EP1002137A1 - Chromatographie par denaturation d'ions apparies de polynucleotide permettant de detecter des mutations - Google Patents

Chromatographie par denaturation d'ions apparies de polynucleotide permettant de detecter des mutations

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Publication number
EP1002137A1
EP1002137A1 EP98939218A EP98939218A EP1002137A1 EP 1002137 A1 EP1002137 A1 EP 1002137A1 EP 98939218 A EP98939218 A EP 98939218A EP 98939218 A EP98939218 A EP 98939218A EP 1002137 A1 EP1002137 A1 EP 1002137A1
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EP
European Patent Office
Prior art keywords
acetate
pcr
mutation
temperature
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98939218A
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German (de)
English (en)
Other versions
EP1002137A4 (fr
Inventor
Douglas T. Gjerde
Paul D. Taylor
Robert M. Haefele
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Precipio Inc
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Transgenomic Inc
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Publication date
Priority claimed from US09/129,105 external-priority patent/US6287822B1/en
Application filed by Transgenomic Inc filed Critical Transgenomic Inc
Publication of EP1002137A1 publication Critical patent/EP1002137A1/fr
Publication of EP1002137A4 publication Critical patent/EP1002137A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the present invention concerns an improved method for detection of mutations in nucleic acids.
  • the ability to detect mutations in double stranded polynucleotides, and especially in DNA fragments, is of great importance in medicine, as well as in the physical and social sciences.
  • the Human Genome Project is providing an enormous amount of genetic information which is setting new criteria for evaluating the links between mutations and human disorders (Guyer et al., Proc. Natl. Acad. Sci. USA 92:10841 (1995)).
  • the ultimate source of disease for example, is described by genetic code that differs from wild type (Cotton, TIG 13:43 (1997)). Understanding the genetic basis of disease can be the starting point for a cure.
  • determination of differences in genetic code can provide powerful and perhaps definitive insights into the study of evolution and populations (Cooper, et.
  • DNA molecules are polymers comprising sub-units called deoxynucleotides.
  • the four deoxynucleotides found in DNA comprise a common cyclic sugar, deoxyribose, which is covalently bonded to any of the four bases, adenine (a purine), guanine(a purine), cytosine (a pyrimidine), and thymine (a pyrimidine), hereinbelow referred to as A, G, C, and T respectively.
  • a phosphate group links a 3'-hydroxyl of one deoxynucieotide with the 5'-hydroxyl of another deoxynucieotide to form a polymeric chain.
  • double stranded DNA two strands are held together in a helical structure by hydrogen bonds between, what are called, complimentary bases.
  • the complimentarity of bases is determined by their chemical structures.
  • double stranded DNA each A pairs with a T and each G pairs with a C, i.e., a purine pairs with a pyrimidine.
  • DNA is replicated in exact copies by DNA polymerases during cell division in the human body or in other living organisms. DNA strands can also be replicated in vitro by means of the Polymerase Chain Reaction (PCR).
  • PCR Polymerase Chain Reaction
  • double stranded DNA is referred to as a duplex.
  • a duplex When the base sequence of one strand is entirely complimentary to base sequence of the other strand, the duplex is called a homoduplex.
  • a duplex contains at least one base pair which is not complimentary, the duplex is called a heteroduplex.
  • a heteroduplex duplex is formed during DNA replication when an error is made by a DNA polymerase enzyme and a non- complimentary base is added to a polynucleotide chain being replicated. Further replications of a heteroduplex will, ideally, produce homoduplexes which are heterozygous, i.e., these homoduplexes will have an altered sequence compared to the original parent DNA strand.
  • the parent DNA has the sequence which predominates in a natural population it is generally called the "wild type.”
  • DNA mutations include, but are not limited to, "point mutation” or “single base pair mutations” wherein an incorrect base pairing occurs.
  • the most common point mutations comprise “transitions” wherein one purine or pyrimidine base is replaced for another and “transversions” wherein a purine is substituted for a pyrimidine (and visa versa).
  • Point mutations also comprise mutations wherein a base is added or deleted from a DNA chain.
  • Such "insertions” or “deletions” are also known as “frameshift mutations”. Although they occur with less frequency than point mutations, larger mutations affecting multiple base pairs can also occur and may be important.
  • frameshift mutations are also known as “frameshift mutations”.
  • Detection of mutations is, therefore, of great interest and importance in diagnosing diseases, understanding the origins of disease and the development of potential treatments. Detection of mutations and identification of similarities or differences in DNA samples is also of critical importance in increasing the world food supply by developing diseases resistant and/or higher yielding crop strains, in forensic science, in the study of evolution and populations, and in scientific research in general (Guyer et al., Proc. Natl. Acad. Sci. USA 92:10841 (1995); Cotton, TIG 13:43 (1997)). These references and the references contained therein are incorporated in their entireties herein.
  • any alterations in the DNA sequence, whether they have negative consequences or not, are called “mutations”. It is to be understood that the method of this invention has the capability to detect mutations regardless of biological effect or lack thereof.
  • the term “mutation” will be used throughout to mean an alteration in the base sequence of a DNA strand compared to a reference strand. It is to be understood that in the context of this invention, the term “mutation” includes the term “polymorphism” or any other similar or equivalent term of art.
  • heteroduplex site separation temperature is defined herein to mean, the temperature at which one or more base pairs denature, i.e., separate, at the site of base pair mismatch in a heteroduplex DNA fragment. Since at least one base pair in a heteroduplex is not complimentary, it takes less energy to separate the bases at that site compared to its fully complimentary base pair analog in a homoduplex. This results in the lower melting temperature of a heteroduplex compared to a homoduplex.
  • the local denaturation creates, what is generally called, a "bubble" at the site of base pair mismatch. The bubble distorts the structure of a DNA fragment compared to a fully complimentary homoduplex of the same base pair length.
  • MIPC Matched Ion Polynucleotide Chromatography
  • Matched Ion Polynucleotide Chromatography is defined as a process for separating single and double stranded polynucleotides using non-polar separation media, wherein the process uses a counter-ion agent, and an organic solvent to release the polynucleotides from the separation media.
  • MIPC separations are complete in less than 10 minutes, and frequently in less than 5 minutes.
  • MIPC systems WAVETM DNA Fragment Analysis System, Transgenomic, Inc. San Jose, CA
  • computer controlled ovens which enclose the columns and column inlet areas.
  • DHPLC can separate heteroduplexes that differ by as little as one base pair.
  • separations of homoduplexes and heteroduplexes can be poorly resolved.
  • Artifacts and impurities can also interfere with the interpretation of DHPLC separation chromatograms in the sense that it may be difficult to distinguish between an artifact or impurity and a putative mutation (Underhill, et al., Genome Res. 7:996 (1997)). The presence of mutations may even be missed entirely (Liu, et al., Nucleic Acid Res. 26:1396 (1998)).
  • the references cited above and the references contained therein are incorporated in their entireties herein.
  • MIPC Denaturing Matched Ion Polynucleotide Chromatography
  • PCR amplification comprises steps such as primer design, choice of DNA polymerase enzyme, the number of amplification cycles and concentration of reagents. Each of these steps, as well as other steps involved in the PCR process affects the purity of the amplified product. Although the PCR process and the factors which affect fidelity of replication and product purity are well known in the PCR art, these factors have not been addressed, heretofore, in relation to mutation detection using MIPC.
  • PCR induced mutations wherein a non-complimentary base is added to a template, are often formed during sample amplification.
  • Such PCR induced mutations make mutation detection results ambiguous, since it may not be clear if a detected mutation was present in the sample or was produced during the PCR process.
  • many workers in the PCR and mutation detection fields make the erroneous assumption that PCR replication is perfect or close to perfect and PCR induced mutations are generally not taken into consideration in mutation detection analyses. This approach can result in false positives.
  • Applicants have recognized the importance of optimizing PCR sample amplification in order to minimize the formation of PCR induced mutations and ensure an accurate and unambiguous analysis of putative mutation containing samples. The use of MIPC by Applicants to identify and optimize the factors affecting PCR replication fidelity will be discussed in the Detailed Description.
  • one object of the present invention is to provide a method for detecting mutations in nucleic acids which is accurate, i.e., practically free of misleading results (e.g. "false positives"), is convenient to use, makes it possible to rapidly obtain results, is reliable in operation, is simple, convenient and inexpensive to operate.
  • Another object of the present invention is to provide a method for detecting mutations which utilizes a chromatographic method for separating polynucleotides with improved and predictable separation and efficiency.
  • An additional object of the present invention is to provide an improved method for preparing a sample of nucleic acids (e.g. DNA or RNA) prior to analysis for mutation.
  • nucleic acids e.g. DNA or RNA
  • Still another object of the instant invention is to provide a method for optimizing PCR for use in mutation detection.
  • Yet another object of the invention is to provide an improved method for selecting the temperature for conducting a chromatographic separation of nucleic acids for mutation detection.
  • An additional object of the invention is to provide an improved method for determining the optimal mobile phase for eluting nucleic acids in screening for mutations.
  • Still yet another object of the invention is to provide a method which can be automated.
  • a further object of the invention is to provide a method which can be used in basic research to test for unknown mutations and which can be used to rapidly screen numerous samples for a known mutation.
  • the present invention is an improved method for separating a sample mixture of polynucleotides by Matched Ion Polynucleotide Chromatography in which the concentration of polynucleotides (e.g., double stranded DNA) in the sample mixture is below a determined threshold concentration (e.g., the lower limit of detection of the polynucleotides).
  • the improvement includes applying the sample to the column whereby the polynucleotides are accumulated on the column.
  • the method includes comprises applying the sample in a mobile phase having a concentration of organic solvent less than a concentration necessary to elute the polynucleotides in the mixture.
  • the mobile phase preferably also includes a counterion agent.
  • the method further includes applying the mixture to a Matched Ion Polynucleotide Chromatography column and flowing an aqueous mobile phase under isocratic conditions through said column wherein impurities are removed from said mixture. If the sample mixture is applied to the column in an aliquot of greater than 10 ⁇ L, the solvent mixture preferably includes a counterion reagent.
  • the present invention is a method for preparing a double stranded DNA fragment for mutation detection and is also a method for mutation detection of a double stranded DNA fragment in which each method uses Denaturing Matched Ion Polynucleotide Chromatography (DMIPC).
  • DMIPC Denaturing Matched Ion Polynucleotide Chromatography
  • the double stranded DNA fragment corresponds to a wild type double stranded DNA fragment having a known nucleotide sequence.
  • the steps of the methods include (a) analyzing the sequence of the wild type double stranded DNA fragment to segment the double stranded DNA fragment into sample sequences, e.g., constant melting domains, of nucleotides having a melting point range of less than about 15 degrees C, each sample sequence having a first end and a second end opposite thereto; (b) amplifying one of these sample sequences by PCR using a set of primers which flank the first and second ends of this sample sequences, and (c) analyzing the amplified sample by MIPC.
  • the PCR amplification can include an analog of dGTP, e.g., 2,6-aminopurine, and can include a G-C clamp of up to 40 bases in a primer.
  • the mixture of the amplified sample sequence and the corresponding wild type double stranded DNA segment are subjected to a hybridization process in which the mixture is heated to a temperature at which the strands are completely denatured and then cooled until the strands are completely annealed, whereby a mixture comprising two homoduplexes and two heteroduplexes is formed if the sample sequence includes a mutation.
  • the method steps include analyzing the sequence of the wild type double stranded DNA fragment to segment the double stranded DNA fragment into sample sequences of nucleotides having a high melting domain and a low melting domain in which a mutation site is located; and amplifying one of said sample sequences by PCR using a set of primers which flank the first and second ends of said sample sequences.
  • the method comprises the steps of analyzing the sequence of the wild type double stranded DNA fragment to segment the double stranded DNA fragment into sample sequences of nucleotides wherein the mutation site is within twenty-five percent of the total number of base pairs from an end of the fragment; and amplifying one of said sample sequences by PCR using a set of primers which flank the first and second ends of said sample sequences.
  • the invention provides a method for evaluating a
  • the method includes the steps of (a) amplifying a polynucleotide by performing a plurality of PCR process cycles to yield a PCR amplification product, (b) analyzing the PCR amplification product preferably by MIPC to yield a PCR amplification product profile, including a profile of any mutations produced by PCR produced mutation.
  • An example of such a profile is the elution profile obtained from the Denaturing Matched Ion Polynucleotide Chromatography process.
  • the product profile is compared against a reference profile to determine the presence of PCR induced mutations in the PCR amplification product.
  • the invention is a method for identifying deviations of a PCR process from a predetermined reference profile.
  • the method steps include amplifying a polynucleotide by performing a plurality of PCR process cycles to yield a PCR amplification product and analyzing the PCR amplification product by MIPC to yield a PCR amplification product profile, including a profile of any PCR-induced mutations.
  • the PCR amplification product profile can be compared against a reference profile to identify the deviations of the PCR reaction product, including PCR-induced mutations, from a predetermined reference profile.
  • PCR induced mutations are detected by hybridizing the reaction after the last cycle and analyzing the reaction by MIPC.
  • the invention is a method for reducing PCR- induced mutations which includes (a) amplifying a polynucleotide by performing a plurality of PCR amplification process cycles to yield a first PCR amplification product (b) analyzing the first PCR amplification product by MIPC to yield a PCR amplification product profile (c) comparing the PCR amplification product profile against a reference profile to determine the presence of PCR induced mutations, and (d) amplifying a polynucleotide by performing a plurality of PCR amplification process cycles with an adjustment of one or more process variables to form a second PCR amplification product with reduced PCR induced mutations.
  • the method can include the additional steps of analyzing the PCR reaction product obtained in step (d) by MIPC to yield a second reaction product profile followed by (f) comparing the second reaction product profile against a set of standard profiles to determine deviations of the PCR process from a predetermined standard; and (g) performing a plurality of PCR process cycles with an adjustment of one or more process variables to form a third PCR reaction product with reduced deviation of the PCR process from the predetermined standard.
  • the process variables include magnesium concentration, dNTP concentrations, enzyme concentration, temperature, and source of DNA polymerase.
  • a non-proof reading DNA polymerase can be replaced by a proof reading polymerase.
  • the analysis of the PCR products can be used to evaluate primers and redesign primers to minimize artifacts, such as primer dimer formation.
  • the evaluation of the PCR process by MIPC can also be used to increase product yield and minimize byproducts.
  • a PCR product profile is compared to a predetermined standard profile.
  • the PCR is repeated with an increase of one or more of, the nucleotide, magnesium ion, or enzyme concentrations, or an decrease in the temperature or a combination thereof. Additional improvements in the PCR can be made by reducing the number of PCR process cycles when an excessive level of by products is observed.
  • Deviations from a predetermined standard profile can be further reduced by analyzing a second product profile, obtained using MIPC, of a PCR reaction after a reaction variable has been adjusted. This second profile is compared to a set of standard profiles to determine deviations of the PCR process form the predetermined standard. Another set of PCR cycles is then performed with a adjustment of one or more process variables to afford a third PCR reaction product profile with reduced deviation in the PCR products form the predetermined standard.
  • the PCR product 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. The purified PCR product can also be amplified by cloning in a host system.
  • the invention provides a method for determining the 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; (b) cooling the product of step (a) until the strands are completely annealed, whereby a mixture comprising two homoduplexes and two heteroduplexes is formed if the sample segment includes a mutation; (c) determining the heteromutant site separation temperature; (d) analyzing the product of step (b) with MIPC at the heteromutant site separation temperature to identify the presence of any heteromutant site separated components therein.
  • the heteromutant site separation temperature if the sequence of the normal double stranded DNA is known, the heteromutant site
  • T(hsst) X + m • T(w), wherein T(hsst) is the heteromutant site separation temperature, T(w) is the temperature, calculated by software or determined experimentally, at which there is a selected equilibrium between denatured and non-denatured states (e.g., a ratio of 50/50 or 25/75 denatured to non-denatured) of the normal double stranded DNA, m is a weighting factor, and X is the DMIPC detection factor.
  • the heteromutant site separation, temperature is determined by analyzing the product of step (b) by MIPC in a series of incremental MIPC separations in the mutation separation temperature range, each successive separation having a higher temperature than the preceding separation until a mutation separation profile is observed or the absence of any mutation separation profile in the mutation separation temperature range is observed, wherein a mutation separation profile identifies the presence of a mutation and the absence of a mutation separation profile indicates an absence of mutation in the sample.
  • the heteromutant site separation temperature can be determined by performing a series of incremental MIPC separations in the mutation separation temperature range, each successive separation having a lower temperature than the preceding separation until a mutation separation profile is observed or the absence of any mutation separation profile in the mutation separation temperature range is observed, wherein a mutation separation profile identifies the presence of a mutation and the absence of a mutation separation profile indicates an absence of mutation in the sample.
  • determination of a T(hsst) by MIPC is computer controlled and automated, whether the series of MIPC separations is performed at incrementally higher or incrementally lower temperatures.
  • a further aspect of the invention provides a preferred method for detecting DNA genetic mutations comprising the steps of (a) a calculation step for obtaining a calculated heteromutant site separation temperature; (b) a prediction step for obtaining a predicted heteromutant site separation temperature; (c) heating a mixture of a sample double stranded DNA segment and a corresponding wild type double stranded DNA segment to the predicted heteromutant site separation temperature; (d) analyzing the product of step (c) with MIPC at the predicted heteromutant site separation temperature to identify the presence of any heteromutant site separated components therein.
  • the calculation step comprises calculating the calculated heteromutant site separation temperature according to a first mathematical model.
  • the prediction step comprises adjusting the calculated heteromutant site separation temperature according to a second mathematical model.
  • the second mathematical model can be based on a comparison of empirically determined heteromutant site separation temperatures with calculated heteromutant site separation temperatures.
  • the calculated heteromutant site separation temperatures can be calculated using the first mathematical model.
  • determination of a T(hsst) by MIPC is computer controlled and automated.
  • a chromatographic method for separating a mixture of heteroduplex and homoduplex DNA molecules, including a first eluting DNA molecule and a last eluting DNA molecule, under conditions which selectively denature a mutation site present in the heteroduplex DNA molecule, comprising the steps of: (a) applying the mixture to a Matched Ion Polynucleotide Chromatographic column, (b) eluting the molecules of the mixture using a mobile phase comprising a counterion agent and a pre-selected fragment bracketing range of organic solvent concentration, the range comprising an initial concentration and a final concentration of organic solvent, the initial concentration containing an organic solvent concentration up to an amount required to elute the first eluting DNA molecule in the mixture, and the final concentration containing an organic solvent concentration sufficient to elute the last eluting DNA molecule in the mixture.
  • the pre-selected fragment bracketing range is obtained from a reference relating organic solvent concentration required for eluting DNA molecules of different base pair length, and base pair length.
  • a preliminary organic solvent concentration capable of eluting a DNA molecule of a specific base pair length, is obtained from a reference relating organic solvent concentration required for eluting DNA molecules of different base pair length, and base pair length, and the preliminary solvent concentration is used to select a fragment bracketing range.
  • the heteroduplex molecules and the homoduplex molecules can have the same base pair length.
  • the heteroduplex molecules can consist of at least two different heteroduplexes and the homoduplex molecules can be at least two different homoduplexes.
  • 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.
  • the preferred organic solvent is acetonitrile.
  • the counterion agent in this aspect of the invention is selected from the group consisting of lower alkyl primary, secondary, and tertiary amines, lower trialkylammonium salts and lower qauternary ammonium salts.
  • Examples of a counterion agent include octylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammoni
  • a related aspect involves, before step (a) immediately above, the preliminary steps of: (a) deriving a relationship between organic solvent concentration in the mobile phase required for eluting DNA molecules of different base pair length from the column, as a function of base pair length, and (b) determining from this derived relationship a pre-selected fragment bracketing range of organic solvent and a preliminary organic solvent concentration.
  • a critical aspect of the invention is a method for treating a matched ion polynucleotide chromatography column in order to improve the resolution of double stranded DNA fragments separated on the column comprising flowing a solution containing a multivalent cation binding agent through the column, wherein said solution has a temperature of about 50°C to 90°C.
  • the preferred temperature is about 70°C to 80°C.
  • the multivalent cation binding agent is a coordination compound, examples of which include water-soluble chelating agents and crown ethers. Specific examples include acetylacetone, alizarin, aluminon, chloranilic acid, kojic
  • salicylaldoxime dimethylglyoxime, ⁇ -furildioxime, cupferron, ⁇ -nitroso- ⁇ - naphthol, nitroso-R-salt, diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN, SPADNS, glyoxal-bis(2-hydroxyanil), murexide.
  • ⁇ - benzoinoxime mandelic acid, anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids, ⁇ , ⁇ '-bipyridine, 4-hydroxybenzothiazole, ⁇ -hydroxyquinaldine,
  • ⁇ "-terpyridyl 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol, rhodizonic acid, salicylaldoxime, salicylic acid, tiron, 4-chloro-1 ,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid, sodium diethyldlthiocarbarbamate, and zinc dibenzyldithiocarbamate.
  • the most preferred chelating agent is EDTA.
  • the solution preferably includes an organic solvent as exemplified by alcohols, nitriles, dimethylformamide, tetrahydrof uran, esters, and ethers.
  • the most preferred organic solvent is acetonitrile.
  • the solution can include a counterion agent such as lower primary, secondary and tertiary amines, and lower trialkyammonium salts, or quaternary ammonium salts.
  • the counterion agent can be octylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium
  • the invention provides a method for storing a Matched Ion Polynucleotide Chromatography column in order to improve the resolution of double stranded DNA fragments separated on the column.
  • the preferred method includes flowing a solution containing a multivalent cation binding agent through the column prior to storing the column.
  • the multivalent cation binding agent is a coordination compound, examples of which include water-soluble chelating agents and crown ethers. Specific examples include acetylacetone, alizarin, aluminon,
  • furildioxime furildioxime, nioxime, salicylaldoxime, dimethylglyoxime, ⁇ -furildioxime,
  • ethylenediamine glycine, triaminotriethylamine, thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids, ⁇ , ⁇ '-bipyridine, 4-
  • phenanthroline picolinic acid, quinaldic acid, ⁇ , ⁇ ', ⁇ "-terpyridyl, 9-methyl- 2,3,7-trihydroxy-6-fluorone, pyrocatechol, rhodizonic acid, salicylaldoxime, salicylic acid, tiron, 4-chloro-1 ,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid, oxalic acid, sodium diethyldlthiocarbarbamate, and zinc dibenzyldithiocarbamate.
  • the most preferred chelating agent is EDTA.
  • the solution preferably includes an organic solvent as exemplified by alcohols, nitriles, dimethylformamide, tetrahydrofuran, esters, and ethers.
  • the most preferred organic solvent is acetonitrile.
  • the solution can also include a counterion agent such as lower primary, secondary and tertiary amines, and lower trialkyammonium salts, or quaternary ammonium salts.
  • the counterion agent can be octylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium
  • FIG. 1 is a MIPC separation of pUC18 DNA-Haelll digestion fragments on a column containing alkylated poly(styrene-divinylbenzene) beads.
  • FIG. 2 is a chromatogram obtained after applying a 5 ⁇ L sample as in FIG. 1 but flowing a fixed concentration of 35%B through the column under isocratic conditions.
  • FIG. 3 is a chromatogram obtained after applying a second 5 ⁇ L aliquot of the standard pUC18 DNA-Haelll digest to the column of FIG. 2 and eluting with a gradient as described in FIG. 1.
  • FIG. 4 shows the melting profile of three DNA fragments with a mutation site indicated by an arrow.
  • FIG. 5 shows the DMIPC elution profile of fragment 1 of FIG. 4.
  • FIG. 6 shows the DMIPC elution profile of fragment 2 of FIG. 4.
  • FIG. 7 shows the DMIPC elution profile of fragment 3 of FIG. 4.
  • FIG. 8 shows the melting profile of four DNA fragments with a mutation site indicated by an arrow.
  • FIG. 9 shows the DMIPC elution profile of fragment 1 of FIG. 8.
  • FIG. 10 shows the DMIPC elution profile of fragment 2 of FIG. 8.
  • FIG. 11 shows the DMIPC elution profile of fragment 3 of FIG. 8.
  • FIG. 12 shows the DMIPC elution profile of fragment 4 of FIG. 8.
  • FIG. 13 is a schematic diagram showing stepwise melting of a theoretical three domain DNA molecule.
  • FIG. 14 shows a temperature titration curve of two homoduplexes (upper two curves) and two heteroduplexes (lower two curves).
  • FIG. 15 shows the melting profile for DNA fragment sY81.
  • FIG. 16 shows a theoretical melting profile of a three domain DNA fragment in which the 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 end 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 yield obtained after PCR with different DNA polymerase enzymes.
  • FIG. 20 is a comparison of MIPC chromatograms showing fidelity of PCR products obtained using different DNA polymerase enzymes.
  • FIG. 21 shows the effect of post-PCR hybridization on the analysis results of a PCR reaction as analyzed using MIPC.
  • FIG. 22 shows the use of MIPC to collect a pure PCR product.
  • FIG. 23 shows a schematic representation of a hybridization to form homoduplex and heteroduplex.
  • FIG. 24 shows the temperature dependent separation of homo- and heteroduplexes.
  • FIG. 25 shows the change in retention time with temperature of the peaks of the homo-and heteroduplexes from FIG. 24.
  • FIG. 26 is a schematic of a stepwise melting of a theoretical three domain DNA molecule.
  • FIG. 27 shows a temperature titration of two homoduplexes (upper two curves) and two heteroduplexes (lower two curves).
  • FIG. 28 shows the temperature titration for DNA fragment sY81.
  • FIG. 29 shows a theoretical temperature titration of a three domain DNA
  • fragment in which the domains have melting temperatures of 55°C, 60°C and
  • FIG. 30 shows the effect of column temperature on separation of homoduplex and heteroduplex DNA for DYS271 209 bp mutation mixture with a heteroduplex mismatch at position 168.
  • FIG. 31 shows a temperature titration for the wild type homoduplex from FIG. 30 with the inflection points indicated by arrows.
  • FIG. 32 is a DNA melting profile of the DYS271 209 bp mutation mixture.
  • FIG. 33 is a schematic representation of a cooperative approach (Model A) and a non-cooperative approach (Model B) to modeling DNA melting within a fragment.
  • FIG. 34 shows the melting profile of the DYS271 209 bp mutation mixture using a noncooperative weighted model.
  • FIG. 35 shows a profile of the melting of a DYS271 209 bp mutation mixture using a cooperative model where the loop entropy has been changed to mimic a noncooperative model.
  • FIG. 36 shows a melting profile of DYS271 209 bp mutation mixture using a cooperative model with different loop entropies with a temperature offset, a slope, and a fragment size dependent term included.
  • FIG. 37 shows the change in retention time with temperature for heteroduplex and homoduplex species in a DYS271 209 bp mutation mixture.
  • 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 double stranded polynucleotides.
  • FIG. 41 shows an embodiment of a mobile phase gradient for mutation detection by DMIPC.
  • FIG. 42 is an elution profile showing separation of a 209 base pair homoduplex/heteroduplex mutation detection mixture performed by DMIPC at 56°C.
  • FIG. 43 is an elution profile of another injection of the same 209 bp mixture and using the same column as in FIG. 42, but after changing the guard cartridge and replacing the pump-valve filter.
  • FIG. 44 is an elution profile of another injection of the same 209 bp mixture and using the same column as in FIG. 43, but after flushing the column with
  • FIG. 45 is an elution profile of DYS271 209 bp mutation standard using titanium frits from lot A.
  • FIG. 46 is an elution profile of DYS271 209 bp mutation standard using titanium frits from lot B.
  • FIG. 47 is an elution profile of DYS271 209 bp mutation standard using PEEK frits.
  • FIG. 48 is a DMIPC elution profile of a 100 bp PCR product from a wild-type strand of Lambda DNA.
  • FIG. 49 is a DMIPC elution profile of a hybridized mixture containing a
  • the subject matter of the present invention primarily relates to an improved method for separating mixtures homoduplex and heteroduplex DNA fragments having the same base pair (bp) length using MIPC. Since such a separation is performed under partially denaturing conditions, i.e., at an elevated temperature which is sufficient to denature a heteroduplex at the site of bp mismatch, the separation process will be called Denaturing Matched Ion Polynucleotide Chromatography (DMIPC) herein.
  • DMIPC Denaturing Matched Ion Polynucleotide Chromatography
  • a separation process called “Denaturing HPLC” (DHPLC) has been used to detect mutations by separating a heteroduplex (resulting from the presence of a mutation) and a homoduplex having the same bp length.
  • DHPLC can separate heteroduplexes that differ by as little as one base pair under certain conditions.
  • separations of homoduplexes and heteroduplexes can be poorly resolved.
  • Artifacts and impurities can also interfere with the interpretation of DHPLC separation chromatograms in the sense that it may be difficult to distinguish between an artifact or impurity and a putative mutation (Underhill, et al., Genome Research 7:996 (1997). For these and other reasons, which will soon become apparent, the presence of mutations may even be missed entirely (Liu, et al., Nucleic Acid Res. 26:1396 (1998)). For example, if a mutation is located in a high melting domain of DNA fragment, it may not be possible to detect that mutation using the known art.
  • the references cited above and the references contained therein are incorporated in their entireties herein.
  • MIPC Matched Ion Polynucleotide Chromatography
  • DMIPC Denaturing Matched Ion Polynucleotide Chromatography
  • MIPC uses unique non-polar separation media which comprises organic polymers, silica media having a non-polar surface comprising coated or covalently bound organic polymers or covalently bound alkyl and/or aryl groups, continuous non-polar separation media, so called monolith or rod columns, comprising non-polar silica gel and organic polymer.
  • the separation media used in MIPC can be porous or non-porous.
  • MIPC systems and separation media are commercially available (Transgenomic, Inc. San Jose, CA)
  • MIPC separation media are washed with acid prior to column packing. In addition, freshly packed columns are washed with 0.1 M
  • EDTA solution at least 50°C, and preferably at least 70°C to ensure removal of residual traces of multivalent cations from the separation media and the column interior.
  • Columns and all solution contacting surfaces of an MIPC system comprise materials which do not release multivalent metal cations e.g., coated stainless steel, titanium, polyetherether ketone (PEEK) or any combination thereof.
  • PEEK polyetherether ketone
  • the columns and samples are additionally protected from adventitious multivalent cations by placing guard cartridges containing multivalent cation capture resin in-line between the solvent reservoir and the column and/or injection port.
  • Applicants have surprisingly found that, when MIPC is used for mutation detection, the method is even more sensitive to purity of the separation media, the presence of trace levels of multivalent cations, and other separation parameters.
  • a column that operates well for MIPC may not operate for DMIPC until additional cleaning is performed; the cleaning processes can include flushing with organic solvents and/or chelating agents to remove contaminants.
  • the requirement for preventing contamination with multivalent cations is even more stringent for detection of mutations using DMIPC.
  • PCR amplification comprises steps such as primer design, choice of DNA polymerase enzyme, the number of amplification cycles and concentration of reagents. Each of these steps, as well as other steps involved in the PCR process affects the purity of the amplified product. Although the PCR process and the factors which affect fidelity of replication and product purity are well known in the PCR art, these factors have not been addressed, heretofore, in relation to mutation detection using MIPC.
  • PCR induced mutations wherein a non-complimentary base is added to a template, are often formed during sample amplification.
  • Such PCR induced mutations make mutation detection results ambiguous, since it may not be clear if a detected mutation was present in the sample or was produced during the PCR process.
  • many workers in the PCR and mutation detection fields make the erroneous assumption that PCR replication has essentially "perfect" fidelity and PCR induced mutations are generally not taken into consideration in mutation detection analyses. This approach can result in false positives.
  • Applicants have recognized the importance of optimizing PCR sample amplification in order to minimize the formation of PCR induced mutations and ensure an accurate and unambiguous analysis of putative mutation containing samples. The use of MIPC by Applicants to identify and optimize the factors affecting PCR replication fidelity will be discussed herein below.
  • MIPC mutation detection by MIPC
  • Other aspects of mutation detection by MIPC comprise improved methods for treating of materials comprising chromatography system components, improved methods for treating separation media, methods for solvent pre-selection to minimize methods development time, methods for optimum temperature pre-selection to effect partial denaturation of a heteroduplex during DMIPC and optimization for rapid DMIPC analysis using automated high throughput mutation detection screening assays.
  • Another important discovery by Applicants takes advantage of the unique mechanism of MIPC to concentrate the polynucleotides in a sample by a plurality of applications onto a MIPC column. This novel method obviates the need to concentrate samples by solvent evaporation which may cause sample degradation or introduce contaminants.
  • Applicants have devised a novel and comprehensive protocol which addresses the problems in the prior art described above.
  • This protocol comprises all the steps necessary to ensure the accuracy, reproducibility and speed of mutation detection using MIPC.
  • Such a comprehensive approach to mutation detection using MIPC has not been previously described.
  • An optimal embodiment of the present invention includes implementation of all of the aspects described herein in order to achieve unambiguous, accurate and reproducible mutation detection results using MIPC.
  • the following presentation is divided into sections: Sample Preparation; Primer Design; Optimization of PCR; Temperature Selection; Mobile Phase Selection; Column Preparation and Maintenance.
  • the sample can be subjected to a process for reducing the volume until a polynucleotide concentration is reached which is sufficient to detect.
  • a precipitating agent e.g., ethanol, acetonitrile or other organic solvents.
  • solid media such as those based on ion exchange (e.g., as available from Qiagen, Valencia, CA), silica gel (e.g., as sold by CPG, Inc. Lincoln Park, NJ), and polymers (e.g., as sold by Hamilton, Inc., Reno, NV).
  • MIPC Matched Ion Polynucleotide Chromatography
  • the change of concentration of bulk acetonitrile from which there is complete adsorption of a 102 bp DNA fragment to the separation media to complete desorption is less than 2%. Larger fragments require a larger range, but the total range of acetonitrile change for the separation of a range of 100 - 600 bp of DNA is 7.5 % acetonitrile, which can be performed over a 5 minute gradient.
  • the DNA is released from the top of the column. The release may be gradual, but as the concentration of the acetonitrile is increased by the gradient elution process, the fragment will travel faster until it is traveling at the linear velocity of the mobile phase.
  • the release process with the conditions reported in these examples, the release length is 1 cm or less.
  • the separation of the fragments is based mostly on the top 20% of a 5 cm column and especially on the top thin section of the column bed. This means that the integrity or uniformity of the top of the column bed is much more important the length of the column for achieving high resolution separations. This is not to say that the length of the column cannot be made to be important when elution conditions are changed to small gradients or isocratic separation conditions.
  • an organic solvent concentration in the mobile phase sufficient to elute a polynucleotide of known base pair length can be predetermined from a reference relating organic solvent concentration and base pair length. For a selected concentration of organic solvent in the mobile phase, only a single base pair length polynucleotide (and all shorter polynucleotides) will elute form the column in a tight band.
  • the present invention is an improved method for separating a sample mixture of polynucleotides by MIPC wherein the concentration of polynucleotides in the sample mixture is contained in a large volume in which the sample concentration is below a determined threshold concentration.
  • a threshold is the limit of detection of a UV absorbance signal which is at or below the background signal.
  • a particular example is a 3
  • the improvement comprises applying the sample to an MIPC column in more than one aliquot or by a large aliquot (e.g., greater than about 20 ⁇ L) whereby the sample accumulates and is concentrated on the column.
  • Polynucleotide samples generally double stranded DNA, are applied in a solvent or mobile phase which has a concentration of organic component less than a concentration necessary to elute the polynucleotides from the MIPC column. Since the organic solvent concentration in the mobile phase is not sufficient to elute the polynucleotides, the polynucleotides applied to the column from a plurality of aliquots, simply accumulate and concentrate, at the top of the column.
  • This improvement obviates the need to concentrate the sample by evaporation and, therefore, eliminates a step which can degrade the sample. This is extremely important, since eliminating a step which can degrade the sample concomitantly eliminates a source of ambiguity in the analysis.
  • counterion agent e.g. TEAA
  • the plurality of sample aliquots is applied to the MIPC column automatically by means of a sample autoinjector.
  • a large dilute sample (e.g., greater than 20 ⁇ L) is injected and preconcentrated on the column.
  • the sample contains a counterion agent such as TEAA to facilitate binding of the sample on the column.
  • the column when multiple aliquots of a sample are applied, the column can be subjected to a wash process under isocratic conditions in which an aqueous mobile phase containing a fixed concentration of organic solvent which is not sufficient to elute any of the polynucleotides of interest. This process washes away impurities such as salts, nucleotide bases, buffers, and other debris, but leaves the polynucleotide sample in a concentrated band at the top of the column.
  • the mobile phase preferably comprises a counterion agent and an organic solvent selected from the group consisting of acetonitrile, ethanol, methanol, 2-propanol and ethyl acetate.
  • the preferred organic solvent in the mobile phase is acetonitrile.
  • the concentration of acetonitrile in the isocratic mobile phase is preferably greater than or equal to 2%.
  • the counterion agent in the mobile phase is selected from the group consisting of lower alkyl primary, secondary, and tertiary amines, lower trialkylammonium salts, and lower quaternary ammonium salts.
  • the preferred counterion agent is triethylammonium acetate due to its volatility.
  • FIG. 1 is an MIPC chromatogram of a standard pUC18 DNA-Haelll digest. A 5 ⁇ L sample
  • FIG. 2 shows that no DNA fragments eluted as represented by the flat baseline of the chromatogram.
  • FIG. 3 a second 5 ⁇ L pUC18 DNA-Haelll digest was injected onto the same MIPC column and the column was eluted with 35% B followed by the gradient described above in relation to FIG. 1.
  • the peaks had essentially identical retention times and twice the height as the reference chromatogram shown in FIG. 1. The fact that there was neither a shift in retention time nor peak broadening, demonstrates that the first sample injection remained in a tight band at the top of the column despite isocratic washing for the ten minutes of FIG. 2 and the subsequent application of the second sample.
  • the present method can be used in the case where a sample is contained in a volume which is too small to be accurately injected onto a MIPC column, e.g., less than about 1 ⁇ L. In this situation, the present method can be used in the case where a sample is contained in a volume which is too small to be accurately injected onto a MIPC column, e.g., less than about 1 ⁇ L. In this situation, the present method can be used in the case where a sample is contained in a volume which is too small to be accurately injected onto a MIPC column, e.g., less than about 1 ⁇ L. In this situation, the
  • sample can be diluted and then injected in multiple aliquots as described hereinabove.
  • Large volume samples can also be loaded onto a MIPC column as a single continuous application, e.g., by using a pump or syringe.
  • PCR polymerase chain reaction
  • a fragment such as an exon
  • the sample sequences can be from about 150 to 450 base pairs. It is possible to detect a single base mutation in long fragments, e.g. 1.5 kbase. However, if in such a fragment a mutation occurred in a sample sequence having a high melting point (e.g. a G-C rich region) then it might not be detectable, since high temperatures would be needed to partially denature at the mutation site, and all the other lower melting sequences would denature first.
  • a high melting point e.g. a G-C rich region
  • Applicants have found that mutation detection of dsDNA using MIPC is more reliable and accurate if the mutation is located within a sample sequence having a narrow melting point range.
  • a range of less than about 20°C is preferred in the present invention, i.e. any one base in the sample sequence has a melting point that is within about ⁇ 10°C of any other base in the sequence. In a more preferred embodiment, the range is less than about 15°C.
  • An example of a sample sequence is the constant melting domain as described by Lerman et al. (Meth. Enzymol. 155:482 (1987)) .
  • a heteroduplex having a base pair mismatch within a sample sequence will denature at the site of the mismatch, while the rest of the sample sequence will remain intact.
  • the partially denatured heteroduplex can be separated and detected using
  • the present invention is a method for preparing the sequence of the normal dsDNA fragment to segment, i.e., mark off, the dsDNA fragment into sample sequences of nucleotides having a melting point range of less than about 15°C, each sample sequence having a first end and a second end opposite thereto.
  • a selected sample sequence is amplified by PCR using both forward and reverse primers which flank the first and second ends of the sequence.
  • a fragment can be constructed using software such as MacMelt® (BioRad Laboratories, Hercules, CA), MELT (Lerman et al. Meth. Enzymol. 155:482 (1987)), or WinMeltTM (BioRad Laboratories).
  • the present invention is a method for analyzing the PCR amplified sequence (amplicon) by MIPC.
  • a standard such as a wild type homoduplex DNA
  • the mixture is subjected to a hybridization process in which the mixture is heated and reannealed to form a mixture of homoduplexes and heteroduplexes.
  • the present invention concerns a method for improved primer design for mutation detection analysis by MIPC.
  • the overall design process design consists of both long range and short range primer design.
  • long range primer design the objective is to design primers that produce good quality PCR products.
  • Good quality PCR products are defined herein to mean PCR products produced in high yield and having low amounts of impurities such as primer dimers and PCR induced mutations.
  • Good quality PCR can also be affected by other reaction parameters, such as the enzyme used, the number of PCR cycles, the concentration and type of buffer used, temperature thermal cycling procedures and the quality of the genomic template.
  • Methods for producing good quality PCR products are discussed by Eckert et al. (PCR: A Practical Approach, McPherson, Quirke, and Taylor eds., IRL Press, Oxford, Vol. 1 , pp. 225-244, 1991). This reference and the references therein are incorporated herein in their entireties.
  • Short range primer design should fulfill two requirements. First, It should fulfill all the requirements of long range primer design and give good quality PCR products.
  • the MIPC method must produce fragments that allow the MIPC method to detect a mutation or polymorphism regardless of the location of the mutation or polymorphism within the amplified fragment.
  • large DNA fragments having up to several thousand base pairs, can be amplified by PCR. If the only goal of the amplification is to replicate the desired fragment, then there is a large latitude in the design of primers which can be used for this purpose.
  • primers must be designed such that the fragment produced in the PCR process is capable of being detected, and will produce a signal, when analyzed by DMIPC.
  • the fragment length is 150-600 bp. In the most preferred embodiment, the fragment length for DMIPC mutation detection analysis is 150-400 bp.
  • primer design is if the analysis is used as a "screening" test. Another goal is in analysis for research or diagnostic purposes. "Screening” is defined herein as the study or analysis of DNA fragments to determine if the fragments contain variations (polymorphisms) in a population and correlate that variation to disease. It is to be understood that, within the context of this invention, the term "mutation" includes polymorphism.
  • DMIPC is used as a screening technique, then an important aspect of the present invention is a method for designing primers to produce a fragment in which a putative mutation can be detected, regardless of where the mutation site is located within the fragment.
  • the primer design can be further refined so that the analysis is optimized, i.e., the resolution of the homoduplex and the hetroduplex peaks is maximized.
  • the resolution for the analysis of known mutations, accuracy of analysis can be performed. Improved resolution is required for diagnostic mutation applications.
  • automatic identification of the positive presence of mutation can be more easily implemented with appropriate software and an algorithm that overlays and comparatively measures the peaks of the wild type and mutant DNA samples.
  • the method of the present invention allows the determination of whether the amplified fragment contains a region within which it would be difficult to detect a putative mutation.
  • a mutation can be detected by DMIPC even if located in a position within a fragment in which it would be difficult to detect by other methods, e.g., in the middle of a fragment or in a high melting domain. Mutations so located can be detected by DMIPC in three ways.
  • a "peak overlay" technique can be used, wherein a wild type standard peak is overlayed onto a partially resolved mutation-containing sample peak. The area of the standard peak is subtracted from the area of the sample peak.
  • the sample is considered to contain a mutation.
  • the DMIPC analysis can be performed at two or more temperatures, each temperature corresponding to a different melting domain, as further described hereinbelow. It has been surprisingly discovered by Applicants that for a multi-domain fragment, that changing the selection of primers has a dramatic effect on the melting profile of the amplified sequence predicted by a software program. This observation is advantageously used in a third embodiment of this aspect of the present invention in which primers are re-selected to change the melting profile of the fragment of interest to lower the differences between the Tm's of the domains in the fragment.
  • short range primer selection there are two situations under which short range primer selection is performed. One is if the mutation is to be used for screening for variation in a genome. The other is a diagnostic or clinical application where the presence of a particular mutation is measured in a set of samples. The following summarizes the options for preferred short range primer selection in each of these situations.
  • primer design for screening applications is to design the primers so that the region of interest is at a lower melting domain within the fragment.
  • the primers are preferred to be designed so that the fragment being measured will overlap the regions of interest as the analysis is performed traveling down the exon.
  • the temperature difference between the higher melting domain and the lower melting domain is preferred to be greater than 5°C and most preferred to be greater than 10°C.
  • primers can be redesigned for R&D diagnostic or clinical applications.
  • the mutation is preferably located within 25% or 25 bases of the end which ever is closer to the end.
  • the other end of the fragment contains a higher melting domain of preferably 5°C, more perferably 10°C higher, and most perferably 15°C higher than the lower domain where the mutation is located.
  • a G-C clamp can be applied. The size of the clamp can be up to 40 bp, but can be as little as 4-5 bp, with 10-20 bp most preferred.
  • primers can be redesigned for research and development diagnostic or clinical applications.
  • the primers are selected to produce a fragment having the mutation near one of the ends. This could be within about 25 bases of one of the ends for fragments having similar length to the examples described herein, or this could be within about 25% of the total length from either end.
  • the primers are selected to produce a fragment having a domain that has at least a 5°C higher Tm at the end opposite to the end containing the mutation.
  • the primers are selected so that the mutation is located in a "lower melting" domain of the fragment.
  • a mutation can also be detected by DMIPC in a high melting domain of the fragment either if the high melting domain does not have a melting temperature that is too different from other domains in the fragment or if a higher column temperature is used that is optimized for the higher melting domain of the fragment.
  • Example 2 The method of the invention for the design of primers is illustrated by Example 2.
  • a p53 DNA template containing a mutation was amplified by means of PCR processes in which three different sets of primers were used. Each primer set was designed to produce amplicon fragments having the mutation located in a different melting domain. The melting profiles,
  • FIG. 5 shows the effects of primer design on the DMIPC mutation detection analysis results of three selected amplicon fragments.
  • Example 3 provides a further illustration of the use of the method of the present invention.
  • Primer design which located the mutation within about 20% of either end gave the best resolution upon DMIPC analysis.
  • Fragment 1 (in FIG. 8), with the mutation located in a constant melting domain, gave the best resolution (FIG. 9), while the poorest resolution was seen (FIG. 12) when the mutation was located near the middle of a fragment (fragment 4 in FIG. 8).
  • a G-C clamp can be applied to increase the melting temperature at the desired end (Myers et al., Nucleic Acids Res. 13:3111 (1985)).
  • G-C clamping is a technique in which additional G or C bases are included on the 5' end of one or both of the primers. The polymerase enzyme will extend over these additional bases incorporating them into the amplified fragment thereby raising the melting temperature of the end(s) of the fragment relative to that in the vicinity of the mutation.
  • the size of the G-C clamp can be up to 40 bp and as little as 4 or 5 bp.
  • the most preferred G-C clamp for mutation detection by DMIPC is 10 to 20 bp.
  • G-C clamps are required (Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232 (1989)) for almost all fragment mutation analysis whereas in DMIPC, G-C clamps are rarely needed.
  • An exception is perhaps where the mutation is in the center of the fragment and the length is less than 100 bp and the melting profile is flat or in cases where the mutation in a high melting region of the fragment and a higher melting region is in effect a G-C clamp. In these cases, proper primer selection will result in a fragment in which the mutation can be detected.
  • the long range primer design described above can be further refined by local primer design in which several other factors should be considered. For example, primers with non-template tails, such as universal sequencing primers or T7 promoters, should be avoided.
  • the preferred primer has a Tm
  • primers is preferably about 1°C.
  • Tm The difference in Tm between primer and
  • the 3'-pentomer of each primer should be more
  • Any possible primer dimers should be less stable than the 3'-pentomer by at least 5 kcal/mol (i.e., 5 kcal more positive).
  • Any primer self annealing loops should have a Tm of
  • a long fragment e.g., an exon
  • Such long fragments generally contain multiple melting temperature domains.
  • Double-stranded DNA fragments melt in a series of discontinuous steps as different regions with differing thermal stabilities which denature in response to increasing temperature. These different regions of thermal stability are referred to as "domains", and each domain is approximately 50-300 bp in length.
  • Each domain has its own respective Tm and will exhibit thermodynamic behavior which is related to its respective Tm.
  • the presence of a base mismatch within a domain will destabilize it, resulting in a decrease in the Tm of that domain in the heteroduplex relative to its fully hydrogen-bonded counterpart found in the homoduplex.
  • the presence of a base mismatch will lower the Tm by approximately 1 ° - 2°C.
  • FIG. 13 depicts the melting of a theoretical three domain fragment in schematic form.
  • every DNA fragment is comprised of one or more regions of independent thermal stability or domains.
  • the Tm of a domain serves as a thermodynamic signature and determines the thermodynamic behavior of a domain. As depicted in the schematic in FIG. 13, as the temperature is gradually increased domain A will denature first because its Tm is lower than that of domain B or C. Domain B has an intermediate Tm and would melt next, and domain C would be the last to melt because its domain has the highest Tm within this fragment.
  • the base pairs within a domain melt in unison over a very narrow temperature range.
  • the denaturing of a domain is characterized by a sigmoidal profile (FIG. 14) which indicates "cooperativity" among the base-pairs comprising the domain.
  • the midpoint of the inflection (slope) is the Tm and corresponds to a temperature at which the domain exists in equilibrium between single and double stranded states. As the temperature is increased beyond the Tm, the entire domain will rapidly convert to a completely single-stranded conformation.
  • a putative point mutation could be present in any of the domains: A, B or C.
  • DMIPC DMIPC
  • the MIPC system is capable of automatically profiling the melting behavior of a DNA fragment by running a series of separations at incremental
  • FIG. 14 depicts the melting of the four related homo- and heteroduplex forms of a DNA fragment (the homoduplexes are represented by dashed lines). These melting profiles illustrate how the midpoints of the heteroduplex inflections are shifted to the left, indicating lower Tms and more rapid elution from the MIPC column compared to the homoduplexes. It is also apparent that the Tms of the heteroduplexes are approximately 1 °-2°C lower than the homoduplexes.
  • FIG. 15 depicts the melting profile of a 230 bp restriction fragment designated sY81. Any domains present in this fragment are now represented by a single sigmoidal curve extending between approximately 54°-59°C. The temperature at this midpoint of the inflection is the Tm of the melting profile of the homoduplex fragment or Tm homo . Determining the Tm homo from the melting profile is necessary for selecting an appropriate temperature at which to carry out mutation screening. Since the presence of a base mismatch will lower the Tm of the corresponding heteroduplex domain being scrutinized by approximately 1°-2°C, a fairly accurate estimation can be made of the Tm of
  • Tm h ⁇ t ⁇ ro Tm homo - 1°C.
  • the appearance of the melting profile indicates that
  • the Tm homo is approximately 56°C. Therefore, the preferred temperature for the Tm homo is approximately 56°C. Therefore, the preferred temperature for the Tm homo is approximately 56°C. Therefore, the preferred temperature for the Tm homo is approximately 56°C. Therefore, the preferred temperature for the Tm homo is approximately 56°C. Therefore, the preferred temperature for the Tm homo is approximately 56°C. Therefore, the preferred temperature for the Tm homo is approximately 56°C. Therefore, the preferred temperature for
  • any domains present in this fragment will be partially denatured at that temperature.
  • the temperature selected must be less than or equal to the Tm of that domain which has the lower Tm. If an intermediate temperature is selected, the lower Tm domain in both the heteroduplex and homoduplex fragments will be denatured and the ability to detect mutations in that domain will be lost. If the DNA fragment melts over a temperature range greater than 5°C, more than one temperature must be used to screen the fragment.
  • Domains A and B can be simultaneously screened at a temperature of 54°C and domains B and C can be
  • FIG. 16 depicts a theoretical melting profile for a three domain fragment with Tms of 55°C, 60°C and 65°C.
  • PCR Polymerase Chain Reaction
  • the 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 the PCR process, and the larger amount so obtained is subsequently analyzed.
  • DNA samples obtained from genetic material may be amplified and sequenced, or studied to determine its biological effects.
  • Apparatus for performing PCR amplifications e.g. Air Thermo Cycler (Idaho Technologies) and reagents are commercially available from numerous sources, e.g. Perkin-Elmer Catalog "PCR Systems, Reagents and Consumables” (Perkin-Elmer Applied Biosystems, Foster City, CA).
  • PCR is typically run in a buffer at pH 5-8.
  • the buffer contains a double stranded DNA sample to be amplified, a first primer, a second primer, magnesium chloride (MgCI 2 ), and the four deoxynucieotide triphosphates (dATP, dTTP, dCTP, and dGTP) generally referred to as "bases", the building blocks of DNA.
  • the reaction mixture is heated to a temperature (typically 90° C) sufficient to denature the DNA sample, thereby separating its two complimentary polynucleotide strands.
  • the DNA may be denatured enzymatically at ambient temperature using a helicase enzyme.
  • DNA polymerase is added before the reaction is started. If denaturing is effected by heat and a thermolabile DNA polymerase is used, the DNA polymerase is added after the denaturing step. If denaturing is effected by helicases at ambient temperature, a thermolabilie DNA polymerase may be included with the other reagents before the start of the reaction. Other denaturing conditions are well known to those skilled in the art and are described in U.S. Patent No. 5,698,400 to Cotton (1997). This reference and the references cited therein are incorporated in their entirety herein. DNA polymerases are commercially available from a variety of sources, e.g.
  • the primers are an oligonucleotide sequence typically consisting of 7 - 25 nucleotide bases. Primers are usually synthesized chemically in a predetermined defined sequence. The primer sequence is designed to be complimentary to an identified portion of the denatured DNA strands to be replicated by PCR. Primers are commercially available from a variety sources, e.g., Synthetic Genetics (San Diego, CA). Upon cooling the reaction to about 50° C, each of the primers anneals to its complimentary base sequence in each strand of the denatured DNA sample to be replicated.
  • dNTPs are commercially available from a variety of sources, e.g. Pharmacia (Piscataway, NJ). By repeating this process numerous times, a geometric increase in the number of desired DNA strands is achieved in the initial stages of the process or as long as a sufficient excess of reagents are present in the reaction medium. Thus, the amount of the original DNA sample is amplified. PCR is well known in the biotechnology art and is described in detail in
  • the PCR process is limited in its ability to replicate DNA strands by the specificity of the DNA polymerase used, as well as other features of the reaction.
  • the primers may bind to portions of a DNA strand which are only partially complimentary. Such nonspecific primer binding will produce products with an undesired sequence.
  • the first and second primers may also bind to complimentary portions of each other, producing primer dimers.
  • the specificity of DNA polymerases varies with the reaction conditions employed as well as with the type of enzyme used. No enzyme affords completely error-free extensions of a primer. A non- complimentary base will be introduced from time to time. Such enzyme related errors produce double stranded DNA products which are not exact copies of the original DNA sample, that is, the products contain PCR induced mutations.
  • PCR process variables which may degrade the accuracy or fidelity of DNA replication include reaction temperature, primer annealing temperature, enzyme concentration, dNTP concentration, Mg" concentration, source of the enzyme and combinations thereof.
  • Most applications of PCR require the highest level of replication fidelity which can be achieved.
  • detection of mutant genes the construction of genetically engineered monoclonal antibodies, analysis of T- cell receptor allelic polymorphism, the study of HIV variation in vivo and cloning of individual DNA molecules from the PCR amplified population depend upon high fidelity amplification for their success.
  • PCR products and processes have been monitored by gel electrophoresis or capillary electrophoresis. These methods separate DNA fragments by size but cannot detect PCR induced mutations.
  • PCR induced mutations is defined to mean an insertion, during the PCR process, of one or more bases which are not complementary to their corresponding base in the template. Thus, a PCR induced mutation is a deviation from replication fidelity.
  • Such mutations have been heretofore separated from their normal counterparts by gradient gel electrophoresis or gradient capillary electrophoresis.
  • Capillary electrophoresis analysis takes at least 30 minutes. A gel electrophoresis analysis takes several hours.
  • Minimizing deviations in the PCR replication process can be achieved by modifying a reaction condition or reagent which causes the deviations if the cause of the deviation can be identified.
  • One aspect of this invention is based on the discovery that the product profile obtained from application of the Matched Ion Polynucleotide Chromatography (MIPC) method to PCR reaction products can be used to identify the sources of the deviations from accurate replication.
  • MIPC Matched Ion Polynucleotide Chromatography
  • MIPC Matched Ion Polynucleotide Chromatography
  • MIPC separates double stranded polynucleotides by size or by base pair sequence and is therefore a preferred separation technology for evaluating and analyzing PCR.
  • MIPC when mixtures of DNA fragments are applied to an MIPC column, they are separated by size, the smaller fragments eluting from the column first.
  • MIPC when performed at a temperature which is sufficient to partially denature a heteroduplex, is referred to as "Denaturing Matched Ion Polynucleotide Chromatography" (DMIPC).
  • DMIPC Denaturing Matched Ion Polynucleotide Chromatography
  • heteroduplexes elute from the column faster than the corresponding homoduplexes during DMIPC.
  • the parameters which optimize PCR fidelity of replication or yield can be predicted by creating and analyzing a PCR product profile and comparing the profile to a standard product profile to predict which of the many possible PCR parameters require adjustment in order to achieve an optimum fidelity of DNA replication and yield.
  • the reaction parameter which is responsible for producing the undesired product can be determined and modified in a subsequent PCR process in order to eliminate or minimize said undesired product.
  • PCR product profile as used herein is defined to mean the data generated by MIPC as applied to the product of a PCR process.
  • the MIPC data can distinguish the expected product and other components of the reaction mixture from one another. These components comprise desired product(s), byproducts and reaction artifacts.
  • the PCR product profile can be in the form of a visual display, a printed representation of the data or the original data stream.
  • the preferred method of this invention for generating a PCR product profile is MIPC.
  • the preferred display is a separation chromatogram output of the MIPC process as seen on a video screen or on printed hard copy or the data stream corresponding thereto.
  • MIPC is an efficient analytical method which can separate all the potential products of a PCR process in an accurate, reproducible manner required to quantify the results. Furthermore, the method is easy to implement. MIPC has not previously been used to analyze and optimize PCR processes.
  • standard profile is defined to mean the data generated by the MIPC method when this method was used to separate reference standards related to the PCR process.
  • Reference standards can comprise the expected product of the PCR process, DNA fragments of known base pair length which can be used to calibrate the display for base pair length, primers, primer dimers, heteroduplexes of the expected PCR product or combinations of more than one of these.
  • the standard profile can also comprise an actual PCR process which has been separated by MIPC.
  • the standard profile can be in the form of a visual display, a printed representation of the data or the original data stream. MIPC is easy to implement, provides reproducible results, and is capable of effectively separating single and double stranded polynucleotides on the basis of both size and base sequence.
  • MIPC can separate mixtures of single and double stranded polynucleotides in general and DNA fragments in particular, with essentially none of the limitations of the previously known gel based methods described above.
  • MIPC separations are typically complete in less than 10 minutes, and frequently in less than 5 minutes.
  • MIPC systems Transgenomic, Inc. San Jose, CA
  • the system used for MIPC separations is rugged and provides reproducible results. It is computer controlled and the entire analysis of multiple samples can be automated.
  • the system offers automated sample injection, data collection, choice of predetermined eluting
  • SUBST ⁇ UTE SHEET (RULE 26) solvent selection based on the size of the fragments to be separated, and column temperature selection based on the base pair sequence of the fragments being analyzed.
  • the separated PCR mixture components provide a reaction product profile which can be displayed either in a gel format as a linear array of bands or as an array of peaks.
  • the display can be stored in a computer storage device.
  • the display can be expanded and the detection threshold can be adjusted to optimize the product profile display.
  • the reaction profile may be displayed in real time or retrieved from the storage device for display at a later time.
  • the product profile display can be viewed on a video display screen or as 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 a product profile of a PCR process (described in Examples 4-8) in the form of a separation chromatogram, wherein the separation was performed at three different temperatures by
  • the product fragment contained base pairs. At 62° C, two poorly resolved peaks are seen. When the temperature of the separation process was raised to 64° C a broad shoulder, representing heteroduplexes resulting form PCR induced mutations, is seen at a lower retention time than the main, sharp peak. The latter peak represents the desired product of the PCR. The appearance of the chromatogram at 64° C indicates that the heteroduplexes
  • FIG. 18 also shows a primer dimer peak at very low retention time, near the void volume. As can be seen in FIG. 18 the entire separation was complete in 6 - 8 minutes.
  • the injection of the sample and temperature of each run were pre-programmed and automatically performed by a computer controlled sample auto-injector and computer controlled column oven.
  • the series of steps described in Example 4 represent one cycle of the
  • the degree of specificity of DNA polymerases varies with the reaction conditions employed as well as with the type of enzyme used. No enzyme affords completely error free extension of a primer. Therefore, a non- complimentary base may be introduced from time to time. Such enzyme related errors produce double stranded DNA products which are not exact copies of the original DNA sample, but contain PCR induced mutations. Other PCR process features, such as reaction temperature, primer annealing temperature, enzyme concentration, dNTP concentration, Mg concentration, and combinations thereof, all have the potential to contribute to the degradation of the accuracy or fidelity of DNA replication by the PCR process. The degree of fidelity of replication of DNA fragments by PCR depends on many factors which have long been recognized in the art.
  • a change in the PCR product profile caused by an increase or decrease in the quantity or concentration of one factor can be offset, or even reversed by a change in a different factor.
  • an increase in the enzyme concentration may reduce the fidelity of replication, while a decrease in the reaction temperature may increase the replication fidelity.
  • An increase in magnesium ion concentration or dNTP concentration may result in an increased rate of reaction which may have the effect of reducing PCR fidelity.
  • PCR reaction product profiles generated by MIPC can be analyzed and evaluated. By comparing a PCR reaction product profile to that of a standard profile, deviations from the standard product profile can be identified and one or more PCR process variables known to cause the observed deviations can be adjusted.
  • PCR reaction products were routinely treated with rYaelll endonuclease to cleave the product fragments near each end, creating a 405 base pair fragments having blunt ends, as described in Example 6 and shown in FIG. 17.
  • any products which separate from the main product peak when the MIPC separation is conducted at 66° C would have to be PCR induced mutations and not simply the result of "overhangs".
  • the 405 base pair product refers to the product obtained after treatment of the 500 base pair PCR amplification product with Haelll endonuclease.
  • FIG. 19 shows three PCR reaction product profiles which demonstrate the use of MIPC to optimize the yield of a desired product produced by a PCR process by analysis and evaluation of said reaction product profiles and adjusting PCR process variables to optimize the yield of the desired product as compared to a predetermined standard profile.
  • the product profile at the top of FIG. 19 represents a PCR process (described in Example 4) in which AmpliTaq® DNA polymerase (Perkin-Elmer Applied Biosystems, Foster City, CA) was used to produce a 405 base pair DNA fragment.
  • the MIPC separation was conducted at 66° C, a temperature which is known not to denature the entire 405 base pair DNA fragment, but sufficient to cause denaturing at a site of a PCR induced mutation, i.e. a base pair mismatch. Since such locally denatured fragments containing a PCR induced mutation have a lower retention time than fragments which contain no base pair mismatch (and are therefore not denatured), the replication
  • the top profile of FIG. 19 shows the 405 base pair homoduplex product peak at a retention time of just over 6 minutes and a broader peak having a retention time of less than 6 minutes.
  • the lower retention time peak was obtained under DMIPC conditions known to separate PCR induced mutations from their corresponding homoduplex.
  • This lower retention time peak is a heteroduplex PCR induced mutation. Integration of the 405 base pair peak having a retention time of just over 6 minutes compared to that of a standard of known concentration showed the 405 base pair product to contain 10 ng.
  • the only other peaks in the reaction product profile were a primer dimer peak near the void volume and a PCR induced mutation peak having a retention time just under 6 minutes.
  • the profile was evaluated to determine how the reaction variables could be adjusted to improve the yield of the desired 405 base pair product.
  • the primer dimer artifact could have no influence on product yield since the primers were present in very large excess relative to the template. Therefore, improving the fidelity of replication should cause a reduction in the amount of heteroduplex PCR induced mutation product and also cause an increase in the yield of desired 405 base pair product. Since the DNA polymerase enzyme has the greatest influence over fidelity of replications, the DNA polymerase reaction variable was adjusted. As a result of this analysis and evaluation, the PCR process cycles were then
  • FIG. 20 shows three PCR reaction product profiles which demonstrate the use of MIPC to provide a means for optimizing PCR fidelity by analysis and evaluation of reaction product profiles and adjusting PCR process variables to minimize deviations from replication fidelity as compared to a predetermined standard profile.
  • the product profile at the top of FIG. 20 represents a PCR process
  • the replication fidelity of the AmpliTaq® induced PCR process can be evaluated and quantitated by MIPC analysis of the reaction mixture.
  • the top profile of FIG. 20 shows the 405 base pair product peak at a retention time of just over 6 minutes and a broader peak having a retention time of less than 6 minutes.
  • the lower retention time peak was obtained under MIPC conditions known to separate PCR induced mutations from their corresponding homoduplex. This lower retention time peak is, therefore, a PCR induced mutation. Integration of the product profile shows that the PCR induced mutation is present to the extent of 62%, indicating very poor replication fidelity.
  • MIPC analysis of the PCR process performed in the presence of Pfu gave the middle reaction product profile of FIG. 20. Quantitation of the product profile showed a large decrease in the amount of undesired PCR induced mutation product to 25%. This improvement was predicted as a result of the analysis and identification of products in the PCR reaction product profile and adjusting the reaction variable, AmpliTaq®, known to be responsible for the formation of undesired products.
  • FIG. 21 The top trace of FIG. 21 shows a chromatogram depicting the separation of a 405 base pair fragment, before post-process hybridization, by MIPC at 66° C, i.e., a temperature sufficient to cause denaturation at a site of PCR induced mutation.
  • the heteroduplex PCR induced mutation product is seen as a small peak having a retention of just under 6 minutes.
  • the large, sharp 405 base pair product peak is seen at a retention time of just over 6 minutes. Integration of these peaks indicates that the PCR induced mutation product was present at an 8% level.
  • the lower trace of FIG. 21 shows an identical MIPC separation chromatogram except that the 405 base pair product was hybridized (post- process) as described above, before separation.
  • the lower retention time heteroduplex which represents PCR induced mutations increased to 23.1%. Therefore, the most preferred embodiment includes a post-process hybridization step in order to obtain an accurate representation of the true degree of PCR induced mutation.
  • a PCR process was analyzed and the products were separated by MIPC to provide a PCR reaction product profile.
  • the separated products were identified and quantitated.
  • the reaction variable most likely responsible for the observed poor replication fidelity was adjusted and the PCR process was repeated using the adjusted conditions.
  • the degree of PCR replication fidelity improved as predicted.
  • the degree of improvement was quantitated by integration of the reaction product profiles using MIPC. Having predicted and demonstrated the improvement in the PCR process, the process was further optimized by again adjusting the previously identified reaction variable.
  • a pure homoduplex fragment, separated and isolated by the method of this invention can be used in a variety of ways.
  • Non-limiting examples of these uses include the use of a relatively large amount of pure fragment as a template in a PCR process. The purity and relatively large amount of such a template in a PCR process would yield a large amount of pure amplified product.
  • a pure fragment could be incorporated into a plasmid and reproduced in a cell. Because of its high initial purity, large amounts of the fragment would be reproduced and isolated from the reproduced plasmids at a very high level of purity since little to no undesired fragments would be present and available for reproduction in the cell.
  • Highly purified PCR products are of great value to the scientific community.
  • the ability to detect mutations in double stranded polynucleotides, and especially in DNA fragments, is of great importance in medicine, as well as in the physical and social sciences.
  • the Human Genome Project is providing an enormous amount of genetic information which is setting new criteria for evaluating the links between mutations and human disorders (Guyer, et al. Proc. Natl. Acad. Sci., USA 92:10841 (1995)).
  • the ultimate source of disease for example, is described by genetic code that differs from wild type (Cotton, TIG 13:43 (1997)). Understanding the genetic basis of disease can be the starting point for a cure.
  • determination of differences in genetic code can provide powerful and perhaps definitive insights into the study of evolution and populations (Cooper et al., Human Genet.
  • the “melting temperature” is defined herein to mean the temperature at which 50% of the base pairs in a DNA fragment have separated.
  • a “homoduplex” is defined herein to mean, a double stranded DNA fragment wherein the bases in each strand are complimentary relative to their counterpart bases in the other strand.
  • heteroduplex is defined herein to mean a double stranded DNA fragment wherein at least one base in each strand is not complimentary to at least one counterpart base in the other strand. Since at least one base pair in a heteroduplex is not complimentary, it takes less energy to separate the bases at that site compared to its fully complimentary base pair analog in a homoduplex. This results in the lower melting temperature at the site of a mismatched base of a hetroduplex compared to a homoduplex.
  • hybridization refers to a process of heating and cooling a dsDNA sample, e.g., heating to 95°C followed by slow cooling. The heating process causes the DNA strands to denature. Upon cooling, the strands re- combine into duplexes in a statistical fashion. If the sample contains a mixture of wild type and mutant DNA, then hybridization will form a mixture of hetero- and homoduplexes.
  • heteromutant site separation temperature T(hsst) is defined herein to mean the temperature which will selectively denature the heteroduplex DNA at a site of mutation. This is a temperature which is optimal to effect a chromatographic separation of heteroduplexes and homoduplexes by MIPC and hence, detect mutations.
  • MIPC separations can be completed in less than 10 minutes, and frequently in less than 5 minutes.
  • MIPC systems WAVETM DNA Fragment Analysis System, Transgenomic, Inc. San Jose, CA
  • Mutation detection at the temperature required for partial denaturation (melting) of the DNA at the site of mutation can therefore be easily performed.
  • the system used for MIPC separations is rugged and provides reproducible results.
  • the system offers automated sample injection, data collection, choice of predetermined eluting solvent composition based on the size of the fragments to be separated, and column temperature selection based on the base pair sequence of the fragments being analyzed.
  • the separated mixture components can be displayed either in a gel format as a linear array of bands or as an array of peaks.
  • the display can be stored in a computer storage device.
  • the display can be expanded and the detection threshold can be adjusted to optimize the product profile display.
  • the reaction profile can be displayed in real time or retrieved from the storage device for display at a later time.
  • a mutation separation profile, a genotyping profile, or any other chromatographic separation profile display can be viewed on a video display screen or as hard copy printed by a printer.
  • MIPC separates double stranded polynucleotides by size or by base pair sequence and is therefore a preferred separation technology for detecting the presence of particular fragments of DNA and RNA of interest.
  • a separation system for mutation detection having the convenience, automation, sensitivity, and range of capabilities of MIPC has not been previously described.
  • MIPC Denaturing Matched Ion Polynucleotide Chromatography
  • heteromutant is defined herein to mean a DNA fragment containing a polymorphism or non-complimentary base pair.
  • mutation separation temperature range is defined herein to mean the temperature range between the highest temperature at which a DNA segment is completely non-denatured and the lowest temperature at which a DNA segment is completely denatured.
  • mutant separation profile is defined herein to mean a DMIPC separation chromatogram which shows the separation of heteroduplexes from homoduplexes. Such separation profiles are characteristic of samples which contain mutations or polymorphisms and have been hybridized prior to being separated by DMIPC.
  • the DMIPC separation chromatograms shown in FIG. 24 which were performed at 51 °C to 61 °C exemplify mutation separation profiles as defined herein.
  • temperature titration of DNA as used herein is an experimental procedure in which the retention-time from DMIPC is plotted as the ordinate against column temperature as the abscissa.
  • a reliable way to detect mutations is by hybridization of the putative mutant strand in a sample with the wild type strand (Lerman, et al., Meth. Enzymol., 155:482 (1987)). If a mutant strand is present, then two homoduplexes and two heteroduplexes will be formed as a result of the hybridization process, as shown in FIG. 23. Hence separation of heteroduplexes from homoduplexes provides a direct method of confirming the presence or absence of mutant DNA segments in a sample.
  • DNA separation methods separate DNA fragments based on the number of base pairs when the separations are performed below the denaturing (melting) temperature of the mismatched base pair in a heteroduplex.
  • DNA fragments of the same number of base pairs can be separated when the separations are performed at the T(hsst).
  • Such separations have been accomplished by denaturing gradient gel electrophoresis or denaturing gradient capillary electrophoresis.
  • denaturing gradient capillary electrophoresis analysis takes at least 30 minutes per run plus setup time.
  • An advantage of the present invention is the ability to automate the determination of T(hsst) by DMIPC for the purpose of mutation detection.
  • MIPC Matched Ion Polynucleotide Chromatography
  • Example 15 The temperature dependent separation of 209 base pair homoduplexes and heteroduplexes by DMIPC is shown in FIG. 24 as a series of separation chromatograms and the separation process is described in Example 15.
  • the hybridization process created 2 homoduplexes and 2 heteroduplexes as shown schematically in FIG. 23. This mixture was separated as described in Example 16.
  • FIG. 24 when MIPC
  • the 2 homoduplexes separate because the A-T base pair denatures at a lower temperature than the C-G base pair.
  • the results are consistent with a greater degree of denaturation in one duplex and/or a difference in the polarity of one partially denatured heteroduplex compared to the other, resulting in a difference in retention time on the MIPC column.
  • a temperature titration of the homoduplex and heteroduplex species from the elution profiles of FIG. 24 is shown FIG. 25. As seen in FIG. 24, the temperature range of 57° to 58° C was optimal for this separation. The appearance of four distinct peaks was observed when a mutation was present in the original sample, in agreement with the expected results, based on the hybridization schematic in FIG.
  • the double stranded fragments are completely denatured, rather than being denatured only at the site of base pair mismatch. This is evidenced by the single peak, representing 4 single polynucleotide strands, seen at low retention time when the separation was carried out at 59° C and above.
  • the single peak representing 4 single polynucleotide strands, seen at low retention time when the separation was carried out at 59° C and above.
  • only two peaks or a partially resolved peak(s) are observed in DMIPC analysis.
  • the two homoduplex peaks may appear as one peak or a partially resolved peak and the two heteroduplex peaks may appear as one peak or a partially resolved peak. In some cases, only a broadening of the initial peak is observed under partially denaturing conditions.
  • the determination of a mutation can be made by hybridizing the homozygous sample with the known wild type fragment and performing a DMIPC analysis at a partially denaturing temperature. If the sample contained only wild type fragments then a single peak would be seen in the DMIPC analysis since no heteroduplexes could be formed. If the sample contained homozygous mutant fragments, then analysis by DMIPC would show the separation of homoduplexes and heteroduplexes as seen in FIG. 24.
  • the temperature at which 50% of a constant melting domain is denatured may also be determined experimentally by plotting the UV (UV) absorbance of a DNA sample against temperature. The absorbance increases with temperature and the resulting plot is called a melting profile (Breslauer et al., Proc. Natl. Acad. Sci. USA 83:3746 (1986); Breslauer, Calculating Thermodynamic Data for Transitions of any Molecularity, p. 221 , Marky et al. eds., J. Wiley and Sons (1987)). The midpoint of the absorbance 99/07899
  • Tm melting temperature
  • this observed Tm is used as a starting temperature for performing DMIPC for mutation detection.
  • software such as
  • MELT (Lerman, et al., Meth. Enzymol. 155:482 (1987)) or WinMeltTM, version 2.0, is used to obtain a calculated Tm which is used as a starting temperature for performing DMIPC for mutation detection.
  • These software programs show that despite individual differences in base pair stability, the melting temperature of nearby base pairs is closely coupled, i.e., there is a cooperative effect. Thus, there are long regions of 30 to 300 base pairs, called “domains", in which the melting temperature is fairly constant.
  • the software MELTSCAN (Brossette, et al., Nucleic Acid Res. 22:4321 (1994)) calculates melting domains in a DNA fragment and their corresponding melting temperatures. The concept of a constant temperature melting domain is important since it makes possible the detection of a mutation in any portion of the domain at a single heteromutant site selective temperature.
  • T(hsst) X + m • T(w), wherein T(hsst) is the heteromutant site separation temperature and where X is the DMIPC detection factor, and m is a weighting factor; both factors are used to adjust T(w) to the T(hsst).
  • X can have a positive or negative value.
  • T(w) is the melting temperature determined from a UV melting profile of the normal (i.e. wild type) DNA duplex.
  • T(w) is calculated by software.
  • the values of m are preferably between 0 and 2. Since X depends on the sequence of the fragment to be analyzed, its value can vary by up to about 10°C.
  • T(hsst) is refined experimentally from the calculated melting temperature as described in the Examples, wherein a DMIPC analysis is initiated at the melting temperature calculated by software (Example 17) or determined from a melting profile of UV absorbance vs. temperature (Example 18). Subsequent samples are then injected and analyzed at incrementally lower and higher temperatures until an optimum separation is achieved. In a preferred embodiment of the invention, all aspects of the analysis are automated and the temperature increments are selected, e.g. 2°C increments.
  • the T(hsst) is generally only 1-2°C lower than the melting temperature as determined from a UV absorbance vs. temperature melting curve, or as determined from a temperature titration curve. After the temperature titration curve is fomred, the T(hsst) can usually be determined in just 1 or 2 runs. Furthermore, Applicants have discovered that the mutation separation temperature range for DNA fragments of about 200-400 bp over which denaturation occurs is about 5° C. Therefore, even if the procedure described hereinabove only approached the T(hsst), it would be obvious in which direction to alter the temperature to achieve an optimum separation.
  • the increments are set at about 0.3°-0.5°C. In a preferred embodiment, the increments are set at 0.1 °C allowing a very accurate determination of T(hsst).
  • FIG. 26 depicts in schematic form the melting of a theoretical three domain fragment.
  • every DNA fragment is comprised of one or more regions of independent thermal stability or domains.
  • the Tm of a domain serves as a thermodynamic signature and determines the thermodynamic behavior of a domain. As depicted in the schematic in FIG. 26, as the temperature is gradually increased, domain A will denature first because its Tm is lower than that of domain B or C. Domain B has an intermediate Tm and would melt next, and domain C would be the last to melt because its domain has the highest Tm within this fragment.
  • the base pairs within a domain melt in unison over a very narrow temperature range.
  • the denaturing of a domain is characterized by a sigmoidal profile (FIG. 27) which indicates "cooperativity" among the base-pairs comprising the domain.
  • the midpoint of the absorbance range is the Tm and corresponds to a temperature at which the domain exists in equilibrium between single and double stranded states. As the temperature is increased beyond the Tm, the entire domain will rapidly convert to a completely single-stranded conformation.
  • a putative point mutation could be present in any of the domains: A, B or C.
  • DMIPC DMIPC
  • the DMIPC system is capable of automatically profiling the melting behavior of a DNA fragment by running a series of separations at incremental temperature increases over the entire likely denaturation range (e.g. 50°-
  • FIG. 27 depicts the melting of the four related homo- and heteroduplex forms of a DNA fragment (the homoduplexes are represented by dashed lines). These melting profiles illustrate how the midpoints of the heteroduplex inflections are shifted to the left, indicating lower Tms and more rapid elution from the DMIPC column compared to the homoduplexes. It is also apparent that the Tms of the heteroduplexes are approximately 1 °-2°C lower than the homoduplexes.
  • FIG. 28 depicts the melting profile of 230 bp restriction fragment designated sY81. Any domains present in this fragment are now represented
  • Tm of the melting profile of the homoduplex fragment or Tm homo The temperature at this midpoint of the inflection is the Tm of the melting profile of the homoduplex fragment or Tm homo . Determining the Tm homo from the melting profile is necessary for selecting an appropriate temperature at which to carry out mutation screening. Since the presence of a base mismatch will lower the Tm of the corresponding heteroduplex domain being scrutinized by
  • Tm h ⁇ t ⁇ ro Tm homo - 1°C.
  • Tm het ⁇ ro Tm homo - 1°C.
  • the appearance of the melting profile indicates that the Tm homo is approximately 56°C. Therefore, the ideal temperature for the melting profile.
  • Domains A and B can be simultaneously screened at a temperature of 54°C and domains B and C can be
  • FIG. 29 depicts a theoretical melting profile for a three domain fragment with Tms of 55°C, 60°C and 65°C.
  • the melting of DNA causes the retention time on a liquid chromatography column to decrease as the temperature of the separation is increased.
  • a sample containing the mutation is examined at a series of temperatures using a heuristic optimization approach.
  • the optimum temperature obtained by this procedure is the temperature at which the mutant DNA fragment is most easily distinguished from the wild-type DNA by the difference in the pattern of peaks.
  • This approach is not systematic and relies on the knowledge on whether a heteroduplex is present in the sample. However, prior knowledge of a mismatch is not always available.
  • a preferred embodiment of the present invention is a method for selection of the T(hsst) is based on the temperature titration.
  • This temperature titration can be obtained by experimental observation or obtained from a theoretical analysis of thermodynamic information.
  • the optimum temperature for mutation detection corresponds to the early stages of denaturation of the segment of the wild type DNA fragment containing the mutation.
  • a plot of retention time vs. temperature shows a parallel relationship between wild type and heteroduplex such that the retention time of both fragments is decreasing with about the same slope. This surprising and consistent relationship discovered by Applicants essentially eliminates the necessity of collecting data on the heteropdupiex sample in order to select T(hsst).
  • T(hsst) melting characteristics of the wild-type fragment can be used to determine T(hsst). This relationship is illustrated in the temperature titration of Example 19, in which both homoduplexes and heteroduplexes in a mixture obtained from a 209 bp DYS217 mutation, gave a slope of about -0.9min/°C.
  • FIG. 30 a temperature titration for a DYS271 209 bp mutation standard mixture with a heteroduplex mismatch at the 168 bp position, shows how temperature titration information may be obtained experimentally.
  • the data show that the 2 heteroduplex and 2 homoduplex peaks from a mismatch are well resolved at 56°C. As the temperature is increased, they become broad peaks (60 °C - 63 °C) and then as the temperature is further increased the peaks merge into single stranded DNA. Since under these conditions, single stranded DNA is separated under sequence as well as size parameters, the peak is split. It is possible to miss this region if the separation is optimized for the mutation at 168 bp because elution conditions for rapid separation would cause the single stranded (melted) peaks to be merged into the first part of the gradient.
  • FIG. 31 is a temperature titration for the latest eluting wild type homoduplex from the data of FIG. 30.
  • the plot shows two inflection points. The first is at 56°C and it is notable that this is the temperature where the two heteroduplex peaks and the two homoduplex peaks are well resolved as seen in FIG. 30.
  • the retention times for the two wild type homoduplex peaks track the two heteroduplex peaks with a slope of approximately -0.9 minutes/°C.
  • FIG. 31 there is a second inflection point at 61.5°C indicating that there is a high melting region within the fragment, but the mismatch is not in this high melting region.
  • the T(hsst) is selected from the temperature titration graph of the wild type homoduplex by first determining a range of temperature in which retention time is decreasing by about 0.9min/°C, and second, obtaining the inflection point on the temperature titration plot within that region and subtracting 1°C.
  • the T(hsst) is selected to correspond with a point in which the melting of the homoduplex is 25% complete. Generally, one would run DMIPC of the actual hybridized sample at three different temperatures, e.g., about 2°C on either side of the T(hsst) as well as the predicted T(hsst).
  • thermodynamic mathematical model of the melting behavior of known fragments can be used to predict the melting behavior of new fragments without any experimental work on the sample itself.
  • the model can be used to predict optimum temperatures for mutation detection and also to assess the suitability of the fragment to the technique.
  • a temperature titration can be determined using the sequence information of the fragment and behavior predicted by thermodynamic data and models, and the fitting of these models to chromatography behavior.
  • the hydrogen bonding energies of nucleic acids can be measured. For example, information of this type is reported by Breslauer et al. (Proc. Natl. Acad. Sci. 83:3746 (1986)).
  • a simple melting model can be used in which neighboring bases (or pairs of bases) do not exert a long range influence beyond the boundary of the unit.
  • an intrinsic helical tendency is combined with a conditional probability such that the probability of a base being in the helical state is strongly affected by its neighbors.
  • a recursive algorithm is required such as the Fixman-Freire implementation of Tru's model (Poland, Biopolymers 13:1859 (1974) and Fixman et al., Biopolymers 16:2693 (1977)).
  • the parameters used in the Fixman-Freire algorithm have been optimized to predict melting behavior at equilibrium, in aqueous solution.
  • An example of implementation of a thermodynamic approach to DNA melting is shown in FIG. 32 with a DNA melting profile of the DYS271 209 bp mutation.
  • the plot shows that there are two melting domains. This approach is called a cooperative melting prediction since the melting of any particular base pair is influenced by its neighbors. This influence extends as far as the neighbors contain a similar GC content.
  • the lower domain contains the heteroduplex mismatch at 168 bp. The plot correlates well with experiment data with two domains and the lower melting domain containing the mismatch.
  • WinMeltTM or any similar program cannot be used to predict the optimum temperature for performing mutation detection due to fact that the column, buffer and solvent can affect the melting temperature of the DNA. Since these programs use the cooperative model for melting, programs do not predict well the "temperature titrations" observed in DMIPC separations of DNA without selecting the coefficients and offsets that have been correlated with the DMIPC performance.
  • the noncooperative thermodynamic approach to modeling of DNA melting can be used.
  • the preferred thermodynamic model is based on a modification of the cooperative approach.
  • a calculated melting temperature is derived using a first mathematical model such as the Fixman- Freire implementation of Tru's model.
  • a predicted melting temperature is then derived by adjusting the calculated melting temperature according to a second mathematical model.
  • a preferred example of a second mathematical model is an adjustment equation developed by comparing calculated temperatures based on the first model with empirically-determined temperatures observed from temperature titrations. The adjustment equation can be used to predict the T(hsst) of melting for DMPIC using only the sequence information of the wild type or homoduplex DNA.
  • An adjustment of the Fixman-Freire calculated temperature is necessary to account for differences between the conditions used in obtaining the thermodynamic data (Breslauer et al. Proc Natl. Acad. Sci USA 83:3746 (1986)) and the conditions used in DMIPC.
  • FIG. 33 shows the difference between a cooperative approach and a noncooperative approach to DNA melting.
  • FIG. 33 employs an analogy in which the bases in a DNA sequence are represented by pontoons (the horizontal gray rectangles) on water, and the melting temperatures are represented by ballast (the black vertical bars, with the heavier ballast represented by longer bars) with a lower melting temperature represented by a heavier ballast.
  • each nucleic acid base pair in a sequence has particular stability determined by each particular hydrogen bonding energy. The stability or the melting of each base pair is independent of any surrounding base pairs. Model A shows that those base pairs that contain a high melting propensity will melt, but will not affect the melting of base pairs that have a lower propensity to melt.
  • FIG. 34 shows a modified noncooperative approach to measuring the melting profile of the 209 bp mutation standard. The plot starts at base 16 and ends at base 192 because a moving weighted window of 30 bases was used to generate each point and smooth out the curve.
  • thermodynamic parameters that relate the extent of melting to retention time are used so that the temperature titrations are modeled more accurately.
  • a temperature offset, a slope, a fragment size dependent term, and the loop entropy and in principal even the 10 nearest neighbor free energies can be optimized. For example, the effect of loop entropy is shown in FIG. 36.
  • FIGs. 38and 39 show how an adjustment equation for obtaining the predicted melting temperature can be obtained.
  • FIG. 38 is a graph of calculated melting temperature versus empirically determined melting temperature.
  • the abscissa represents a melting temperature corresponding to 75% helical content, Tm 075 , i.e. a point on the melting profile where denaturation is just beginning (75% helical which is equivalent to 25% melting) calculated by the Fixman-Freire model using a loop entropy of 0.0001.
  • the ordinate represents empirically determined melting temperatures which are preferably those previously optimized for use in high throughput screening by DMIPC for known mutations.
  • the data plotted on the graph indicate the empirically determined melting temperatures for each of 40 experimental temperature titrations of hybridized fragments.
  • FIG. 39 is a graph of calculated melting temperature versus predicted melting temperature.
  • the solid line represents a linear fit of the 40 points from FIG. 38.
  • the line Tm 075 is defined by the equation
  • dashed lines representing one degree above and below the line representing Tm 075 '. These dashed lines indicate that the accuracy of the predicted Tm 075 ' may be expected to be within about one degree of the empirically determined values.
  • a predicted melting temperature can be obtained by first calculating Tm 075 according to the Fixman-Freire algorithm, and then by adjusting Tm 075 according to the above equation.
  • the equation above is used to predict a point on the melting profile where denaturation is just beginning (75% helical which is comparable to 25% melting on an experimental titration curve), where Tm' 075 is the predicted temperature corresponding to 75% helical content and Tm 075 is the temperature calculated from the Fixman-Freire algorithm using a loop entropy of 0.0001.
  • Tm' 075 is the predicted temperature corresponding to 75% helical content
  • Tm 075 is the temperature calculated from the Fixman-Freire algorithm using a loop entropy of 0.0001.
  • This has improved predictive power at calculating the optimal temperature for mutation detection compared with the unmodified algorithm.
  • the loop entropy term is increased, the melting profiles become less dominated by domain like stepwise melting (FIG. 36). This term can therefore be optimized to model the experimental curves more accurately.
  • the melting temperature in the above embodiment was selected at 75% helical content.
  • thermodynamic data and equations to predict temperature titrations can be performed in many different ways. The above approach is preferred for this invention. However, since the approach is an empirical one with the calculated temperatures fitted to actual optimum temperatures for mutation detection, the use of the equations can be performed in other ways with different weighted coefficients.
  • the present invention provides a method for detecting mutations in a DNA sample using MIPC.
  • MIPC Magnetic Ink Characterization
  • a mixture of homoduplexes and heteroduplexes is formed prior to the MIPC analysis.
  • a standard polynucleotide homoduplex is added to the sample and the mixture is
  • the standard polynucleotide is the "wild type" polynucleotide.
  • a heteroduplex strand will denature selectively at the site of base pair mismatch, creating a "bubble", at a lower temperature than is necessary to denature the remainder of the heteroduplex strand, i.e., those portions of the heteroduplex strand which contain complimentary base pairs.
  • This phenomenon generally referred to as partial denaturation, occurs because the hydrogen bonds between mismatched bases are weaker than the hydrogen bonds between complimentary bases. Therefore, less energy is required to denature the heteroduplex at the mutation site, hence the lower temperature required to partially denature the hetroduplex at the site of base pair mismatch than in the remainder of the strand.
  • MIPC separates DNA fragments by base pair length
  • homoduplex and heteroduplex fragments having the same base pair length are separated when the chromatography is conducted under partially denaturing temperature conditions, i.e., at a temperature which partially denatures a heteroduplex as described above.
  • partially denaturing temperature conditions i.e., at a temperature which partially denatures a heteroduplex as described above.
  • the heteroduplexes usually elute ahead of the homoduplexes.
  • An important aspect of the invention is the surprising, and heretofore unreported discovery by Applicants, that there exists a highly reproducible relationship between the concentration of organic solvent in the mobile phase required to elute DNA fragments from an MIPC column and the base pair length of the DNA fragments.
  • a "preliminary organic solvent concentration" required to elute a DNA fragment of known size can be obtained from a reference which relates the concentration of organic solvent in the mobile phase required to elute a given base pair length fragment to the base pair length of DNA fragments, obviating the need to develop methods for elution conditions.
  • This reference is used in the invention to determine the preliminary solvent concentration.
  • the "preliminary solvent concentration” is defined to mean the concentration of organic solvent, obtained from a reference, which is required to elute a fragment of corresponding base pair
  • the reference relating the organic solvent concentration in the mobile phase required to elute DNA fragments having different base pair lengths is represented by the graph in FIG. 40. It is to be understood that the relationship depicted in the graph in FIG. 40 can be expressed over different ranges of base pair length and solvent concentrations.
  • the data used to generate the reference depicted in FIG. 40 can be represented as a graph or a table. The data can be used to obtain an equation of a best-fit curve. For example, the following equation gave the curve shown in FIG. 38: 99/07899
  • FIG. 40 The reference graph, FIG. 40, was derived by Applicants as described in Example 20. Standard fragments of known base pair lengths were applied to an MIPC column and the concentration of organic solvent in the mobile phase sufficient to elute each fragment was determined when the chromatography was conducted at 50° C. The concentrations of organic solvent so determined, were plotted against their respective base pair length fragments to create FIG. 40.
  • the standard fragments of known base pair length were obtained from a pUC18 DNA-Hae ⁇ digest (S6293, Sigma- Aldrich). The fragments used to prepare the reference of FIG.
  • Example 21 An example of a procedure for pre-selection of organic solvent concentration in the mobile phase for mutation detection by MIPC is described in Example 21 , and shown in FIG. 41.
  • two buffers are prepared: A first buffer, "A” containing only the counterion agent (e.g., 0.1 M TEAA) and a second buffer, "B", containing the counterion agent and organic solvent (e.g., 0.1 M TEAA, 25% acetonitrile). These buffers are mixed to achieve the desired concentration of organic solvent in the mobile phase during the separation.
  • a first buffer containing only the counterion agent (e.g., 0.1 M TEAA)
  • B containing the counterion agent and organic solvent
  • the %B corresponding to the base pair length fragment of interest is obtained from the reference graph of mobile phase concentration vs. base pair length (FIG. 40).
  • a "fragment bracketing range" of organic solvent is selected.
  • the fragment bracketing range has an initial concentration of organic solvent and a final concentration of organic solvent.
  • the initial concentration contains an organic solvent concentration up to an amount required to elute the first eluting DNA molecule in the mixture.
  • the final concentration contains an organic solvent concentration sufficient to elute the last eluting DNA fragment in the mixture.
  • the initial solvent concentration of the pre-selected fragment bracketing range is less than or equal to about 15 percentage units below the %B of the preliminary solvent concentration.
  • the final solvent concentration of the preselected fragment bracketing range is at least about 5 percentage units higher than the %B of the preliminary solvent concentration.
  • the chromatography system is controlled by a computer and is run in an automated fashion.
  • the chromatography column is equilibrated using the initial solvent concentration.
  • the solvent concentration is increased at the rate of 2% minute over 5 - 15 minutes.
  • the gradient is run over 10 minutes to reach the final concentration of the pre-selected fragment bracketing range.
  • the solvent concentration is then immediately increased to 100%B for 2 minutes to wash the column.
  • the solvent concentration is then reduced to the initial solvent concentration and the column is equilibrated for two minutes in preparation for the next sample injection. This entire process is automated and the entire time span between samples, including column washing and equilibration, is less than 15 minutes.
  • the MIPC process described above can be optimized to increase throughput in mutation detection assays or other analyses which require screening a large number of samples.
  • the process an be optimized by adjusting the slope of the solvent gradient to effect earlier elution of heteroduplexes, so long as the separation of homoduplexes is maintained.
  • the solvent gradient can be programmed to ramp up to 100% for column washing immediately after the retention time of the heteroduplex is passed, without waiting for the homoduplex to appear. Following washing, the solvent concentration can be immediately ramped down to the initial concentration to equilibrate the column for the next analysis. In this manner the entire chromatography time for a sample can be reduced from about 15 minutes to less than 10 minutes, and preferably, to less than about 5-7 minutes.
  • the fragment bracketing range is selected automatically by software residing in the computer.
  • the user enters the base pair length of the fragment to be analyzed into a user interface screen.
  • Software using reference data which relates base pair length to solvent concentrations and the equation shown above, calculates the injection conditions, the initial and final solvent concentrations of the pre-selected fragment bracketing range required to effect the desired separations, and the column wash conditions.
  • the organic solvent in the mobile phase is selected from the group consisting of methanol, ethanol, acetonitrile, ethyl acetate, and 2-propanol.
  • the preferred organic solvent in the mobile phase is acetonitrile.
  • the mobile phase contains a counterion agent selected from the group consisting of lower alkyl primary, secondary, and tertiary amines, lower trialkyammonium salts and lower quaternary alkyalmmonium salts.
  • counterion agents include, but are not limited to octylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium
  • anion in the above examples is acetate
  • other anions may also be used, including carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, and bromide, or any combination of cation and anion.
  • carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, and bromide or any combination of cation and anion.
  • the preferred counterion agent is triethylammonium acetate.
  • the pH of the mobile phase is typically maintained between about 7 and 9.
  • the mobile phase is maintained at a pH of about 7.3.
  • a DNA sample is hybridized with a wild type DNA fragment by denaturing and annealing the mixture as described herein above.
  • the DNA sample can be hybridized with wild type directly.
  • the DNA sample can also be amplified by PCR and then hybridized with wild type. Alternatively, a wild type fragment may be added to the sample prior to PCR amplification.
  • the amplified mixture can then be hybridized following amplification. In each of these three hybridization scenarios, a mixture of homoduplexes and heteroduplexes is produced if a mutation is present in the sample.
  • the sample, so prepared, is analyzed by MIPC under partially denaturing conditions, preferably at 56° to 58° C, for the presence of a mutation using the method of the invention for pre-selecting the preliminary organic solvent concentration and the fragment bracketing range as described hereinabove.
  • the throughput of samples may be increased significantly by speeding up the analysis for each sample using a steeper gradient for the fragment bracketing range.
  • the polynucleotide fragments are detected as they are separated and eluted from the column. Any detector capable of detecting polynucleotides can be used in the MIPC mutation detection method.
  • the preferred detector is an online UV detector.
  • the DNA fragments are tagged with fluorescent or radioactive tags, then a fluorescence detector or radioactivity detector can be employed, respectively.
  • the separated fragments are displayed on a video display screen or printed by a printer.
  • the fragments so displayed appear either as peaks or as bands in a lane, i.e., in a virtual gel display format as described in U.S. Patent Application No. 09/039,061 filed March 13, 1998. The choice of format is selectable by the user.
  • the mutation detection method of the invention can also be used to detect mutations in DNA samples when the base pair length of the fragment is unknown. Although the base pair length of such samples can be determined by the method of the invention, the presence of a mutation can only be determined if the sample is from a heterozygous source. Hybridization of a heterozygous sample will result in the formation of heteroduplexes and homoduplexes, which can be detected by DMIPC. However, a homozygous mutant will not produce heteroduplexes after hybridization. A homozygous mutant in an unknown fragment will, therefore not be detected. Since the sequence of a DNA fragment of unknown length is also unknown, hybridization with wild type to produce heteroduplexes is not possible.
  • the sample is applied to an MIPC column without PCR amplification.
  • An unknown sample cannot be amplified since primers cannot be designed for an unknown sequence.
  • the sample is hybridized prior to analysis in order to create a mixture of heteroduplexes and homoduplexes if the sample was from a heterozygous source.
  • the chromatography is conducted under non-denaturing conditions
  • the chromatography is then repeated under denaturing conditions (56° to 58° C) using a fragment bracketing range of solvent concentration in a linear 2% per minute gradient as described hereinabove.
  • the base pair length corresponding to the solvent concentration which effected elution of the unknown fragment can be determined, thereby establishing the base pair length of an unknown fragment.
  • MIPC Matched Ion Polynucleotide Chromatography
  • DMIPC DMIPC is typically performed at a temperature between 52°C and
  • the optimum temperature for performing DMIPC is 54°C to 59°C.
  • the previously described precautions taken to remove multivalent metal cations were adequate for maintaining column life, as demonstrated by good separation efficiency, under non-denaturing conditions.
  • Applicants have surprisingly found that when performed at partially denaturing temperature, conditions for effective DMIPC separations become more stringent. For example, a separation of a standard pUC18 Hae ⁇ digest on a
  • an aqueous solution of multivalent cation binding agent is flowed through the column to maintain separation efficiency.
  • the column is preferably washed with multivalent cation binding agent solution after about 500 uses or when the performance starts to degrade.
  • Non-limiting examples of multivalent cation binding agents which can be used in the present invention are selected from the group consisting of acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid, morin, rhodizonic acid, thionalide, thiourea, ⁇ -furildioxime, nioxime, salicylaldoxime,
  • the multivalent cation binding agent is water soluble.
  • the solubility in water can be enhanced by attaching covalently bound ionic functionality, such as, sulfate, carboxylate, or hydroxy.
  • the cation binding agent must be easily removed from the column by washing with water, organic solvent or mobile phase. The cation binding agent must not interfere with the use of the column.
  • a preferred multivalent cation binding agent is EDTA.
  • the concentration of a solution of the cation binding agent can be between 0.01 M and 1 M.
  • the column washing solution contains EDTA at a concentration of about 0.03 to 0.1 M.
  • the solution contains an organic solvent selected from the group consisting of acetonitrile, ethanol, methanol, 2- propanol, and ethyl acetate.
  • a preferred solution contains at least 2% organic solvent to prevent microbial growth.
  • a solution containing 25% acetonitrile is used to wash a MIPC column.
  • the multivalent cation binding solution can, optionally, contain a counterion agent.
  • the counterion agent is selected from the group consisting of lower primary, secondary and tertiary amines, and lower trialkyammonium and quaternary ammonium salts.
  • counterion agents include, but are not limited to octylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, butylethylammonium acetate, methylhexyiammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropyl
  • anion in the above examples is acetate
  • other anions may also be used, including carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride, and bromide, or any combination of cation and anion.
  • the MIPC separation column is washed with the multivalent cation binding solution at an elevated temperature in the range of 50° to 80° C.
  • the column is washed with a
  • the solution contains 0.032 M EDTA, 0.1 M TEAA, and 25% acetonitrile.
  • Column washing can range from 30 seconds to one hour.
  • the column in a high throughput DMIPC assay, can be washed for 30 seconds after each sample, followed by equilibration with mobile phase. Since DMIPC can be automated by computer, the column washing procedure can be incorporated into the mobile phase selection program without additional operator involvement.
  • the column is washed with multivalent cation binding agent for 30 to 60 minutes at a flow rate preferably in the range of about 0.05 to 1.0 mL/min.
  • a DMIPC column is tested with a standard mutation detection mixture of homoduplexes and heteroduplexes after about 1000 sample analyses. If the separation of the standard mixture has deteriorated compared to a freshly washed column, then the column can be washed for 30 to 60 minutes with the multivalent cation binding solution at a temperature above about 50°C to restore separation performance.
  • Applicants have discovered that column separation efficiency can be preserved by storing the column separation media in the column containing a solution of multivalent cation binding agent therein.
  • the solution of binding agent may also contain a counterion agent.
  • Any of the multivalent cation binding agents, counterion agents, and solvents described hereinabove are suitable for the purpose of storing a MIPC column.
  • a column packed with MIPC separation media is stored in an organic solvent containing a multivalent cation binding agent and a counterion agent.
  • An example of this preferred embodiment is 0.032 M EDTA and 0.1 M tetraethylammonium acetate in 25% aqueous acetonitrile.
  • a solution of multivalent cation binding agent as described above, is passed through the column for about 30 minutes.
  • the column is then disconnected from the HPLC apparatus and the column ends are capped with commercially available threaded end caps made of material which does not release multivalent cations.
  • Such end caps can be made of coated stainless steel, titanium, organic polymer or any combination thereof.
  • FIG. 43 shows some improvement in the separation of homoduplexes and heteroduplexes of the standard mutation detection mixture when a guard cartridge containing cation capture resin was deployed in line between the solvent reservoir and the MIPC system.
  • the chromatography shown in FIG. 43 was performed at 56° C.
  • the column used in FIG. 43 was the same column used in the separation shown in FIG. 42 and for separating the standard pUC18 Haelll digest.
  • FIG. 44 shows the separation of homoduplexes and heteroduplexes of the standard mutation detection mixture at 56°C on the same column used to generate the chromatograms in FIGs. 42 and 43. However, in FIG. 44 the column was washed for 45 minutes with a solution comprising 32 mM EDTA and 0.1 M triethylammonium acetate in 25% acetonitrile at 75° C prior to sample application.
  • FIG. 44 shows four cleanly resolved peaks representing the two homoduplexes and the two heteroduplexes of the standard 209 bp mutation detection mixture. This restoration of the separation ability, after washing with a solution containing a cation binding agent, of the MIPC column under DMIPC conditions compared to the chromatograms of FIGs.
  • DMIPC as used herein, is defined as a process for separating heteroduplexes and homoduplexes using non-polar beads in the column, wherein the process uses a counterion agent, and an organic solvent to desorb the nucleic acid from the beads, and wherein the beads are characterized as having a Mutation Separation Factor (MSF) of at least 0.1.
  • MSF Mutation Separation Factor
  • the beads have a Mutation Separation Factor of at least 0.2.
  • the beads have a Mutation Separation Factor of at least 0.5.
  • the beads have a Mutation Separation Factor of at least 1.0.
  • the performance of the column is demonstrated by high efficiency separation by DMIPC of heteroduplexes and homoduplexes.
  • a Mutation Separation Factor as described in Example 23. This is measured as the difference between the areas of the resolved heteroduplex and homoduplex peaks.
  • a correction factor may be applied to the generated areas underneath the peaks. Factors, such as the following listed below, may affect the calculated areas of the peaks and reproducibility of the same: baseline drawn, peak normalization, inconsistent temperature control, inconsistent elution conditions, detector instability, flow rate instability, inconsistent PCR conditions, and standard and sample degradation.
  • the Mutation Separation Factor (MSF) is determined by the following equation:
  • MSF (area peak 2 - area peak 1 )/area peak 1
  • area peak 1 is the area of the peak measured after DMIPC analysis of wild type and area peak 2 is the total area of the peaks or peaks measured after DMIPC analysis of a hybridized mixture containing a putative mutation, with the hereinabove correction factors taken into consideration, and where the peak heights have been normalized to the wild type peak height.
  • Separation particles are packed in an HPLC column and tested for their ability to separate a standard hybridized mixture containing a wild type 100 bp Lambda DNA fragment and the corresponding 100 bp fragment containing an A to C mutation at position 51.
  • the pH 7 mobile phase comprised component A, 0.1 M triethylammonium acetate (TEAA) and component B, 0.1 M TEAA, 25% acetonitrile. Gradient conditions used in the separation shown in FIG.
  • FIG. 2 shows that no DNA fragments eluted as represented by the flat baseline of the chromatogram.
  • a second 5 ⁇ L pUC18 DNA-Haelll digest was injected onto the same MIPC column and the column was eluted (FIG. 3) with 35% B followed by the gradient described above in relation to FIG. 1.
  • p53 exon 6 genomic DNA having a C to T mutation at location 13346 was amplified by PCR in which three different sets of primers were used. Each primer set was designed to produce DNA fragments (amplicons) having the mutation located in a different melting domain. The melting profiles,
  • the melting profiles of the three fragments in FIG. 4 are presented relative to the point of mutation.
  • the temperature and sequence positions do not refer to a specific temperature and base pair length, but rather, refer to relative temperature and sequence position.
  • the primer sets used are indicated in the following table:
  • cycling program parameters are shown in the table below:
  • PCR products produced using the above protocol were analyzed by DMIPC using a Transgenomic WAVETM DNA Fragment Analysis System (Transgenomic, Inc., San Jose, CA). Following initial DMIPC analysis, the
  • 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 during DMIPC was 61 °C for fragment 1 , 61 °C for fragment 2, and 62 °C for fragment 3.
  • the template was bacteriophage Lambda (base pairs 31500-32500) with a mutation at position 32061 (available from FMC Corp. BioProducts, Rockland, Maine) was amplified by means of PCR processes in which four different sets of primers were used.
  • the Lambda sequence has been published by O'Conner et al. in Biophys. J74:A285 (1998) and by Garner et al at the Mutation Detection 974 th International Workshop, Human Genome Organization, May 29-June 2, 1997, Bmo, Czech Republic, Poster no. 29.
  • Each primer set was designed to produce amplicon fragments such that each would have the mutation located in a different melting domain.
  • the primers which produced the 248 bp fragment 1 were designed to locate the mutation at position 198, near the 3'- end of the fragment.
  • the primers which produced the 253 bp fragment 2 were designed to locate the mutation at position 50, near the 5'-end of the fragment.
  • the primers which produced the 400 bp fragment 3 were designed to locate the mutation at position 199, near the middle of the longest fragment.
  • the primers which produced the 100 bp fragment 4 were designed to locate the mutation at position 51 , near the middle of the shortest fragment.
  • the melting profiles of the 4 fragments in FIG. 8 are presented relative to the point of mutation. The temperature and sequence positions do not refer to a specific temperature and base pair length, but rather, refer to relative temperature and sequence position.
  • the primer sets used are indicated in the following table:
  • the cycling program parameters are shown in the table below:
  • PCR products produced using the above protocol were analyzed by DMIPC using a Transgenomic WAVETM DNA Fragment Analysis System (Transgenomic, Inc., San Jose, CA). Following initial DMIPC analysis, the
  • 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 mlJmin.
  • the column temperature during DMIPC was 62°C for fragment 1 , 62°C for fragment 2, 63°C for fragment 3, and 60°C for fragment 4.
  • PCR Polymerase chain reaction
  • Samples for PCR amplification were purchased from Perkin-Elmer Applied Biosystems (Foster City, CA) in the GeneAmp (PCR Reagent Kit (Part No. N801-0055), which included AmpliTaq® (DNA polymerase,
  • GeneAmp (10X PCR Buffer, dNTP's as well as DNA template and primers.
  • additional samples were prepared which substituted Cloned Pfu DNA Polymerase (Cat. No. 600153, Stratagene, La Jolla, CA, USA) and PFUTurboTM ONA Polymerase
  • a 500-bp product was amplified from the DNA control template (bacteriophage Lambda DNA) from Control Primer #1 (5'- GATGAGTTCGTGTCCCTACAACTGG-3') and Control Primer #2 (5'- GGTTATCGAAATCAGCCACAGCGCC-3').
  • PFUTurboTM DNA Polymerase 1 ⁇ M Control Primer #1 , 1 ⁇ M Control Primer #2, and 1 ng DNA control template to total 100 ⁇ L.
  • Amplification was performed on the MJ Research (Watertown, MA, USA) PTC-100 Thermocycler using 15 or 35 PCR cycles.
  • the 500 bp PCR product has four cleavage cites for Hae ⁇ endonuclease (R5628, Sigma-Aldrich, St. Louis, MO, USA) at bases 37, 47, 452 and 457, producing a 405 bp blunt ended product.
  • Haelll was diluted 1 :30 with ddH20.
  • diluted Haelll was added to PCR product (1 part diluted Haelll:2 parts PCR sample), vortexed, then incubated at room temperature.
  • the PCR samples were analyzed before, during and after digestion with Haelll to ensure the cleavage was complete.
  • a 405 bp blunt ended product was produced after a 500 bp PCR product was cleaved with Haelll endonuclease. At 0 minutes, a full 500 bp product peak was seen before cleavage with Haelll. After 15 minutes, the 500 bp product had been partially cleaved, showing 3 species: uncleaved 500 bp, partially cleaved intermediate species with portion of the ends removed, and cleaved 405 bp product. Finally, after 30 minutes, cleavage of all 4 end fragments was complete. MIPC analysis conditions using the WAVETM DNA Fragment Analysis System, were as follows: Solvent
  • SUBST1TUTE SHEET (RULE 26) A: 0.1 M TEAA, Solvent B: 0.1 M TEAA, 25% Acetonitrile, linear gradient from 31-53% solvent B in 0.1 minute, 53-77% B in 12 minutes; flow rate: 0.9mL min; temperature: 50° C; detection; UV, at 254nM.
  • PCR-induced mutation analysis by MIPC and corresponding reaction product profiles As a baseline, a sample was run under conditions that would intentionally enhance the mutation rate, but could be found in a typical PCR lab. This control sample, containing PCR mutations in 61.7% of its fragments, was amplified using AmpliTaq® DNA polymerase with 35 PCR cycles. Additional samples were run to study factors which effect the PCR fidelity. These samples were identical to the control except for a change in the cycle number, or type of DNA polymerase. Samples which were PCR amplified using 15 cycles of AmpliTaq® had a 51.6% mutation rate. This 10.1 % decrease from the 35 cycle amplification is expected since with fewer cycles there are fewer opportunities for mutations to occur.
  • a 405 bp PCR product was amplified with AmpliTaq®, Pfu and
  • PFUTurboTM Substituting Pfu or PFUTurboTM for AmpliTaq® DNA polymerase had a positive effect on the yield of the PCR product. The yield of homoduplex product was determined by integration of the peak area and that of a standard of known quantity. AmpliTaq® gave the lowest yield (10 ng) and a large quantity of primer dimer (peak at 2 mins). Pfu or PFUTurboTM gave yields of 35 ng and 93 ng, respectively, and lower amounts of primer dimers (FIG. 19).
  • MIPC analysis conditions using the WAVETM DNA Fragment Analysis System were as follows: Solvent A: 0.1 M TEAA, Solvent B: 0.1 M TEAA, 25% Acetonitrile; linear gradient from 31-53% solvent B in 0.1 min, 53-71% B in 9 min; flow rate: 0.9mUmin; temperature: 66° C; detection:
  • MIPC analysis conditions using the WAVETM DNA Fragment Analysis System were as follows: Solvent A: 0.1 M TEAA, Solvent B: 0.1 M TEAA, 25% Acetonitrile; linear gradient from 31-53% solvent B in 0.1 min, 53-71 % B in 9 min; flow rate: 0.9 mlJmin; temperature: 66° C; detection: UV, 254nM.
  • MIPC analysis conditions using the WAVETM DNA Fragment Analysis System were as follows: Solvent A: 0.1 M TEAA, Solvent B: 0.1 M TEAA, 25% Acetonitrile; linear gradient from 43-57% solvent B in 0.5 min, 29-
  • each dNTP 0.2 ⁇ M primers, 2.5 mM MgCI 2 2.5U Perkin-Elmer AmpliTaq®.
  • Taq per 100 ⁇ L of Pfu, Taq (or Taq GoldTM); about 50 ng template.
  • One can experimentally manipulate the probability of DNA sequence changes by altering the number of cycles (n) and/or the polymerase error rate per nucleotide (p): f np/2, where f is the error frequency, n is the number of per cycles and p is the error rate.
  • the number of cycles should be kept to a minimum required to produce a feasible amount of product DNA.
  • EXAMPLE 15 Analyte hybridization procedure A PCR process is terminated by addition of 5 mM EDTA, 60 mM NaCI, 10 mM TrisHCI pH 8.0 to the reaction mixture. The reaction mixture is heated 95° C for 3 min. then cooled to 25° C over 45 min. Homozygous mutant must be combined with wild type in approximately 1 :1 ratio prior to hybridization.
  • EXAMPLE 16 Description of temperature dependent DMIPC separation process The following Example refers to FIG. 24 (heteroduplex separations over a 51 ° to 61 ° C temperature range).
  • a 209 base pair fragment from the human Y chromosome, locus DYS271 with an A to G mutation at position 168 was hybridized with wild type as described in Example 15 above and the sample was injected onto an
  • MIPC column 50 mm X 4.6 mm i.d.
  • the column was eluted at 0.9 mL/min with a gradient of acetonitrile in 0.1 M TEAA over 7 minutes.
  • the chromatography was monitored 260nm using an UV detector.
  • heteroduplex present in the mixture was not denatured at 51° C; therefore, a single peak was observed.
  • T(w) determined by MELT was 52° C. The sample is applied to
  • T(hsst) is determined to be 56°C.
  • EXAMPLE 18 Determination of T(hsst) by starting a DMIPC analysis at a melting temperature determined from a UV melting profile The T(hsst) is determined in a similar manner as for Example 17, but starting with a T(w) based on a UV melting profile obtained as described S. Lim (Varian Technical Note "DNA denaturation using the Cary 1/3 Thermal Analysis System", No. UV-51 , pp. 1-5, June 1991 , Varian Associates, Palo Alto, CA) and using a pH 7.3 buffer comprising 0.1 M TEAA, and acetonitrile at a concentration as obtained from FIG. 38 for a 209 bp fragment.
  • the DMIPC retention times of a DYS271 209 bp mutation standard mixture of heteroduplex and homoduplex species was measured as a function of oven temperature starting at 50°C and continuing in 0.5 and 0.3 degree increments up to 57.5°C (FIG. 37) in a temperature titration.
  • the HPLC instrument was a unit controlled via RS232 interface from customized system software.
  • the software control was from Transgenomic Inc. (San Jose, CA) custom prototype front-end software package (an extensively modified version of WAVEMakerTM). This oven was produced from a Model PTC200 M J Research thermocycler that was modified to contain a
  • DNASepTM column and preheat lines (150cm x 0.007"i.d.) made of PEEK tubing.
  • the preheat tubing was interwound between the PCR tube wells (i.e., physically placed around the wells themselevs and in thermal contact with the 96-well heating block) and then was connected to the column placed in a cavity machined out of the thermocycler.
  • the oven response was high with approximately 10 seconds required to reach a set temperature. It took about 2 minutes for the fluid to reach the set temperature. This response was much faster than conventional ovens for liquid chromatography.
  • the oven was peltier cooled, so that increases and decreases in temperature were reached rapidly.
  • the mobile phase used in the separation comprised 0.1 M TEAA (solvent A) and 0.1 M TEAA in 25% acetonitrile (solvent B).
  • the MIPC column was eluted with the gradient shown below at a flow rate of 0.9 mL/min.
  • FIG. 37 illustrates the critical dependence of separations on oven temperature and, more importantly, on the mobile phase fluid temperature. Even a 0.1° temperature change will be reflected by a change in retention time and peak pattern. For genotyping of mutations, it is therefore critical that temperature is reproducible between runs and between instruments preferably to at least O. C and most preferably to al least 0.05°C.
  • the data here shows the effect of temperature control based on the resolution of the homoduplexes and heteroduplexes is critical within a range of better than 0.1 °C.
  • the actual gradient d(retention time (min))/d(temperature (°C)) from the plot was measured to be -0.875 min/°C.
  • EXAMPLE 20 Preparation of a Reference Graph of Mobile Phase vs.
  • Nucleotide Base Pair Length A standard pUC18 Haelll restriction enzyme digest containing DNA fragments having base pair lengths of 80, 102, 174, 257, 267, 298, 434, 458 and 587 was applied to an MIPC column at non-denaturing temperature, 50° C. 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 by UV at 260nm. The gradient is shown below:
  • the reference curve (FIG. 38) was constructed by taking the retention time of each fragment and finding the corresponding %B from the gradient. The %B was then plotted against base pair as shown in FIG. 38.
  • the %B corresponding to the base pair length fragment of interest is obtained from the reference graph (FIG. 40).
  • the preliminary solvent concentration of 65%B is required to elute the 500 base pair fragment from an MIPC column under non-denaturing conditions of 50° C.
  • the mobile phase solvent concentration is augmented by 5 percentage units, to 70%B in this example. This is the final mobile phase organic solvent concentration.
  • the initial mobile phase solvent concentration is set by subtracting 15 percentage units from the preliminary concentration B determined from the reference. In this example, the initial mobile phase solvent concentration is set at 50%B (65% minus 15%).
  • the mobile phase gradient for the fragment bracketing range used in the mutation detection by MIPC is 2% per minute increase in the percent of B in the mobile phase over 10 minutes. This is followed by an immediate increase to 100%B for about 2 min to wash the column. After this wash, the mobile phase concentration is brought back to the initial 50%B for 2 minutes to equilibrate the column in preparation for the next sample injection.
  • a graphical representation of the gradient described above is depicted in FIG. 39.
  • EXAMPLE 22 Use of PEEK and titanium frits in mutation detection The resin lots used in this experiment were shown to be suitable for mutation detection having passed the Mutation Separation Factor test, with a value of > 0.1 , as described in Example 23.
  • the types of titanium frits used for columns have a significant effect on the capability to resolve heteroduplexes from homoduplexes. Work was performed with titanium frits from two separate lots. The source of all frits used in this example has been obtained from Isolation Technologies (Hopedale, MA). The elution conditions used in this example were identical to those used in FIG. 37.
  • FIG. 45 shows the separation of the DYS271 209 bp mutation standard using titanium frits from lot A.
  • Four peaks appear with the two heteroduplex peaks clearly separated from the two homoduplex peaks, with retention times of 3.07 and 3.24 minutes for the heteroduplexes, and 3.65 and 3.82 minutes for the homoduplexes.
  • FIG. 46 the same 209 mutation standard using titanium frits from lot B yields a different result: only three peaks appear, and the second homoduplex peak has disappeared altogether.
  • the type of titanium frit used may affect such resolution of heteroduplexes from homoduplexes.
  • FIG. 47 shows that the optimum temperature at which the heteroduplexes are observed to separate from the homoduplexes is 56°C. Only one peak appears at a retention time of 3.45 minutes.
  • MSF (area peak 2 - area peak 1 Varea peak 1
  • area peak 1 is the area of the peak measured after DMIPC analysis of wild type and area peak 2 is the total area of the peaks or peaks measured after DMIPC analysis of a hybridized mixture containing a putative mutation, with the hereinabove correction factors taken into consideration, and where the peak heights have been normalized to the wild type peak height.
  • Separation particles are packed in an HPLC column and tested for their ability to separate a standard hybridized mixture containing a wild type 100 bp Lambda DNA fragment and the corresponding 100 bp fragment containing an A to C mutation at position 51.
  • the procedure requires selection of the driving solvent concentration, pH, and temperature.
  • Any one of the solvents can be used: acetonitrile, tetrahydrof uran, methanol, ethanol, or propanol.
  • a counterion agent is selected from triaikylamine acetate, triaikylamine carbonate, triaikylamine phosphate, or any other type of cation that can form a matched ion with the polynucleotide anion.
  • FIG. 49 shows the resolution of the separation of the hybridized DNA mixture into heteroduplexes and homoduplexes.
  • the cycling program parameters are shown in the table below:
  • the DMIPC conditions used for the mutation detection separations are shown below:
  • the Lambda sequence has been published by O'Conner et al. in Biophys. J74:A285 (1998) and by FMC Corp. at the Mutation Detection 97 4 th International Workshop, Human Genome Organization, May 29-June 2, 1997, Brno, Czech Republic, Poster no. 29.
  • the 100 bp Lambda fragment sequence (base positions 32011 - 32110) used as a standard (available from FMC Corp.), the mutation was at position 32061.
  • Figure 48 is a chromatogram of the wild type strand analyzed under the above conditions. The peak appearing has a retention time of 4.78 minutes and an area of 98621.
  • Figure 49 is the Lambda mutation analyzed in identical conditions as Figure 48 above. Two peaks are apparent in this chromatogram, with retention times of 4.32 and 4.68 minutes and a total area of 151246.
  • the Mutation Separation Factor may be calculated by applying these various peak areas to the above MSF equation.
  • 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 measures on sample resolution by DMIPC The separation shown in FIG. 42 was obtained using a WAVETM DNA Fragment Analysis System (Transgenomic, Inc., San Jose, CA) under the following conditions: Column: 50 x 4.6 mm i.d.
  • aikylated poly(styrene-divinylbenzene) beads DNASep®, Transgenomic, Inc.
  • mobile phase 0.1 M TEAA (1 M concentrate available from Transgenomic, Inc.) (Eluent A), pH 7.3; gradient: 50-53% 0.1 M TEAA and 25.0% acetonitrile (Eluent B) in 0.5 min; 53-60% B in 7 min; 60-100% B in 1.5 min; 100-50% B in 1 min; 50% B for 2 min.
  • the flow rate was 0.9 mL/min, detection UV at 254 nm, and column temp. 56°C.
  • FIG. 43 is the same separation as performed in FIG. 42, but after changing the guard cartridge (20 x 4.0 mm, chelating cartridge, part no.
  • the guard cartridge had dimensions of 10 x 3.2 mm, containing iminodiacetate chelating resin of 2.5 mequiv/g capacity and 10 ⁇ m particle size, and was positioned directly in front of the injection valve.
  • FIG. 44 is the same separation as performed in FIG. 43, but after flushing the column for 45 minutes with 0.1 M TEAA, 25% acetonitrile, and 32 mM EDTA, at 75°C.

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Abstract

La présente invention concerne des procédés améliorés de détection des mutations de l'ADN, au moyen d'une chromatographie par dénaturation d'ions appariés de polynucléotide (DMIPC). L'invention recouvre les aspects suivants: analyse des produits d'amplification par réaction en chaîne de la polymérase (PCR) afin d'identifier les facteurs affectant la fidélité de la réplication par PCR; conception des amorceurs de PCR; sélection d'une température adaptée à la réalisation de la DMIPC; sélection de la composition en phase mobile pour une élution graduée; procédés de préparation et d'entretien des colonnes; et procédés de préparation d'échantillons polynucléotidiques avant toute analyse par chromatographie.
EP98939218A 1997-08-05 1998-08-05 Chromatographie par denaturation d'ions apparies de polynucleotide permettant de detecter des mutations Withdrawn EP1002137A4 (fr)

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US129105 1998-08-04
US09/129,105 US6287822B1 (en) 1997-08-05 1998-08-04 Mutation detection method
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CA2308314A1 (fr) * 1997-10-30 1999-05-14 Transgenomic, Inc. Procede de separation des polynucleotides par chromatographie liquide a haute resolution
US7225079B2 (en) 1998-08-04 2007-05-29 Transgenomic, Inc. System and method for automated matched ion polynucleotide chromatography
AU7384100A (en) * 1999-09-17 2001-04-17 Transgenomic, Inc. Improved column for dna separation by matched ion polynucleotide chromatography
WO2001046687A2 (fr) * 1999-12-22 2001-06-28 Transgenomic, Inc. Systeme et procede de chromatographie de polynucleotides par ions apparies automatisee
AU2001245650A1 (en) * 2000-03-10 2001-09-24 Ana-Gen Technologies, Inc. Mutation detection using denaturing gradients
EP1299166A4 (fr) * 2000-05-01 2003-07-09 Transgenomic Inc Separation de polynucleotides sur supports de separation polymeres
EP1316049A2 (fr) * 2000-06-02 2003-06-04 TRANSGENOMIC, Inc. Analyse de donnees issues de la separation chromatographique en phase liquide d'adn
GB0108349D0 (en) * 2001-04-03 2001-05-23 Medinnova Sf Method
GB0114632D0 (en) * 2001-06-15 2001-08-08 Sec Dep Of The Home Department Improvements in and relating to the provision of DNA investigating tools
US7135289B2 (en) 2001-10-05 2006-11-14 Transgenomic, Inc. Methods and compositions for mutation analysis of polynucleotides by liquid chromatography

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US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
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Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BAKALYAR S R ET AL: "Choosing sample volume to achieve maximum detection sensitivity and resolution with high-performance liquid chromatography columns of 1.0, 2.1 and 4.6 mm I.D" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 762, no. 1-2, 21 February 1997 (1997-02-21), pages 167-185, XP004116710 ISSN: 0021-9673 *
COTTON R G H: "Slowly but surely towards better scanning for mutations" TRENDS IN GENETICS, ELSEVIER SCIENCE PUBLISHERS B.V. AMSTERDAM, NL, vol. 13, no. 2, 1 February 1997 (1997-02-01), pages 43-46, XP004034140 ISSN: 0168-9525 *
HAYWARD-LESTER A ET AL: "RAPID QUANTIFICATION OF GENE EXPRESSION BY COMPETITIVE RT-PCR AND ION-PAIR REVERSED PHASE HPLC" BIOTECHNIQUES, EATON PUBLISHING, NATICK, US, vol. 20, no. 2, February 1996 (1996-02), pages 250-257, XP001041581 ISSN: 0736-6205 *
HUBER C G ET AL: "DETECTION OF PARTIAL DENATURATION IN AT-RICH DNA FRAGMENTS BY ION-PAIR REVERSED-PHASE CHROMATOGRAPHY" ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 68, 1996, pages 2959-2965, XP002911659 ISSN: 0003-2700 *
HUBER C G ET AL: "HIGH-RESOLUTION LIQUID CHROMATOGRAPHY OF DNA FRAGMENTS ON NON-POROUS POLY(STYRENE-DIVINYLBENZENE) PARTICLES" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 21, no. 5, 1993, pages 1061-1066, XP002911655 ISSN: 0305-1048 *
LI ANZHI Z ET AL: "DNA end-to-end distance change due to divalent counterion condensation studied by pulse gel electrophoresis" PROCEEDINGS OF THE 1996 MRS FALL MEETING;BOSTON, MA, USA DEC 2-6 1996, vol. 463, 2 December 1996 (1996-12-02), pages 67-72, XP008009905 Mater Res Soc Symp Proc;Materials Research Society Symposium Proceedings; Statistical Mechanics in Physics and Biology 1997 Materials Research Society, Pittsburgh, PA, USA *
MILLS M J ET AL: "Assessment of injection volume limits when using on-column focusing with microbore liquid chromatography" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 759, no. 1, 24 January 1997 (1997-01-24), pages 1-11, XP004049969 ISSN: 0021-9673 *
See also references of WO9907899A1 *
VISSERS J P C ET AL: "Optimised injection techniques for micro and capillary liquid chromatography" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER SCIENCE, NL, vol. 746, no. 1, 4 October 1996 (1996-10-04), pages 1-7, XP004020339 ISSN: 0021-9673 *

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