replaced by a proofreading enzyme. 20. The PCR cycle of step (a) is performed using a proofreading enzyme, the bias of the first PCR reaction product from a predetermined standard is the presence of a polymorphism, and step (d). 18. The method of claim 17, wherein the plurality of PCR cycles comprises reducing the concentration of one or more nucleotides, magnesium ions or enzymes, increasing the temperature, or a combination thereof. 21. The method according to claim 19, wherein the first PCR reaction product has a low bias from a predetermined standard with low product yield, and wherein the plurality of PCR cycles of step (d) have one or more nucleotide, magnesium ion or enzyme concentrations. Increase or decrease the temperature,
18. The method according to claim 17, wherein the method is performed by combining them. 22. The method of claim 17, wherein the bias of the first PCR reaction product from the predetermined standard is an excessive level of by-product, wherein the number of cycles in step (d) is reduced. 23. e) analyzing the PCR reaction product obtained in step (d) by matched ion polynucleotide chromatography to obtain a second reaction product profile; f) a second reaction product profile G) determining the bias of the PCR method from a predetermined standard by comparing it with a set of standard profiles; g) performing multiple PCR cycles by adjusting one or more method variables to obtain a predetermined standard 18. The method of claim 17 for further reducing PCR bias from a predetermined standard, comprising: forming a third PCR reaction product having reduced PCR bias from the standard. 24. The analysis of the PCR reaction product by matched ion polynucleotide chromatography of step (b) is performed at a temperature that causes partial denaturation of the polymorphism, and the analysis is performed in a final PCR cycle. 18. The method of claim 17, which is performed after hybridization of the product. 25. The method of claim 17, wherein the purified PCR reaction product is separated from impurities during the analysis of the PCR reaction product by matched ion polynucleotide chromatography to form a pure product. Method. 26. The method according to claim 25, wherein step (d) is performed using a pure product. 27. The method of claim 25, wherein the pure purified product is further amplified by cloning in a host system. 28. A method for detecting DNA gene mutations, comprising: a) heating a mixture of double-stranded DNA segments of a sample and corresponding wild-type double-stranded DNA segments to a temperature at which the strands are completely denatured; b) cooling the product of step (a) until the strands have completely annealed, whereby a mixture comprising two homoduplexes and two heteroduplexes if the sample segment contains a mutation C) determining the heteromutant site separation temperature; d) analyzing the product of step (b) using denatured matched ion polynucleotide chromatography at the heteromutant site separation temperature; Identifying the presence of a component in the product in which the site of the heteromutant has been separated. 29. The sequence of a normal double-stranded DNA is known, and the heteromutant site separation temperature has the formula: T (hsst) = X + mT (w), where T (hsst) is Mutant site separation temperature, where T (w) is a software calculated or experimentally determined temperature at which normal double-stranded DNA denatures between denatured and non-denatured states. Selected equilibrium is present, X is a detection factor for denaturing matched ion polynucleotide chromatography, and m is a weighting factor selected between 0 and 2]. the method of. 30. A heteromutant site separation temperature wherein the product of step (b) is subjected to denaturing matched ion polynucleotide chromatography in a series of incremental denatured matched ion polynucleotide chromatography separations in the mutation separation temperature range. Determined by analysis, each successive separation has a higher temperature than the previous separation until a mutation separation profile is observed or the absence of a mutation separation profile is observed in the mutation separation temperature range. 29. The method of claim 28, wherein the mutation segregation profile identifies the presence of the mutation, and wherein the absence of the mutation segregation profile indicates the absence of the mutation in the sample. 31. The heteromutant site separation temperature is determined by denaturing the product of step (b) over a range of mutational separation temperatures by a series of incremental denatured matched ion polynucleotide chromatography separations. Determined by analysis, each successive separation has a lower temperature than the previous separation until a mutation separation profile is observed or the absence of the mutation separation profile in the mutation separation temperature range is observed. 29. The method of claim 28, wherein the mutation segregation profile identifies the presence of the mutation, and wherein the absence of the mutation segregation profile indicates the absence of the mutation in the sample. 32. The method of claim 30, wherein the determination of T (hsst) by denaturing matched ion polynucleotide chromatography is computer controlled and automated. 33. The method of claim 31, wherein the determination of T (hsst) by denaturing matched ion polynucleotide chromatography is computer controlled and automated. 34. A method for detecting a DNA gene mutation, comprising: a) a calculating step for obtaining a calculated heteromutant site separation temperature; b) a calculating step for obtaining a predicted heteromutant site separation temperature. C) heating the mixture of the sample double-stranded DNA segments and the corresponding wild-type double-stranded DNA segments to the expected heteromutant site separation temperature; d) the product of step (c). Analyzing at a predicted heteromutant site separation temperature using denatured matched-ion polynucleotide chromatography to identify the presence of the heteromutated site-separated component in the product. The above method. 35. The method of claim 34, wherein the calculating step comprises calculating a heteromutant site separation temperature calculated according to a first mathematical model. 36. The method of claim 34, wherein the predicting step comprises adjusting a heteromutant site separation temperature calculated according to a second mathematical model. 37. The method of claim 36, wherein the second mathematical model is based on a comparison of the empirically determined heteromutant site separation temperature and the calculated heteromutant site separation temperature. 38. The method of claim 37, wherein the calculated heteromutant site separation temperature is calculated using a first mathematical model. 39. A mixture of heteroduplex and homoduplex DNA molecules containing the first and last eluting DNA molecules is converted to the heteroduplex D
A chromatographic method for separating mutation sites present in NA molecules under conditions that selectively denature, comprising: (a) adding the mixture to a matched ion polynucleotide chromatography column; Eluting the molecules of the mixture using a mobile phase comprising an organic solvent concentration range enclosing the agent and the preselected fragment, wherein the range comprises an initial concentration and a final concentration of the organic solvent; Is the DN that elutes first in the mixture
Containing an organic solvent concentration up to the amount required to elute the A molecule, and the final concentration containing sufficient organic solvent concentration to elute the last eluting DNA molecule in the mixture. The above method comprising: 40. The range of bracketing the preselected fragments is obtained from a reference relating to the organic solvent concentration required to elute DNA molecules of various base pair lengths and base pair lengths. The method described in. 41. Preliminary organic solvent concentrations that can elute DNA molecules of a particular base pair length are obtained from references relating to the organic solvent concentration required to elute DNA molecules of various base pair lengths and base pair lengths. 40. The method of claim 39, wherein the preliminary organic solvent concentration is used to select a range that brackets the fragments. 42. The method of claim 39, wherein said heteroduplex molecule and said homoduplex molecule have the same base pair length. 43. The method of claim 39, wherein the heteroduplex molecule comprises at least two different heteroduplexes, and wherein the homoduplex molecule comprises at least two different homoduplexes. the method of. 44. The method of claim 39, comprising detecting the molecule after the elution. 45. The method according to claim 39, wherein said organic solvent is selected from the group consisting of methanol, ethanol, acetonitrile, ethyl acetate and 2-propanol. 46. The method according to claim 39, wherein said organic solvent is acetonitrile. 47. The method of claim 39, wherein said counter ionic agent is selected from the group consisting of lower alkyl primary, secondary and tertiary amines, lower trialkyl ammonium salts and lower quaternary ammonium salts. The described method. 48. The method according to claim 48, wherein the counter ionic agent is octyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate,
Pyridinium ammonium acetate, cyclohexylammonium acetate, diethyl ammonium acetate, propylethyl ammonium acetate,
Butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethyldiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate 40. The method of claim 39, wherein the method is selected from the group consisting of, tetrapropyl ammonium acetate, tetrabutyl ammonium acetate, carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, bromide, and mixtures of one or more of the foregoing. . 49. The method according to claim 39, wherein the counter ionic agent is triethylammonium acetate. 50. Before step (a): (a) the relationship between the concentration of organic solvent in the mobile phase required to elute DNA molecules of various base pair lengths from the column is a function of base pair length. 40. The method of claim 39, comprising: (b) determining from the resulting relationship an organic solvent range that brackets the preselected fragment. 51. Before step (a): (a) the relationship between the concentration of organic solvent in the mobile phase required to elute DNA molecules of various base pair lengths from the column is a function of base pair length. 42. The method of claim 41, comprising: (b) determining the preliminary organic solvent concentration from the obtained relationship. 52. Increasing the resolution of a double-stranded DNA fragment separated on the column, comprising flowing a solution containing a polyvalent cation binding agent through the column, wherein the solution comprises about 50 A method for treating a matched ion polynucleotide chromatography column having a temperature of ~ 90 ° C. 53. The method of claim 52, wherein said temperature is about 70-80 ° C. 54. The method of claim 52, wherein said polyvalent cation binder comprises a coordination compound. 55. The method of claim 54, wherein said coordination compound is a member selected from the group consisting of a water-soluble chelating agent and a crown ether. 56. The chelating agent is acetylacetone, alizarin, aluminone, chloranilic acid, kojic acid, morin, rhodizonic acid, thionalide, thiourea, α-furyldioxime, nioxime, salicylaldoxime, dimethylglyoxime, α-furyl. Dioxime, cuperon, α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone, diphenylcarbazone, eriochrome
Black T, PAN, SPADNS, glyoxal-bis (2-hydroxanyl), murexide, α-benzoin oxime, mandelic acid, anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, thionalide, triethylenetetraamine, EDTA, metalphthalein, Arsonic acid, α, α'-bipyridine, 4-hydroxybenzothiazole, β-hydroxyquinaldine, β-hydroxyquinoline, 1,10
-Phenanthroline, picolinic acid, quinardic acid, α, α ', α''-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol, rhodizonic acid, salicylaldoxime, salicylic acid, tiron 56. The method according to claim 55, wherein the compound is selected from the group consisting of, 4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid, sodium diethyldithiocarbamate and zinc dibenzyldithiocarbamate. Method. 57. The method of claim 52, wherein said chelating agent is EDTA. 58. The method according to claim 52, wherein said solution further comprises an organic solvent. 59. The method according to claim 58, wherein said organic solvent is selected from the group consisting of alcohols, nitriles, dimethylformamide, tetrahydrofuran, esters and ethers. 60. The method according to claim 59, wherein said organic solvent is acetonitrile. 61. The method of claim 59, wherein said solution further comprises a counter ionic agent. 62. The method of claim 61, wherein the counter ionic agent is selected from the group consisting of lower primary, secondary and tertiary amines, and lower trialkyl ammonium salts and quaternary ammonium salts. The described method. 63. The method according to claim 63, wherein the counter ionic agent is octyl ammonium acetate, decylammonium acetate, octadecyl ammonium acetate,
Pyridinium ammonium acetate, cyclohexylammonium acetate, diethyl ammonium acetate, propylethyl ammonium acetate,
Butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethyldiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate 62. The method according to claim 61, wherein the compound is selected from the group consisting of, tetrapropyl ammonium acetate, tetrabutyl ammonium acetate, carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, bromide, and mixtures of any one or more of the foregoing. Method. 64. The method according to claim 61, wherein said counter-ionic agent is triethylammonium acetate. 65. A matched ionic polynucleotide comprising flowing a solution containing a polyvalent cation binding agent through the column prior to storage of the column to improve the resolution of the double-stranded DNA fragment separated by the column. How to store chromatography columns. 66. The method of claim 65, wherein said polyvalent cation binder comprises a coordination compound. 67. The method of claim 66, wherein said coordination compound is a member selected from the group consisting of a water-soluble chelating agent and a crown ether. 68. The polyvalent cation binder is acetylacetone, alizarin, aluminone, chloranilic acid, kojic acid, morin, rhodizonic acid, thionalide,
Thiourea, α-furyldioxime, nioxime, salicylaldoxime, dimethylglyoxime, α-furyldioxime, cuperon, α-nitroso-β-naphthol, nitroso-R-salt, diphenylthiocarbazone, diphenylcarbazone, Eriochrome Black T, PAN, SPADNS, glyoxal-bis (2-hydroxanyl), murexide, α-benzoin oxime, mandelic acid, anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, thionalide, triethylenetetraamine, EDTA, metal Phthalein, arsonic acid, α, α'-bipyridine, 4
-Hydroxybenzothiazole, β-hydroxyquinaldine, β-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldiic acid, α, α ', α''-terpyridyl, 9-methyl-2,3,7 -Trihydroxy-6-fluorone, pyrocatechol, rhodizonic acid, salicylaldoxime, salicylic acid, thyrone, 4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid, sodium diethyldithiocarbamate and 66. The method of claim 65, wherein the method is selected from the group consisting of zinc dibenzyldithiocarbamate. 69. The method of claim 65, wherein said multivalent cation binder is EDTA. 70. The method of claim 65, wherein said solution further comprises an organic solvent. 71. The method of claim 70, wherein said organic solvent is selected from the group consisting of alcohols, nitriles, dimethylformamide, tetrahydrofuran, esters and ethers. 72. The method according to claim 70, wherein said organic solvent is acetonitrile. 73. The solution of claim 6, wherein the solution further comprises a counter ionic agent.
5. The method according to 5. 74. The method according to claim 74, wherein the counter ionic agent is a lower primary, secondary, or tertiary agent.
74. The method of claim 73, wherein the method is selected from the group consisting of tertiary amines, and lower trialkyl ammonium salts and quaternary ammonium salts. 75. The counter ionic agent may be octyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate, pyridinium ammonium acetate, cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, butyl ethyl ammonium acetate, methyl hexyl ammonium acetate, tetramethyl ammonium acetate, Methylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate,
Dimethyldiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate,
73. It is selected from the group consisting of tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, bromide and a mixture of any one or more of the foregoing. The method described in. 76. The method according to claim 73, wherein said counter-ionic agent is triethylammonium acetate. 77. A mixture of polynucleotides having up to 1500 base pairs is placed on a separation column containing polymer beads having an average diameter of 0.5 to 100 microns, wherein the beads have a mutational separation factor of at least 0.01. Separating the mixture of double-stranded polynucleotides, comprising flowing and separating the mixture of polynucleotides. 78. A method for preparing a double-stranded DNA fragment for detecting mutations by denaturing matched-ion polynucleotide chromatography, comprising the steps of:
The fragment corresponds to a wild-type double-stranded DNA fragment having a known nucleotide sequence. (A) Analyzing the sequence of the wild-type double-stranded DNA fragment, (B) amplifying one of the sample sequences by PCR using a set of primers flanking the first and second ends of the sample sequence The above method comprising the steps of: 79. A method for preparing a double-stranded DNA fragment for detecting mutations by denaturing matched-ion polynucleotide chromatography, comprising the steps of:
The fragment corresponds to a wild-type double-stranded DNA fragment having a known nucleotide sequence, and (a) analyzing the sequence of the wild-type double-stranded DNA fragment, Splitting into a sample sequence of nucleotides within 25 percent of total base pairs, and (b) separating one of the sample sequences using a set of primers flanking the first and second ends of the sample sequence The above method comprising the step of amplifying by PCR.