JP2003534798A - Apparatus and method for separating and purifying polynucleotides - Google Patents

Abstract

(57) Abstract: A method for removing a target DNA fragment having a predetermined base pair length from a mixture of DNA fragments comprises the following steps. The mixture of DNA fragments, which may contain the target DNA fragment, is applied to a separation column containing a medium having a non-polar, non-porous surface, the mixture of DNA fragments containing the counterion and the DNA binding concentration of the driving solvent in the co-solvent. In a first solvent mixture. The target DNA fragment is contacted with a second solvent solution containing a predetermined concentration of a driving solvent in a co-solvent to remove a DNA fragment having a counter ion and a target DNA fragment base pair length from the medium. Is separated from the medium. The target DNA fragment can be collected and optionally amplified. If present, when applying the method to collect putative fragments, DNA fragments having the base pair length of the target DNA may not be present in the mixture. Alternatively, a DNA fragment having the base pair length of the target DNA is present in the mixture. The present disclosure also relates to an ambient pressure for separating polynucleotide fragments from a mixture of polynucleotide fragments, comprising an upper solution input chamber, a lower eluent receiving chamber, and a tube having a fixed unit of separation medium supported therein. Or describe lower pressure devices. The separation medium has a non-polar separation surface free of polyvalent cations that reacts with counterions to form an insoluble polar coating on the surface of the separation medium.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to devices and methods for separating, isolating and purifying polynucleotides. In particular, the invention provides an apparatus for separating target polynucleotide fragments having a predetermined base pair length or stretch of base pairs, as well as for separating and purifying polynucleotides in both high and low pressure equipment. And method.

BACKGROUND OF THE INVENTION Single-stranded oligonucleotides and single-stranded DNA fragments, RNA single-stranded DN
There is a need for a rapid and efficient method for isolating, isolating and purifying A fragments, plasmids, etc. Traditional methods such as ion exchange chromatography, high performance reverse phase chromatography, gel electrophoresis, capillary electrophoresis, etc. are time consuming, tedious and inefficient, and the operation of highly skilled chromatography experts. Need. Moreover, many methods are not able to provide separation based on base pair length size of these fragments, and can only yield very small amounts of separated material.

Mixtures of single-stranded nucleic acid fragments with different base pair lengths are separated for a number of and various reasons. In general, DNA fragments containing mutations are the same length as their corresponding wild type (defined herein below) but differ in base sequence, so that they will be present in single-stranded polynucleotides, especially by PCR. The ability to detect mutations in DNA fragments that are being amplified presents somewhat different problems.

DNA isolation and mutation detection are of great importance in medicine, and in physical science and sociology, and in forensic investigations. The Human Genome Project provides a vast amount of genetic information that sets new standards for assessing the association between mutations and human disease (Guyer, et al., Proc. Nat).
l. Acad. Sci. USA 92: 10841 (1995)). For example, the underlying cause of the disease is indicated by a genetic code different from wild type (Cotton, T
IG 13:43 (1997)). Understanding the genetic basis of the disease can be a starting point for treatment. Similarly, determination of differences in the genetic code can provide powerful and perhaps clear insights into evolutionary and population studies (C
ooper, et al. , Human Genetics 69: 201 (1
985)). An understanding of these and other problems with the genetic code is based on the ability to identify abnormalities, or mutations, in DNA fragments relative to the wild type. Therefore, it is possible to separate DNA fragments based on size differences in an accurate, reproducible and reliable manner and also DNAs of the same length but with different base pair sequences (from wild type to There is a need for a methodology that can also isolate mutations). Ideally, such methods are efficient and adaptable to routine high throughput sample screening applications.

DNA molecules are polymers comprising subunits called deoxynucleotides. The four deoxynucleotides present in DNA are common cyclic sugars,
Deoxyribose, which is shared by any of four bases, hereinafter referred to as A, G, C and T, adenine (purine), guanine (purine), cytosine (pyrimidine) and thymine (pyrimidine) respectively. It is connected associatively. The phosphate group links the 3'-hydroxyl of one deoxynucleotide with the 5'-hydroxyl of another deoxynucleotide to form a polymer chain. Single-stranded DNA
In, the two strands are organized into a helical structure by so-called complementary base hydrogen bonding. The complementarity of bases is determined by their chemical structure. Single chain DN
In A, each A is paired with T, and each G is paired with C, ie the purine is paired with pyrimidine. Ideally, DNA is replicated in exact copy by a DNA polymerase during cell division in the human body or in other living organisms. DNA strands can also be replicated in vitro by the polymerase chain reaction (PCR).

Occasionally, correct replication fails and incorrect base pairing occurs, resulting in single-stranded DNA progeny containing a heritable difference in base sequence from the parent after further replication of the new strand. . Such heritable variations in the base pair sequence are called mutations.

In the present invention, single-stranded DNA is referred to as double-stranded. When the base sequence of one strand is completely complementary to the base sequence of the other strand, the double strand is called a homoduplex. A duplex is said to be a heteroduplex if it contains at least one base pair that is not complementary. Heteroduplexes are formed during DNA replication when the DNA polymerase enzyme makes an error, and non-complementary bases are added to the replicated polynucleotide chain. Ideally, further replication of heteroduplexes produces homoduplexes that are heterologous, that is, these homoduplexes have a modified sequence compared to the original parent DNA strand. .. If the parental DNA has a sequence that predominates in the naturally occurring population, it is commonly referred to as "wild type."

Many different types of DNA mutations are known. Examples of DNA mutations include, but are not limited to, "point mutations" or "single base pair mutations" that result in one incorrect base pairing. The most common point mutations comprise a "transition" in which one purine or pyrimidine base is replaced by another and a "transversion" in which the purine is replaced by a pyrimidine (and vice versa). . Point mutations also comprise mutations in which one base is added or removed from the DNA strand. Such "insertions" or "deletions" are also known as "frameshift mutations." Although they occur less frequently than point mutations, larger mutations affecting multiple base pairs can also occur and can be significant. For a more detailed description of mutations, see US Pat. No. 5,459,039 to Modrich (1995).
And US Pat. No. 5,698,400 (1997) to Cotton. All of these references and the references contained therein are incorporated herein in their entirety.

The sequence of base pairs in DNA encodes the production of proteins. Especially DN
The DNA sequence in the exon portion of the A chain encodes the corresponding amino acid sequence in the protein. Thus, mutations in the DNA sequence can result in alterations in the amino acid sequence of the protein. Such a modification in the amino acid sequence may be entirely benign, or it may inactivate the protein or modify its function to be life-threatening or lethal. on the other hand,
Since the intron portion does not contain the code for protein production, mutations in the intron portion of the DNA strand are not expected to have biological consequences. Nevertheless, mutation detection in the intron portion can be important, for example, in forensic investigations.

Therefore, the detection of mutations is of great interest and importance in diagnosing disease, understanding the origin of disease and developing potential treatments. Also, D
Detection of mutations and identification of similarities or differences in NA samples has been identified in forensic science in increasing global food supply by developing crop varieties that are disease resistant and / or have higher yields. It is also of crucial importance in evolutionary and population studies, and in scientific studies in general (Guyer
, Et al. , Proc. Natl. Acad. Sci. USA 92:10
841 (1995); Cotton, TIG 13:43 (1997)).

Modifications in DNA sequences that are benign or have no negative consequences are sometimes referred to as “polymorphisms”. In the present invention, any modification in the DNA sequence, whether they have a negative result or not, is designated as "mutation".
It should be understood that the methods and systems of the present invention have the ability to detect mutations regardless of biological effect or lack thereof. For simplicity, the term "mutation" is used throughout to mean a modification in the base sequence of a DNA strand relative to a reference strand (generally, but not necessarily wild-type).
It is to be understood that in the context of the present invention, the term "mutation" includes the term "polymorphism" or any other similar or equivalent term in the art.

There is a need for accurate and reproducible analytical methods for mutation detection that are easy to perform. Ideally, the method is automated and provides high throughput sample screening with minimal operator attention, and is also highly desirable.

Prior to the present invention, size-based analysis of DNA samples was performed by gel electrophoresis (G
EP). Capillary gel electrophoresis (CGE) has also been used to separate and analyze mixtures of DNA fragments having different lengths, eg, different lengths resulting from restriction enzyme cleavage. However, these methods cannot distinguish DNA fragments having different base sequences but having the same base pair length. Therefore, gel electrophoresis cannot be used directly for mutation detection. This is a serious limitation of GEP.

Denaturing gradient gel electrophoresis (DGGE) and denaturing gradient gel capillary electrophoresis (DGGC)
Gel-based analytical methods such as) can detect mutations in heteroduplex DNA strands under "partially denaturing" conditions. The phenomenon of "partial denaturation" is well known in the art, and heteroduplexes denature at sites of base pair mismatches at the lower temperatures required to denature the rest of the chain. Because it happens. However, these gel-based techniques are operationally difficult to implement and require highly skilled personnel. Moreover, the analysis is very long and requires a great deal of preparation time. Denaturing capillary gel electrophoresis analysis is limited to smaller fragments. Separation of 90 base pair fragments takes more than 30 minutes. Gradient denaturing gels run overnight and require about 1 day of set up time.
A further deficiency of gradient gels is the isolation of discrete DNA fragments (which requires specialized techniques and equipment) and analytical conditions must be experimentally developed for each fragment (Laboratory Methods). f
or the Detection of Mutations and Po
lymorphisms, ed. G. R. Taylor, CRC Press,
1997). The long analysis times of gel methodologies are further exacerbated by the migration of DNA fragments in the gel being inversely proportional to their length in a geometric relationship.
Therefore, longer DNA fragment analysis times may often not hold.

Recently, HPLC-based ion-pairing chromatography methods have been introduced to effectively separate mixtures of single-stranded polynucleotides in general, and DNA in particular, where the separation is based on base pair length. This method is described in the following references, all of which are incorporated herein: US Pat. No. 5,795 to Oefner.
, 976 (1998); US Patent No. 5,585,236 to Bonn (19).
96); Huber, et al. , Chromatographia 37:
653 (1993); Huber, et al. , Anal. Biochem.
212: 351 (1993)).

As the use and understanding of HPLC evolves, HP is partially denatured, that is, at a temperature sufficient to denature the heteroduplex at sites of base pair mismatches.
LC analysis revealed that homoduplexes could be separated from heteroduplexes having the same base pair length (Hayward-Lester, et al.
, Genome Research 5: 494 (1995); Underhi.
ll, et al. , Proc. Natl. Acad. Sci. USA 93:
193 (1996); Doris, et al. , DHPLC Worksho
p, Stanford University, (1997)). All of these references and the references contained therein are incorporated herein in their entirety. Therefore, the use of denaturing HPLC (DHPLC) has been applied to mutation detection (
Underhill, et al. , Genome Research 7: 9
96 (1997); Liu, et al. , Nucleic Acid Res
． , 26; 1396 (1998)).

DHPLC can separate heteroduplexes that differ by as little as one base pair. However, in some cases the separation of homoduplexes and heteroduplexes cannot be well separated. Artifacts and impurities may also interfere with the interpretation of DHPLC separation chromatograms in that it may be difficult to distinguish the artifact or impurity from the putative mutation (Underhill, et al., Genome. Res.
7: 996 (1997)). The presence of the mutation can even be completely overlooked (
Liu, et al. , Nucleic Acid Res. 26: 1396 (
1998)). The references cited above and the references contained therein are incorporated herein in their entirety.

Accuracy of HPLC-based DNA fragment separation and mutation detection assays,
Reproducibility, convenience and speed have heretofore been compromised due to problems associated with HPLC systems. The present invention addresses these problems and provides separation methods and apparatus for use in connection with the present invention in "matched ion polynucleotide chromatography" (Mat
The term ched Ion Polynucleotide Chromatography (MIPC) applies. MIPC, when used under partially denaturing conditions, is defined herein as denaturing matched ion polynucleotide chromatography (DMIPC).

MIPC system (WAVE ^™ DNA fragment analysis system, Transgenom
ic, Inc. San Jose, CA) is equipped with a computer controlled oven that seals the column and the column inlet area. Non-limiting examples of important characteristic aspects of MIPC include: a) use of hardware having a liquid contact surface that does not release polyvalent cations, and b) exotic with cartridges containing polyvalent cation capture resin. Protection of liquid contact surfaces from polyvalent cations, c) use of special washing protocols for MIPC separation media, d) automatic selection of optimal solvent gradient for elution of specific base-length DNA fragments, and e) Included is automatic determination of the temperature required to effect partial denaturation of the heteroduplex when MIPC is used under partially denaturing conditions for mutation detection (DMIPC).

The present invention can be used to separate RNA or double-stranded or single-stranded DNA. For the purpose of simplifying the description of the invention, and not by way of limitation, the separation of double-stranded DNA is described in the Examples herein, and all polynucleotides are included within the scope of the invention. It is understood that This invention is MIPC
It applies to size-dependent separations and denaturing separations. Both of these separations can include the separation of DNA fragments with non-polar tags.

[0021] Important aspects of DNA separation and mutation detection by HPLC and DHPLC, which have not been investigated so far, are: a) processing and materials of materials comprising chromatographic system components, b) materials comprising separation media. Processing and materials, c) Method development time (
solvent preselection to minimize methods development time), d) preselection of optimal temperature to effect partial denaturation of the heteroduplex during HPLC, and e) automated high throughput mutation detection screening assay Comprising DHPLC optimization for These factors, which comprise MIPC / DMIPC but not HPLC / DHPLC, have demonstrated chromatographic methods to obtain clear, accurate, reproducible and high-throughput DNA separation and mutation detection results. Mandatory when used. A comprehensive description of MIPC systems and separation media, including the critical importance of maintaining a multivalent cation-free environment, can be found in Gje.
U.S. Pat. No. 5,772,889 (1998) to Rde and August 4, 1998.
U.S. Patent Application No. 09 / 129,105 filed on May 18, 1998 09 / 081,040 filed May 18, 1998; No. 09 / 080,547 filed May 18, 1998 No. 09/058, filed April 10, 1998,
No. 580; No. 09 / 058,337, filed April 10, 1998; 19
No. 09 / 065,913 filed April 24, 1998; May 13, 1998
No. 09 / 039,061 filed on May 28, 1998; No. 09 / 081,039 filed on May 18, 1998. All of these references and the references contained therein are incorporated herein in their entirety.

All of the liquid chromatographic separations described herein above comprise gradient elution, that is they utilize a multi-component mobile phase, where the concentration of the driving component, which is usually an organic solvent. Increases during the course of chromatography. This method reduces the time required to complete the analysis. However, the separation of mixture components can be compromised. Efforts are being made to improve the resolution of MIPC. These efforts focus on improving the gradient process, changing the column particle size, or changing the length of the column. However, these efforts have yielded only marginal improvements. Therefore, there is a need to improve the separation of components that are not well separated or that flow closely. Such improvements are particularly useful where it is important to isolate the components in pure form, such as for PCR amplification, sequencing, mutation detection and numerous other applications.

Many tasks within molecular biology require pre-purification of nucleic acids. Current methods involve the use of gel electrophoresis or solid phase extraction (typically on silica gel or anion exchange resin). These methods provide an overall improvement in nucleic acid purity over the initial crude material, but suffer from negative properties. Hecker demonstrating the limitations of prior art separation methods and the excellent purity obtained with the method of the present invention
, Kari et al, "Optimization of cloning"
efficiency by pre-cloning DNA Fragmen
t Analysis ", Biotechniques, 26: 216-222.
See February 1999. Gel electrophoresis suffers from lack of automation, poor separation of different fragments, and poor recovery of fragments. Although solid phase extraction methods lend themselves to automation, they too can suffer from poor separation of different fragments and poor recovery of fragments.

Numerous methods for examining or assessing genetic material require enzymatic cleavage of the material and isolation of a particular DNA fragment or stretch of DNA fragments from a mixture of DNA fragments produced by the cleavage. . These isolations are of particular importance in the early diagnosis of certain diseases, especially cancer. In the case of cancer and other diseases of genetic origin, early detection often relies on the availability of suitable analytical methods that can accurately and reliably detect mutations in DNA samples.

As described in co-pending Sklar et al US patent application attorney docket no. P-217, filed May 13, 1999, this problem is related to the fact that such samples are not Exacerbated by being able to contain a very small population of cells containing mutant DNA in the presence of a very large population of predominantly normal cells containing type DNA. Prior to the development of the present invention, any isolation technique that could theoretically detect mutant DNA in the presence of wild type would simply detect the concentration of mutant DNA relative to wild type. It was too low for me, so I think I failed. That is, the concentration of mutant DNA may be too low to be detected in absolute terms. Alternatively, the concentration of mutant DNA may be sufficient to detect, but is completely masked by the very high relative amount of wild type in the sample.

Increasing the amount of mutant DNA by PCR amplification of the sample does not solve the above problems. The mutant and wild type DNA in the sample are very similar. In fact, their sequences may differ by only a single base pair. Therefore, the primers used to amplify the mutant DNA will also amplify the wild type since both are present in the sample. As a result, the relative amounts of mutant and wild type DNA are unchanged.

After radiation or chemotherapy, the cancer patient may be treated with residual cancer to determine if the patient is in remission, for example, as described in the co-pending Sklar et al application (supra). Monitored for the presence of cells. The effect of these treatments can be monitored if small levels of residual cancer cells can be detected predominantly in the large wild-type population. Traditionally, remission status is assessed by pathologists who perform histological examination of tissue samples. However, these visual methods are generally qualitative, time consuming, and expensive. At best, the sensitivity of these methods allows the detection of about 1 cancerous cell in 100 cells.

Analysis of DNA samples has historically been performed using gel electrophoresis. Capillary electrophoresis has also been used to separate and analyze mixtures of DNA. However, these methods are unable to distinguish point mutations from homoduplexes with the same base pair length.

In addition to the deficiencies of the denaturing gel method described above, performing these gel slab preparations and analyzes can vary greatly from operator to operator, so these techniques are always reproducible or accurate. Not necessarily. As a result, the mobility of DNA fragments is different on different gel slabs and in one lane compared to another on the same gel slab. The problems and deficiencies of gel-based DNA separation methods are well known in the art and are described in the published literature, eg, G. et al. R. Tayl
or, editor, LABORATORY METHODS FOR THE
DETECION OF MUTATIONS AND POLYMORP
HISMS, CRC Press (1997).

SUMMARY AND OBJECTS OF THE INVENTION Single-stranded oligonucleotides and single-stranded DNA fragments, RNA, for the rapid and efficient base pair length size separation of single-stranded DNA (dsDNA)
It is an object of the present invention to provide methods and devices for effecting size-based separation of plasmids, etc.

It is a further object of the present invention to provide a simple and inexpensive method and device for size-based separation of these fragments which can be easily and reproducibly manipulated by a skilled technician. Is.

It is an even further object of the present invention to provide a method and apparatus for low pressure separation of these fragments.

It is another object of the present invention to provide a sensitive and reproducible method that allows the isolation of small amounts of any target DNA in a mixture of DNA fragments.

It is an even further object of the present invention to provide suitable methods and systems for isolating mutant DNA in the presence of relatively large amounts of wild-type DNA, where such abrupt changes occur. The mutation would otherwise go undetected.

It is the main object of the present invention to provide an analytical method that is reproducible, reliable, inexpensive, can be automated and can be used for high throughput sample screening.

In summary, the method of the present invention for isolating a target DNA fragment having a predetermined base pair length from a mixture of DNA fragments comprises the following steps. The mixture of DNA fragments is applied to a separation column containing a separation medium having a non-polar, non-porous surface, the mixture of DNA fragments containing a counterion and a DNA binding concentration of a driving solvent. In one solution. The target DNA fragment is a DNA having a counter ion and a target fragment.
The fragments are removed from the medium by contacting the medium with a second solution containing a driving solvent concentration that has been previously determined to remove the fragments from the separation medium into separate compartments of the eluent.

In some cases, the target DNA fragment may be recovered. Recovered target DNA
The fragment can be amplified and cloned.

The method can be applied to determine the presence or absence of DNA fragments with a particular base pair length in a sample mixture. The method can be applied when a DNA fragment having the base pair length is present in the sample mixture at a concentration that is too low to be detected in the assay, and the DNA fragment having the base pair length in the sample mixture is Amplification products are analyzed to confirm their presence.

Another method of the invention separates target homoduplex and / or heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments having the same base pair length by DMIPC. The method, wherein the heteroduplex fragment has at least one mismatch site. The method comprises the steps of: applying a mixture of homoduplex and heteroduplex DNA fragments to a separation column containing a separation medium having a non-polar, non-porous surface to form homoduplex and heteroduplex. The mixture of double-stranded DNA fragments is applied in a first solution containing a counterion and a DNA binding concentration of a driving solvent. Separation media for counterions and target homoduplex and / or heteroduplex DNA fragments in separate fractions while maintaining media and solution at temperatures that locally denature heteroduplex fragments at their mismatch sites. A desired solution of homoduplex and / or heteroduplex DNA from the separation medium by contacting the separation medium with a second solution containing a driving solvent concentration that has been previously determined for separation from the separation medium. Separate the fragments. If the desired fragment is a homoduplex fraction, the concentration of the driving solvent is selected to remove one or both homoduplex fractions as a separate fraction from the homoduplex fraction. Alternatively, if the desired fragment is a heteroduplex fraction, the concentration of the driving solvent is chosen to remove one or both of the heteroduplex fractions as a separate fraction from the homoduplex fraction.

The heteroduplex fraction can be collected and amplified. The amount of heteroduplex fragment to homoduplex fragment is less than 1: 1. The method can be used when the amount of heteroduplex in the distinct heteroduplex fraction is below the level of detection.

The retention time used for separation of homoduplex or heteroduplex fractions can be determined in advance from a reference standard. The reference standard can be obtained by matched ion polynucleotide chromatography by separating a standard mixture of homoduplexes and heteroduplexes having the same base pair sequence as the sample.

This method can be used to determine the presence or absence of homoduplex and / or heteroduplex fractions in a mixture of homoduplex and heteroduplex DNA fragments having the same base pair length. , Heteroduplex fragments have at least one mismatch site. The recovered fragment can be cloned. The method comprises the steps of: if the DNA sample contains a large amount of wild-type background, and if the mutant DNA is below the limit of detection, if the DNA sequences of the wild-type and mutant DNA are known, and Mutant DNA is wild-type DNA
Can be applied when at least one base pair differs from.

In summary, the ambient or lower pressure apparatus of the present invention for separating polynucleotide fragments from a mixture of polynucleotide fragments comprises an upper solution input chamber, a lower eluent receiving chamber, and a separation medium supported therein. A tube having a fixed unit of. The separation medium has a non-polar separation surface that is free of polyvalent cations that react with counterions to produce an insoluble polar coating on the surface of the separation medium.

The separation medium can be beads, capillaries or monolith structures. The fixed unit of separation medium can comprise a fixed bed of particles of separation medium, and the particles of separation medium can be organic polymers or inorganic particles having a non-polar surface.

In one aspect, the lower chamber is closed. Alternatively, the lower chamber has an open bottom portion. The tube can be combined with an eluent container adapted to receive the lower chamber. The eluent chamber can be a centrifuge vial.

In one embodiment, the tubes are members of a series of tubes, and the eluent vessels are members of a series of eluent vessels, and the array of tubes and the array of containers have a matching arrangement.

The ambient or lower pressure separation system of the present invention comprises a combination of a multi-lumen separation plate having an outer sealed edge, a multi-well collection plate and a vacuum system. The vacuum system has an outer sealing edge of the multi-cavity separation plate and a separation plate sealing means that creates a sealing engagement with the vacuum cavity that receives the multi-well collection plate. The multi-lumen separation plate comprises a series of tubes, each tube having an upper solution input chamber, a lower eluent receiving chamber having a bottom opening therein, and a fixed unit of separation medium supported therein. The separation medium can have a non-polar separation surface that is free from polyvalent cations that react with counterions to produce an insoluble polar coating on the surface of the separation medium. The multi-well collection plate has a collection well arranged to receive liquid from the bottom opening of the lower eluent receiving chamber. The separation medium can be beads, capillaries or monolith structures. The fixed unit of separation medium comprises a fixed bed of particles of separation medium.
Separation medium particles can be selected from the group consisting of organic polymers having non-polar surfaces and inorganic particles.

DETAILED DESCRIPTION OF THE INVENTION The methods of the present invention generally relate to polynucleotides, but the following description is provided for reference only, and not by way of limitation, single-stranded DNA (dsDNA).

The term “matched ion polynucleotide chromatography”, as used herein, is defined as a method for separating single-stranded and single-stranded polynucleotides using a non-polar separation medium, where the The method uses a counterion agent and an organic solvent to release the polynucleotide from the separation medium. MIPC separation is completed in less than 10 minutes and often less than 5 minutes. MIPC system (WAVE ^™ DNA fragment analysis system, Transgenomic, Inc. San
(Jose, CA) is equipped with a computer controlled oven that seals the column and sample introduction area.

A “gradient”, as defined herein, is a chromatographic mobile phase identified by an initial time point having an initial solvent composition and an end time point having a final solvent composition different from the initial solvent concentration, and a gradient composition Varies continuously (eg, usually increases) from the initial solvent composition to the final solvent composition over the time interval between the initial and final time points. The gradient mobile phase is used to elute the fragments from the chromatography column for the purpose of separating the components in the mixture.

The term “isocratic” is defined herein to describe a chromatographic mobile phase whose composition remains essentially constant over any or all of the duration of the chromatographic separation step. . "Isocratic"
The term is used to maintain a single solvent concentration throughout the separation, step the solvent concentration from one constant to one or more constants in a series of steps, or have a gradient separation and have a constant Methods with one or more moieties performed under solvent concentration conditions are intended to be included. An isocratic mobile phase is used to elute fragments from a chromatography column for the purpose of separating the mixture on the column into its components (Remington: The Science and P.
ractice of Pharmacy, 19 ^th Ed. , A. Gennar
o, Ed. p. 537 (1995)). This reference is incorporated herein in its entirety. The isocratic separation solvent concentration should be kept within ± 1% of the selected isocratic solvent concentration, and preferably within ± 0.5% of the selected isocratic solvent concentration. . Optimally, the isocratic solvent concentration is kept at ± 0.1% of the selected isocratic solvent concentration.

The term “partially denatured” refers to the separation of mismatched base pairs (caused by temperature, pH, solvent or other factors) in a DNA duplex while leaving other parts of the duplex intact. Means

In general, there is a wide range of interactions with the column. Typically, a large number of different size fragments are analyzed. For example, separation of 80-600 base pair fragments on MIPC columns requires acetonitrile concentrations of about 8.75% to 16.25%, respectively. Longer fragments require a further increase in acetonitrile concentration. Therefore, a gradient step is required to achieve the full range of fragment size separations. Otherwise it is not possible to separate the sizes in this range.

The device of the present invention comprises a single-stranded oligonucleotide and a single-stranded DNA fragment,
It provides a new and unique method for isolating and purifying RNA, single stranded DNA fragments, plasmids and the like. The device simplifies the separation method and uses a unique size-based separation method based on our Matched Ion Pair Chromatography (MIPC), which is also referred to herein by the term DNA chromatography. This method takes advantage of the binding properties of a separation medium with a non-polar surface in the presence of counterions and a separating substance. Counterions and substances in aqueous solutions with low stripping solvent concentrations bind to non-polar surfaces, and subsequently release or strip the substance from the surface of the separation medium, releasing the substance from the surface by application of a stripping solvent concentration. The size of the material to be stripped is a function of the stripping solvent concentration. Larger size materials require the application of higher stripping solvent concentrations to effect their release. The ratio of the fragment size desorbed from the medium to the concentration of the stripping solvent can be calibrated and is so reproducible that it can be calculated with high accuracy. The method can be applied in any system capable of retaining a separation medium and providing a means of rapidly passing liquid through the separation medium.

FIG. 1 is a schematic diagram of a high pressure system for carrying out the Matched Ion Pair Chromatography (MIPC) method of the present invention having a proportional valve system for effecting a gradient of solvent concentration in the separation. Chromatographic solutions, such as solvents, counterions, and other solutions that mix with the solvent, are retained in and in communication with the solvent container 2, carrier liquid container 4 and auxiliary liquid (eg, auxiliary solvent) container 6 and dissolved gas. Removal device 14
Each solvent transport pipe 8, carrier transport pipe 10 and auxiliary liquid transport pipe 1 connected to
Have two.

The column wash solution is contained in a wash solution container 16, which also has a wash solution transport conduit 18 communicating with it and leading to the dissolved gas removal device 14. In this aspect, the wash solution can flow by gravity pressure when the vessel 16 is lifted above the dissolved gas remover and injection valve 54. Alternatively, a pump as shown in Figure 2 can be provided to obtain a flow of wash solution.

The degassing solvent conduit 20, the degassing carrier liquid conduit 22 and the degassing auxiliary liquid conduit 24 connected from the dissolved gas removing device 16 are respectively connected to a solvent proportional valve 26, a carrier liquid proportional valve 28 and an auxiliary liquid proportional valve 30. I understand. The settings of these proportional valves are set and modified by a valve operator, such as a stepper motor associated therewith, and these valve operators respond to commands from the valve operator control module, which is described in greater detail below. And react to define the desired set of settings. The setting of these proportional valves controls the ratio of liquid (co-solvent, drive solvent, etc.) through the injector valve and separation column. Conduits 32, 34 and 36 connect from respective proportional valves 26, 28 and 30 to the intake of pump 38. The dissolved gas removing device 14 removes the dissolved gas from the liquid. Its removal is particularly important as the presence of dissolved oxygen oxidizes iron or other oxidizable metals in the system components, thus increasing the risk of introducing corresponding cations into the liquid.

The wash solution transport conduit 31 connects to a wash solution valve 40. Optional wash solution conduit 42
Connects from valve 40 and leads to the inlet of pump 38.

The openings of valves 26, 28 and 30 accurately set the relative ratio of solvent or solvents to carrier liquid, and size-based DNA separation by MIPC is a function of solvent concentration, so that The most important part. As described for various DNA fragment separation steps, the slope of the solvent gradient as a function of time is altered during the separation step, and the most important step is to obtain a very accurate gradient or, in some steps, a very accurate gradient. Isocratic (constant solvent concentration) composition may be required. The setting of valves 26, 28 and 30 is defined by a conventional valve actuator which can be set remotely by a signal to a conventional valve controller. As will be described in greater detail below, the control system of the present invention allows the valve 2 to operate at the appropriate time during system operation.
A computer control command is given to set the 6, 28 and 30 settings to the exact flow rate value.

Similarly, the control system of the present invention provides computer control commands that define operating parameters of the pump 38, such as pump off / on state and pump pressure or flow rate settings.

The pump outlet conduit 44 leads to an in-line mixer 46, which directs the flow of liquid through the mixer 46 for thorough mixing of the components. The mixed liquid outlet conduit 48 is D
It leads to a guard column 50 which processes the mixture to remove polyvalent metal cations and other contaminants that interfere with the separation of NA fragments. The guard column 50 can contain a cation exchange resin in the sodium or hydrogen form for the removal of polyvalent metal cations by conventional ion exchange. The conduit 52 leads to the outlet of the guard column and the inlet of the wash solution injector valve 54. Waste supply conduit 5
6 connects valve 40 with wash solution injector valve 54 and waste outlet conduit 58 leads to waste. Conduit 60 leads from waste solution injector valve 54 to sample injection valve 62.

The sample aliquot selector 64 communicates with the injector valve 62 through the sample conduit 66. The waste conduit 68 connects from the injector valve,
Then remove the waste liquid.

At injector valve 62, the sample is introduced from conduit 60 into the flow of solvent and carrier liquid through the valve. The sample conduit 70 leads to the outlet of the injector valve 62 and a column prefilter 74 in an air bath oven 72. The capillary coil 76 leads to the inlets of the prefilter 74 and the separation column 78. The extended length of the capillary coil 76 is able to transfer sufficient heat from the heated oven air to the liquid passing through the coil, resulting in a liquid within ± 0.05 ° C of the selected temperature. The oven 72 establishes this temperature uniformity in the prefilter 74, coil 76 and separation column 78.

The separation column 78 contains DNA in the presence of a counter ion matching by the MIPC method.
Beads with unique separation surfaces that provide size-based separations of fragments are packed by conventional column construction. Details of the separation method and beads are described in detail below. A stream (eluent) containing DNA fragments separated by base pair length size travels from separation column 78 through eluent conduit 80.

The analyzer 84 leads to a conduit 80. The analyzer cell 84 can be a conventional UV luminometer that measures the UV luminescence level of natural DNA fragment structures in a liquid. The level of absorption is a function of the concentration of DNA fragments in the fluid tested.

Alternatively, if the DNA is labeled with a fluorescent marker, the analyzer continuously measures the level of the fluorescent marker in the liquid by detecting the emission level at the frequency most appropriate for the marker. Similarly, if the DNA is labeled with a mass label, the detector can be a mass spectrophotometer. Any analytical system capable of continuously measuring the properties of the liquid as a function of the concentration of the DNA fragments therein is suitable,
And it is readily apparent that it is considered to be within the scope of the invention.

Eluent travels from analyzer 84 to fragment collector 88. In the fragment collector 88, a selected portion of the eluent containing the separated DNA fraction is collected in a vial for later processing or analysis. Uncollected fractions are removed through waste conduit 90.

The DNA separation step is impaired by the presence of polyvalent cations. In the above description, the liquid flow system is described as a series of conduits. The conduit is a capillary chosen to avoid the introduction of polyvalent cations into the liquid. Preferred capillary materials are titanium and PEEK. For the same reason, the other components of the system are preferably made of titanium or PEEK or exposed to a PEEK coated liquid to protect them from oxidation and prevent the introduction of polyvalent cations into the liquid. Has a surface that is In addition, the titanium and PEEK parts, tubes and surfaces can be further processed to remove contaminants.

Stainless steel can also be used provided it is treated to remove all oxidized surface material and the solution contacting the stainless steel surface does not contain dissolved oxygen.

FIG. 2 is a partial schematic diagram corresponding to the representation of FIG. 1, but with a proportional pump system for producing a gradient of solvent concentration in the separation. This system relies on a proportional pump to control the ratio of solvent to carrier liquid. Proportional pumps 92, 94 and 9
The six inlets lead to the dissolved gas removal device 14 through their respective supply conduits 98, 100 and 102, and their respective outlet conduits 104, 1
06 and 108 lead to an in-line mixer 46. The operating speed of these proportional pumps is calibrated to the flow rate therethrough and is controlled by the proportional pump control module described in greater detail below. The setting of these proportional valves is such that the flow rate of the liquid through the injector valve and the separation column and the liquid (co-solvent,
Drive solvent etc.) ratio is controlled.

The pump 110 can supply the wash solution to the system through an optional conduit 112. Optional conduit 113 can lead from conduit 112 and lead to an in-line mixer 46.

FIG. 3 is a partially exploded view of the physical structure of a typical separation column. The column comprises a column tube 120 with threads on both ends. The tube is filled with a separation medium 122. The frit 124 is held against the upper surface of the separation medium by the frit plug 126. Female thread coupling 128 is secured to the end of tube 120 and receives frit 124 and frit plug 126. The female screw coupling 128 receives the male screw coupling 130 by screw engagement. The external thread coupling 130 has an internal thread end receptor 132 for receiving a capillary end coupler (not shown).

Separation medium 122 is an organic polymeric or inorganic material having the requisite structure and a non-polar surface as described in greater detail below.

The method of the present invention for isolating a target DNA fragment having a predetermined base pair length from a mixture of DNA fragments comprises the following steps. DNA
The mixture of fragments is applied to a separation column containing a separation medium having a non-polar, non-porous surface, the mixture of DNA fragments is
In the first solution containing the binding concentration. The target DNA fragment is then treated with a second solution and medium containing a concentration of driving solvent that has been previously determined to remove the DNA fragment with the counterion and the target fragment from the separation medium into separate compartments of the eluent. Are taken out of the medium by bringing them into contact with each other.

Therefore, the present invention provides for the determination of a predetermined base pair (from a mixture of fragments
bp) relates to the isolation of DNA fragments having a length. It also has the same bp
It also relates to isolation in a mixture of DNA fragments having a length, heteroduplex fragments from homoduplex fragments, heteroduplex fragments from each other and homoduplex fragments from each other. For example, the technique of the present invention is a method similar to Matched Ion Polynucleotide Chromatography (MIPC), which is our modified Matched Ion Polynucleotide Chromatography (DMIPC).
For the unambiguous detection and identification of very small amounts of heteroduplex fragments containing mutant DNA in the presence of relatively large amounts of known wild type S.
It can be applied to the method of klar et al (supra).

MIPC separates DNA fragments based on their base pair length (B
US Patent No. 5,585,236 (1996) to Onn; Huber, et.
al. , Chromatographia 37: 653 (1993); Hub.
er, et al. , Anal. Biochem. 212: 351 (1993)
). All of these references and the references contained therein are incorporated herein in their entirety. If the MIPC analysis is performed at a temperature that partially denatures, the method is called DMIPC. These separation methods avoid the deficiencies of gel-based methods and allow the collection and identification of mutant DNA fragments that have low concentrations relative to wild type and may be below the detection limit of the detector. Alternatively, MIPC and DMIPC allow the collection and identification of relatively large amounts of wild-type hidden mutant fragments in samples as exemplified in the method of Sklar et al (supra). This method is described in detail herein below.

MIPC is a silica medium having an organic polymer, a coated or covalently bonded organic polymer or a non-polar surface comprising covalently bonded alkyl and / or aryl groups, and a continuous non-polar material. Separation medium, ie
Unique non-polar separations comprising beads, monoliths or rod columns, blocks containing capillaries or capillary-sized channels or grooves, or other non-polar surfaces of materials such as organic polymers or silica gel with a non-polar surface finish. Use a medium. The separation medium used in MIPC can be porous or non-porous. For a detailed description of the MIPC separation method, the MIPC separation medium and the MIPC system, see G.
US Pat. No. 5,772,889 (1998) to Jerde and March 1998.
Co-pending U.S. patent application Ser. No. 09 / 058,580 filed March 10, 1;
No. 09 / 058,337, filed March 10, 998; May 1, 1998
No. 09 / 081,040 filed on 8th; 09 / 080,547 filed on May 18, 1998; 09/16 filed on October 9, 1988
No. 9,448; No. 09 / 183,123, filed October 20, 1998; and No. 09 / 183,450, filed October 20, 1998. MIPC systems and separation media are commercially available (Transgenomic,
Inc. WAVE ^™ DNA Separation System from San Jose, CA, WAVEM
AKER ^TM DNA isolation system software and DNASep ^R DNA separation column)
. Total MIPC analysis can be automated by desktop computer and sample autoinjector. The analytical data for each sample can be analyzed in real time or can be collected and stored in computer storage for later analysis.

MIPC at partially denaturing temperatures for detecting mutations, ie D
The use of MIPC is described in co-pending US patent application Ser. No. 09 / 129,105, filed Aug. 4, 1998. This application and the references contained therein are incorporated herein in their entirety.

The MIPC method is very reproducible. Therefore, it is not necessary to calibrate the column for each sample or each day. DNA fragments of a specific base pair length elute from the MIPC column with specific retention times that are reliable and reproducible. This property, coupled with the automation, sample collection and rapid sample analysis capabilities of MIPC, makes this method uniquely suitable for the detection of minor mutations in the presence of large amounts of wild-type background.

The MIPC method of the present invention has been found to involve a unique relationship between the concentration of organic (driving) solvent in the mobile phase and the hydrophobic stationary phase. The strong affinity of the polynucleotide fragment for the stationary phase governs the interaction until the solvent concentration reaches the critical range for each fragment size. Within the critical range, the solvent competes with the stationary phase and the fragments are released or desorbed, rapidly reaching the rate of eluent. In the separation method using a solvent gradient, the fraction released is a simple direct function of solvent concentration, a binding and release relationship that is independent of column length.

When the solvent concentration is kept at a constant level within the critical range of fragment size (isocratic separation), the competitive phase is weakened and the separation of fractions in the eluent is increased with the column. Shows some increase with length. If the solvent concentration changes with a very slight gradient through the critical range of fragment sizes, the rate of complete release of fragments to the eluent will increase and the competitive phase will decrease rapidly.

In other words, if the solvent gradient gives a rapid release of the fragments, the length of the column becomes insignificant as the complete separation step takes place within a very short distance at the top of the column. As the slope of the solvent gradient diminishes and approaches the isocratic process, competition with the stationary phase spreads over increasing lengths of the column, delaying complete release of fractions to the eluent.

In the MIPC separation method, on the other hand, DNA is added to the non-polar, non-porous surface of the separation medium in a dilute solution of the driving solvent, the co-solvent and the counterion. DNA binds at the top of the column. We have found that the separation determined by the base pair length of DNA from the separation medium is entirely a function of the driving solvent concentration in the cosolvent. By controlling the driving solvent concentration as a function of time, the target DNA fragment is isolated from the column as a function of time in a completely predictable manner.

The method can be used to collect and amplify target DNA fragments. The method can be used as an assay to determine if a target DNA fragment is present in a sample. Therefore, the method is directed to target DN
If no DNA fragment with a base pair length of A is present, and the target DNA
It can be applied when there is a DNA fragment having a base pair length of.

Isocratic elution gives a much better separation of component fragments with slight differences in length than can be done with gradient elution. Thus, in one aspect, the invention provides a method of enhancing the separation of dsDNA fragments on a MIPC column by using an isocratic mobile phase to elute the fragments from the column.

Separation of dsDNA fragments by MIPC under isocratic elution conditions is very sensitive to solvent composition. Minor changes in the composition of the solvent mixture contained in the solvent container can occur as a result of evaporation. Such changes can result in different retention times for fragments from the same sample.
It is important to keep the solvent container bottle tightly closed with a lid, parafilm, aluminum foil or similar material to prevent changes in retention time due to changing solvent composition. Furthermore, if helium sparging is used to degas the solvent, gas flow must be minimized to minimize solvent evaporation. The use of an in-line dissolved gas remover is the preferred method used to minimize solvent evaporation and maintain a constant solvent composition. An example of a suitable dissolved gas remover is DEGASET, model 6324 (MetaChem Techn).
LOGIES, Torrance, CA).

There are several types of counterions that are suitable for use in MIPC. These are mono-, to which a proton can be added to generate a positive counter-charge.
, Di- or trialkylamines or quaternary alkyl-substituted amines which already contain a positive countercharge. The alkyl substitution can be homogeneous (eg triethylammonium acetate or tetrapropylammonium acetate) or hybrid (eg propyldiethylammonium acetate). The size of the alkyl groups may be small (methyl) or large (up to 30 carbons), especially when only one of the substituted alkyl groups is large and the rest are small. For example, octyl dimethyl ammonium acetate is a suitable counterion factor. Preferred counterion agents are those containing alkyl groups from the ethyl, propyl or butyl size range.

The mobile phase preferably contains a counterion factor. Typical counterion factors include
Included are trialkylammonium salts, lower trialkylammonium salts and lower quaternary alkylammonium salts of organic or inorganic acids such as lower alkyl primary, secondary and lower tertiary amines. Examples of counterion factors are octyl ammonium acetate, octadimethyl ammonium acetate, decyl ammonium acetate, octadecyl ammonium acetate, pyridinium ammonium acetate, cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, propyl diethyl ammonium acetate, butyl ethyl ammonium acetate. , Methylhexyl ammonium acetate, tetramethyl ammonium acetate, tetraethyl ammonium acetate, tetrapropyl ammonium acetate, tetrabutyl ammonium acetate, dimethyl diethyl ammonium acetate, triethyl ammonium acetate, tripropyl ammonium acetate , Tributylammonium acetate and the like. The anion in the above example is acetate, but other anions including carbonate, phosphate, sulfate, nitrate, propionate, formate, chloride and bromide, or any combination of cations and anions may also be used. it can. These and other factors have been described by Gjerde, et al in Ion Chromatog.
raphy, 2nd Ed. , Dr. Alfred Huethig Verl
Ag Heidelberg (1987). Counterion agents that are volatile are preferred for use in the method of the invention, with triethylammonium acetate (TEAA) and triethylammonium hexafluoroisopropyl alcohol being most preferred.

In order to obtain optimal peak separation during the separation of DNA by MIPC, the method is preferably 20 ° C to 90 ° C; more preferably 30 ° C to 80 ° C; most preferably 50 ° C.
It is performed at a temperature within the range of ℃ to 75 ℃. The flow rate is selected to produce a back pressure not exceeding 5000 psi. In general, separation of single-stranded fragments should be done at higher temperatures.

The temperature at which the separation is carried out affects the selection of the organic solvent used for the separation. One reason is that the solvent influences the temperature at which the single-stranded DNA melts to form two single-stranded or partially melted complexes of single- and single-stranded DNA. One solvent can stabilize the molten structure better than another. Another reason solvent is important is that it affects the distribution of DNA between the mobile and stationary phases. Acetonitrile and 1-propanol are the preferred solvents in these cases. Finally, solvent toxicity (and cost) can be important. In this case, methanol is preferred over acetonitrile, and 1-propanol is preferred over methanol.

When the separation is carried out at a temperature within the above range, preferably a water-soluble organic solvent,
For example, alcohols, nitriles, dimethylformamide (DMF), tetrahydrofuran (THF), esters and ethers are used. A water-soluble solvent is defined as one that exists as a single phase with the aqueous system under all conditions of operation of the present invention. Particularly preferred solvents for use in the method of the invention include methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran (THF) and acetonitrile, with acetonitrile being most preferred.

Size-based separations with an isocratic mobile phase are optimally performed below the temperature at which dsDNA is denatured. This temperature range comprises 25 ° C to about 55 ° C, with about 50 ° C being most preferred.

One aspect of the present invention is the use of DN with a particular base pair length in a sample mixture.
This is a method of determining the presence or absence of the A fragment. The method comprises subjecting the sample mixture to a separation column containing a separation medium having a non-polar, non-porous surface, the sample mixture containing a counterion and a DNA binding concentration of a driving solvent in a co-solvent. In a first solvent mixture. The specific base is then contacted with a second solvent solution containing a concentration of a driving solvent that has been previously determined to remove the counterion and a DNA fragment having the base pair from the separation medium. Any DNA in the sample with pair length is removed. The separated fractions can be amplified by PCR, for example, to increase the amount of the putative fraction or to confirm its lack from the sample.

The present invention applies this discovery to purify and isolate mutant fragments by "blind collection". The term "blind collection" is defined herein to mean the collection of mobile phase flowing through the MIPC column over a specified time interval after addition of the DNA sample to the column. More specifically, "blind collection" refers to DNA
It refers to collecting the mobile phase during a retention time interval corresponding to the predetermined retention time interval of the fragment standard. The relationship between MIPC retention time and base pair length is so reproducible that it is not necessary to detect the desired fragment with a detector to know when to collect the fragment. Simply collect the column mobile phase at a predetermined and expected retention time for the desired fragment.

The use of specific concentrations of organic components in an isocratic mobile phase is referred to herein as the “target range” of base pair length, dsDN within a relatively narrow range of base pair lengths.
Results in enhanced separation of A. Thus, in another embodiment, a gradient mobile phase can be used to separate a mixture of dsDNA to separate fragments within the selected base pair range. Chromatographic fractions containing fragments within the target range can be isolated and rechromatographed using an isocratic mobile phase. The enhanced separation afforded by the use of an isocratic mobile phase allows the isolation of one or more pure fragments within the target range.
Such pure fragments can then be amplified using PCR to provide relatively large amounts of highly pure product. High-purity dsDNA fragments have many uses, eg, sequencing, forensics, cloning and DN.
Sample preparation before A analysis.

In another aspect, the invention can be used to detect the presence or absence of mutations. In this embodiment, sample dsDNA is hybridized with wild type. Hybridization, a standard method in the biotechnology art, is brought about by heating a solution of sample and wild-type DNA to about 90 ° C. for about 5 minutes and then slowly cooling the solution to ambient temperature over 45-60 minutes. . During the heating period, the dsDNA strands denature. On slow cooling, they recombine statistically. Thus, if the sample contains a mutation, the hybridized product will contain a mixture of two homoduplexes and two heteroduplexes. The outline of the hybridization step is shown in FIG.

Another aspect of the invention is to separate target homoduplex and / or heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments having the same base pair length by DMIPC. The method, wherein the heteroduplex fragment has at least one mismatch site. The method comprises subjecting a mixture of homoduplex and heteroduplex DNA fragments to a separation column containing a separation medium having a non-polar, non-porous surface, the homoduplex and heteroduplex. The mixture of single-stranded DNA fragments is applied in a first solution containing a DNA binding concentration of counterion and driving solvent. The counterion and target homoduplex and / or heteroduplex DNA fragments are then separated from the separation medium while maintaining the medium and solution at a temperature that locally denatures the heteroduplex fragment at its mismatch site. A desired homoduplex and / or heteroduplex DNA fragment from the separation medium by contacting the separation medium with a second solution containing a concentration of the driving solvent that has been previously determined to separate in the fractions. To separate.

For example, heteroduplexes and homoduplexes have been separated by HPLC using gradient elution and “partially denaturing conditions” (US Pat. No. 5, to Oefner).
795, 976 (1998)). However, the separations performed by Oefner were not well separated and were not always reproducible.

Gradient elution MIPC under partially denaturing conditions, as described in US patent application Ser. Achieve an excellent separation of. This reference and the references contained therein are incorporated herein in their entirety. MIPC separates dsDNA fragments by base pair length under non-denaturing conditions. However, under partially denaturing conditions, the components likewise lead to a separation. Therefore, homoduplexes and heteroduplexes separate from each other when performed at temperatures that partially denature MIPC. The temperature of partial denaturation will vary according to the sequence of any given DNA fragment. However, preferred partially denaturing temperatures are obtained between 50 ° C. and 70 ° C., most preferred partially denaturing temperatures are 53 ° C. to 62 ° C., and optimal partially denaturing temperatures are about 56 ° C. ℃. Liquid chromatographic separations of homoduplexes and heteroduplexes using an isocratic mobile phase have not been previously reported.

In this aspect of the invention, the hybridized dsDNA mixture is applied to a MIPC column comprising a non-polar separation medium and the fragments are eluted isocratically. The difference in retention times shown in Figure 31 for heteroduplexes and homoduplexes is greater than 2 minutes, a completely clear separation and a significant improvement over similar separations using gradient elution. This wide open separation can be effectively used to collect and isolate the heteroduplex as it elutes from the column. Heteroduplex PCR amplification provides sufficient pure material for sequencing or other applications requiring high purity material.

As a preferred embodiment of the present invention, “kinetic separation (kinetic separation) is performed.
ation) ”for mutation detection, which utilizes MIPC separation at temperatures that result in partial melting of mismatches. This aspect achieves even better separation between heteroduplexes and homoduplexes. MIPC a mixture of DNA fragments
When applied to the column, they are separated by size and smaller fragments elute first from the column. However, heteroduplexes separate from homoduplexes when MIPC is performed at temperatures high enough to denature portions of the DNA fragment containing non-complementary base pairs or polymorphisms.

“Kinematic separation” refers to isocratic or near isothermal selection of temperature conditions to use the hysteresis effect (illustrated in FIG. 32) to enhance the separation of heteroduplex and homoduplex species. This is a separation method performed under isocratic conditions. The temperature is chosen such that the partially melted heteroduplex species moves through the column at approximately the linear velocity of the mobile phase without interacting with the stationary phase. The homoduplex interacts or is adsorbed by the column and later elutes.

During the cooling step of hybridization, the higher melting homoduplexes regenerate rapidly and completely, while the heteroduplexes, which are more denatured during heating, regenerate slowly. Cooling while fixing the melting temperature is equivalent.

FIG. 32 illustrates the effect of higher temperature on the percentage of denaturation with respect to maintaining a constant temperature and raising the melting temperature. Regeneration is a critical degree of denaturation (cri
It is kinematically slower than the physical degree. That is, the higher the temperature used for denaturation, the longer the time required for regeneration to occur.

One aspect of the invention provides a method that can be used to isolate a mutation in a sample containing a relatively high amount of wild type, wherein the concentration of the mutation is the detector. Can be below the limit of detection by. Alternatively, the present invention may be sufficient if the concentration of mutant DNA in the sample is sufficient to be detected but is not found because the mutant DNA is masked by the relatively large amount of wild type in the sample. To provide a method for detecting a mutation. The present invention takes advantage of the unique and surprising properties of MIPC and DMIPC to achieve the goal of detecting mutations in such samples, where wild type and mutants are known.

One aspect of the present invention is a homoduplex and a heteroduplex D having the same base pair length.
A method for determining the presence or absence of homoduplex or heteroduplex fractions in a mixture of NA fragments, wherein the heteroduplex fragment has at least one mismatch site. The method comprises subjecting a mixture of homoduplex and heteroduplex DNA fragments to a separation column containing a separation medium having a non-polar, non-porous surface, the homoduplex and the heteroduplex. The mixture of single-stranded DNA fragments is applied in a first solution containing a DNA binding concentration of counterion and driving solvent.
The counterion and target homoduplex and / or heteroduplex DNA fragments are then separated from the separation medium while maintaining the medium and solution at a temperature that locally denatures the heteroduplex fragment at its mismatch site. A desired homoduplex and / or heteroduplex DNA fragment from the separation medium by contacting the separation medium with a second solution containing a concentration of the driving solvent that has been previously determined to separate in the fractions. To separate.

In detecting mutations, PCR primers are preferably selected to yield a fragment that is capable of undergoing complete separation of the heteroduplex from the homoduplex by MIPC. Details of proper primer selection, August 4, 1998
No. 09 / 129,105, co-pending U.S. Patent Application No. 09 / 129,105, filed Jan. 29, 1994, the entire contents of which are incorporated herein by reference.

In one preferred embodiment, the present invention eliminates any ambiguity regarding the presence or absence of a particular mutant fragment in a sample when the sample contains large amounts of wild-type DNA for a putative mutation. It comprises a number of steps. These steps are described below.

A method for separating homoduplex and heteroduplex DNA fragments from a mixture of homoduplex and heteroduplex DNA fragments by DMIPC is a method of separating a mixture of homoduplex and heteroduplex DNA fragments. Comprising applying to a separation column containing a medium having a non-polar, non-porous surface. The mixture of DNA fragments is run in a first solvent mixture containing the counterion and the DNA binding concentration of the driving solvent in the cosolvent. The desired DNA fragment is separated from the medium by contacting the medium with a second solvent solution containing a counterion and a driving solvent concentration in the cosolvent that has been previously determined to selectively remove the desired fragment. To be done.

The desired fragment can be a homoduplex fraction. The concentration of driving solvent is chosen to remove the homoduplex fraction, and the homoduplex fraction is collected. The collected fractions can be amplified to obtain an increased ratio of heteroduplex to homoduplex. Alternatively, the desired fragment can be a heteroduplex fraction, and the concentration of driving solvent is chosen to remove the heteroduplex fraction, and the heteroduplex fraction is collected.

The method is according to the method of Sklar et al, ie the DNA sample contains a large amount of background wild-type, the mutant DNA is below the detection limit, and the wild-type and mutant DNA It is particularly useful when the DNA sequence is known and the mutant DNA differs from wild-type DNA by at least one base pair.

In these separation methods, the retention time used for separation of the homoduplex or heteroduplex fraction is, for example, by matching ion polynucleotide chromatography, a homoduplex having the same base pair sequence as the sample and Determined from a reference standard that can be obtained by separating a standard mixture of heteroduplexes. The retention times used for separation of homoduplex or heteroduplex fractions in gradient and isocratic separations of DNA mixtures, and in denaturing MIPC methods, can be determined in advance from a reference standard and are highly predictive. It is possible. A reference standard separates a standard mixture of homoduplexes and heteroduplexes having the same base pair sequence as the sample by, for example, matched ion polynucleotide chromatography and identifies the elution time or times of elution of the desired fragment. Can be obtained by the modified MIPC method.

Since the sample wild type DNA and the base sequences of the putative mutations are known, standards for these materials are combined and hybridized. Hybridization involves heating the combined standards to about 90 ° C. and then allowing the reaction to reach ambient temperature for about 45-60.
Provided by slow cooling over minutes. During hybridization, the duplexes in the sample are denatured, that is, separated to form single strands. Upon cooling, these chains recombine. If a mutant strand having at least one base pair difference in sequence from the wild type is present in the sample, the single strands will recombine, producing a mixture of homoduplexes and heteroduplexes. In this way, a standard mixture of homoduplexes and heteroduplexes is produced as shown diagrammatically in FIG. The standard mixture contains the same homoduplexes and heteroduplexes present in the sample containing the putative mutation, but not in the same ratio. This standard mixture contains M under normal conditions because the heteroduplex and homoduplex have the same base pair length.
It cannot be separated by IPC. However, MIPC at temperatures high enough to selectively and partially denature the heteroduplex at sites of base pair mismatches.
(DMIPC), partially denatured heteroduplexes separate from homoduplexes with the same base pair length. Therefore, MIP the hybridized standard mixture.
Apply to C column and perform separation under DMIPC conditions. The chromatogram so produced shows the separation of homoduplexes and heteroduplexes as shown in FIG. The retention times of the separated homoduplex and heteroduplex standards can then be used to predict the retention times of putative mutations that have concentrations that are too low to be detected by the detector. Alternatively, then, the retention times of the separated homoduplex and heteroduplex standards should be used to predict the retention time of putative mutations in samples where the mutation signal is masked by the wild-type signal. You can

Once the standard retention time is determined, PCR is used to amplify the sample containing the putative mutation in order to increase the total amount of sample. Since the sequences are known, the primers can be designed to maximize replication fidelity and minimize reaction artifacts and the formation of byproducts. Methods for primer design and PCR optimization for mutation detection by DMIPC are described in co-pending US patent application Ser. No. 09 / 129,105, filed Aug. 4, 1998. However, the wild type and mutant DNA strands in the sample have almost identical base sequences. Mutations may contain only one base pair difference compared to wild type. Therefore, primers cannot be designed to selectively anneal and preferentially amplify mutant strands in the presence of wild type. Thus, when PCR is used to amplify such a sample, the ratio of mutant to wild type in the amplified product is the same as in the first sample.

Analysis of amplified samples using MIPC reveals a single major peak in the resulting chromatogram. This peak corresponds to the combined wild-type and mutant DNA when mutant DNA is present. Since the mutant and wild type DNA have the same base pair length, no isolation is obtained. Therefore, amplified samples are hybridized and analyzed under conditions that are partially denatured by DMIPC. However, the heteroduplex corresponding to the putative mutation, if present, either has a concentration below the detection limit of the detector, or the wild-type to putative mutation ratio is so high that the wild-type homoduplex is present. The double-stranded peak is not seen by the detector because it either hides the heteroduplex peak.

In any case, the heteroduplex corresponding to the mutant DNA in the original sample need not be seen as the chromatographic peak to be determined. Pre-identification of the retention time of the heteroduplex standard “blinds” the mobile phase from the column at the expected retention time.

The “blindly collected” mobile phase described above is preferably, for example, by evaporation of the mobile phase,
Concentrate. If the mutation was present in the first sample, the residue is now enriched for heteroduplexes. The heteroduplex enriched residue is reamplified by PCR and the products hybridized. The hybridized product of the second round of PCR amplification now contains increased amounts of heteroduplex relative to homoduplex. A chromatogram showing the result of this method is shown in FIG. Evaporation can be accomplished by standard and conventional DNA solution evaporators such as SPEEDVAC evaporator (Model U).
CS 100 Universal Speed Vac System, Savan
t Instruments, Inc, Hayward, CA).

The assembled fragments can be amplified by conventional PCR or cloning and further analyzed by sequencing and restriction digests. If desired, the cycle of separation and amplification can be repeated to increase the amount of the desired fraction available for later analysis.

A process comprising the method of the invention was designed to enrich the sample for heteroduplexes to allow the detection of mutations that normally go undetected.
The steps of the method of the invention can be repeated multiple times to increase the purity and amount of heteroduplex to any desired level. The increased amount of heteroduplex compared to the homoduplex thus obtained can be represented by the "rate of increase".
"Rate of increase" is defined herein as the increase in the ratio of heteroduplex to homoduplex compared to the ratio of heteroduplex to homoduplex in the first hybridized sample, where Where the increase is due to the implementation of the method of the invention. The "rate of increase" depends on the number of iterations performed and can range from 10 to over 1,000.

After the last iteration, PCR products are hybridized and analyzed by DMIPC. If the initial sample contains a mutation, the concentration of the heteroduplex or its concentration relative to wild type is now sufficient for detection. Therefore, D
The MIPC chromatogram shows peaks with retention times for standard heteroduplexes. In this case, it can be clearly concluded that the mutation was present in the first sample.

As a further confirmation of the identity of the mutation, an aliquot of the standard heteroduplex can be mixed with an aliquot of the heteroduplex enriched sample. D of this mixture
The MIPC chromatogram shows an increase in the area of the heteroduplex peak compared to the area of the heteroduplex enriched sample peak alone.

Furthermore, the purification and enrichment methods described above provide sufficient heteroduplexes for their base pair sequencing. Sequencing gives further confirmation of the identity of the mutation.

If no heteroduplex peak is observed in the DMIPC chromatogram after performing multiple iterations according to the method of the invention as described above, it is safe to conclude that the first sample did not contain a mutation. be able to.

Denaturing gradient gel electrophoresis technology capable of separating homoduplexes from heteroduplexes cannot be used as a substitute for DMIPC. Although it is possible to recover the sample from the gel with difficulty, blind mobility is not possible because the mobility of DNA fragments in the gel is not constant. Therefore, the position cannot be predicted with high reliability. Moreover, the shape of DNA fragment bands in gels is often irregular, making sample collection more difficult and detection uncertain. A further problem is that the gel takes a lot of time to develop, making it impractical for routine use.

On the other hand, the highly predictable nature of retention times determined from DMIPC separations makes this method uniquely suitable for mutation detection when blind collection is required. The use of DMIPC for the purpose of mutation detection as described herein has not been previously reported.

Other features of the invention will become apparent during the following description of representative embodiments which are given by way of illustration of the invention and not limitation thereof.

FIG. 7 is a cross-sectional view of the spin vial separation device of the present invention. This system uses a standard laboratory centrifuge to rapidly pass liquid through the separation medium. The system is
A standard cylindrical centrifuge vial or eluent container 142 is used that inserts a separator tube or cylinder 144. The separator cylinder can have a cylindrical body 146, an opening at the top end 148 and a bottom end 150, and is sized to fit within the vial 142. The upper end 148 has an outwardly projecting upper flange 152 that is sized to rest on the upper edge 154 of the cylindrical vial 142. Lower end 150
Has an inwardly projecting lower flange 156 that is sized to support the separation unit 158.

The separation unit comprises a porous support disk 160 mounted on a flange 156,
A separation medium 164 comprises an optional outer cylinder 162 within which is disposed.
The separation unit also includes an optional upper porous disc 1 to prevent destruction of the separation medium.
It may also include 66 and an optional ring 168. The optional ring 168 is preferably of slightly elastic or pliable composition and cylinder 1
It has an outer diameter that is sized to establish a friction interlock with the inner wall of 46. The ring 168, when pressed against the disc 166, holds the disc in place during use of the column.

Separation media is a unique feature of the invention. The surface of the medium must be a non-polar surface and should not contain any traces of metallic contaminants such as polyvalent metal ions. The medium can be in the form of a bead; a monolith; a group of capillaries or a body having parallel capillary passages with one set of ends open to the upper separation chamber 170 and another set of ends open to the lower separation chamber 172. The medium surface can be porous or non-porous. However, in order to provide a quick and accurate separation,
A non-porous medium surface is preferred. Examples of porous media are described in co-pending commonly assigned US patent application Ser. No. 09 / 081,039, filed May 18, 1998. Examples of preferred non-porous media are co-pending commonly assigned US patent application Ser. No. 09 / 183,123 and 1 filed October 30, 1998.
No. 09 / 183,450, filed October 30, 998.

These separation media can be beads or other structures as described above. Preferred separation media are beads made of non-polar materials such as the organic polymers described in US patent application Ser. No. 09 / 183,123, or treated to endblock or coat polar groups. Inorganic polymer beads. The beads of choice are organic polymer beads, such as styrene-divinylbenzene polymer beads, which can have any alkyl substitution described in US patent application Ser. No. 09 / 183,123.

The beads are preferably mono- and di-vinyl substituted aromatic compounds such as styrene, substituted styrenes, alpha-substituted styrenes and divinylbenzenes; acrylates and methacrylates; polypropylene and polyethylenes. Polyolefins; Polyesters; Polyurethanes; Polyamides; Polycarbonates; as well as polymers including substituted polymers that include fluoro-substituted ethylene commonly known under the trademark TEFLON. The base polymer can also be a mixture of polymers, non-limiting examples of which include poly (
Styrene-divinylbenzene) and poly (ethylvinylbenzene-divinylbenzene). The polymer is unsubstituted or 1 to 1,000
It can be replaced by a hydrocarbon such as an alkyl group having 1,000 carbons. In a preferred embodiment, the hydrocarbon is an alkyl group having 1 to 24 carbons. In a more preferred embodiment, the alkyl group has 1-8 carbons.

The separation medium surface should be free of polyvalent metal cations for the most efficient separation.

Separation of materials using the apparatus of FIG. 7 is shown in the examples presented below. Generally, they are carried out by the following sequence of steps.

1. A mixture of single-stranded oligonucleotides, single-stranded DNA fragments, single-stranded DNA fragments, RNA, plasmids, etc. to be separated is diluted in an aqueous solution containing a counterion and a low concentration of a driving solvent.

2. The diluted mixture is placed in chamber 170, and the separator with liquid is placed in a standard laboratory centrifuge and spun until all of the free liquid has transferred to chamber 172. Remove the inner cylinder 144 from the vial and discard the contents of chamber 172. In this step, the substance to be separated is bound to the separation medium.

3. A second aqueous solution containing counterions and a second, more concentrated drive solvent is prepared and placed in chamber 170. The driving solvent concentration is calculated to be the amount that removes all undesired small size material from the separation medium. The exact concentration of the driving solvent is determined from a standard table or curve, or it is calculated using values determined in a calibration of the medium performed with a double-stranded DNA (dsDNA) ladder or standardized enzymatic digestion products. be able to.

4. The separator is spun in a centrifuge until all of the free second solution has been transferred to chamber 172. Remove inner cylinder 144 from vial and remove chamber 17
Remove the contents of 2. This step removes contaminants and small size fragments from the separation medium.

5. A third aqueous solution containing counterions and a third, more concentrated driving solvent is prepared and placed in chamber 170. The third, higher concentration of drive solvent concentration is calculated to be the amount that removes the desired large size material from the separation medium. The exact concentration of driving solvent can be determined from standard tables or curves, or double-stranded DNA (d
It can be calculated using the values determined in the calibration of the medium carried out with sDNA) ladders or standardized enzymatic digestion products.

6. The separator is spun in a centrifuge until all of the free third solution has been transferred to chamber 172. Inner cylinder 144 is removed from the vial and the contents of chamber 172 containing the desired large size fraction or fractions are removed for further processing. This step removes the desired material from the separation medium.

Clearly, the vial 142 can be replaced or washed between steps to prevent contamination of the product fraction or fractions.

The concentration of the driving solvent in the third solution can be selected to remove a single fragment size or a series of fragment sizes.

Steps (5) and (6) can be repeated with successive higher concentrations of solvent to remove a series of successively increasing fragment sizes.

It will be readily apparent to those skilled in the art that other variations can be adapted to remove a series of purified fractions in much the same way as shown above and illustrated in the examples and figures of the present application.

Examples of suitable organic stripping solvents are alcohols, nitrites, dimethylformamide, tetrahydrofuran, esters, ethers and mixtures of one or more thereof, such as methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran, acetic acid. Ethyl, acetonitrile are included. The most preferred organic solvent is acetonitrile. The counterion factor is preferably the group consisting of lower alkyl primary amines, lower alkyl secondary amines, lower alkyl tertiary amines, lower trialkylammonium salts, quaternary ammonium salts and mixtures of one or more thereof. Selected from. Non-limiting examples of counterion factors include octyl ammonium acetate, octadimethyl ammonium acetate, decyl ammonium acetate,
Octadecyl ammonium acetate, pyridinium ammonium acetate,
Cyclohexyl ammonium acetate, diethyl ammonium acetate, propyl ethyl ammonium acetate, propyl diethyl ammonium acetate, butyl ethyl ammonium acetate, methylhexyl ammonium acetate, tetramethyl ammonium acetate, tetrapropyl ammonium acetate, tetrabutyl ammonium acetate, dimethyl diethyl ammonium acetate, triethyl ammonium Acetate, tripropylammonium acetate, tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate and mixtures of any one or more of the above.

Examples of suitable counterion agents are anions such as acetates, carbonates, bicarbonates, phosphates, sulphates, nitrates, propionates,
Contains formate, chloride, perchlorate or bromide. The most preferred counterion agent is triethylammonium acetate or triethylammonium hexafluoroisopropyl alcohol.

FIG. 8 is a sectional view of the vacuum tray separating apparatus of the present invention, and FIG. 9 is a top view of the separating tray of FIG. Separation tray 200 is a single plate having rows and columns of tubular separation grooves 202, preferably with a constant repeating spacing between rows and columns to indicate spacing. The dimensions of the tray 200 and separation groove can correspond to and match the dimensions of a standard multi-well plate such as a 96-well microtiter plate.

The multi-groove plate 200 is supported on a support flange and vacuum seal 204 created in the lumen of the top plate 206 of the vacuum assembly 207. Vacuum assembly 2
07 further comprises a vacuum cavity 208 identified by housing 210.
The top plate 206 is placed on the housing 210 by locating the pins 212, and the top plate 206 and the housing 210 have a sealing engagement with the seal 204. The housing 210 has a vacuum chamber 208 and an exhaust outlet groove 214 leading to a vacuum conduit 216 and a vacuum valve 218. The vacuum conduit 216 and vacuum valve 218 lead to a vacuum source (not shown).

The multi-well collection plate 220 is supported in the vacuum chamber 208. The multi-well collection plate 220 is a single plate having rows and columns of separation grooves 222, preferably with a constant repeating spacing between rows and columns to indicate spacing. The dimensions of the tray 220 and collection groove correspond and can be matched to those of standard multi-well plates such as 96-well microtiter plates. The collection plates 220 are kept in alignment with each of the collection wells 222 in alignment with the corresponding separation groove 202 of the separation plate 200 so that each well 222 can collect liquid that falls from the corresponding separation groove 202.

FIG. 10 is a cross-sectional view of the separation tray of FIG. 9 taken along line AA. Separation groove 2
02 are respectively uniformly spaced upper cavity 224, separation medium 226 and liquid outlet 2.
28.

FIG. 11 is an enlarged view of the separated components of the separation tray of FIG. The bottom of the separation cavity 224 carries the porous disk 230, which in turn carries the separation medium 232. An optional containment disc 234 rests on the separation medium 232, and the containment disc 234 can optionally be held in place by a friction ring 236 or equivalent device.

Separation medium 232 can be the same non-polar medium as described above for medium 122 in FIG.

Separation vial components 142 and 146 of FIG. 7 and plates 200 and 220 of FIGS. 8-11 are made of materials that do not interfere with the separation process, such as polystyrene, polypropylene or polycarbonate. The top plate 206 and housing 210 can be made of any material that has the required strength, such as hard organic polymers, aluminum, stainless steel, and the like. The vacuum chamber walls are preferably covered with Teflon film. Vacuum conduits and valves can also be made of Teflon coated aluminum or the like.

Separation of materials using the apparatus of Figures 8-11 is performed by the following sequence of steps.

1) a single-stranded oligonucleotide containing components that separate from the mixture that separates,
A mixture of single-stranded DNA fragments, single-stranded DNA fragments, RNA, plasmids, etc. is diluted in an aqueous solution containing counterions and a low concentration of driving solvent.

2) Place the diluted mixture in one of the fully assembled vacuum chambers 202. The other chamber 202 contains another mixture which is separated by the same method.

3) Apply vacuum to vacuum chamber 208 by opening vacuum valve 218 until all of the liquid from the mixture contained in each chamber has collected in chamber 222. The vacuum device is disassembled and the contents of chamber 222 are discarded. In this step, the material to be separated binds to the separation medium 232 in each chamber 202.

4) Reassemble the vacuum apparatus and plate and prepare a second aqueous solution containing counterions and a second, more concentrated drive solution and place in chamber 202. The driving solvent concentration is calculated to be the amount that removes all low molecular weight material from the separation medium. The exact concentration of the driving solvent is determined from a standard table or curve or calculated using values determined in a calibration of the medium performed with a double stranded DNA (dsDNA) ladder or standardized enzymatic digestion products. can do.

5) Apply vacuum to vacuum chamber 208 by opening vacuum valve 218 until all of the liquid from the mixture contained in each chamber has collected in chamber 222. The vacuum system is disassembled and the contents of chamber 222 are removed. In this step, low molecular weight and small size material is removed from the column and collected, and large base pair length material remains on the separation medium 232.

6) Reassemble the vacuum apparatus and plate and prepare a third aqueous solution containing counterions and a second, higher concentration of driving solvent and place in chamber 202.
The driving solvent concentration is calculated to be the amount that removes the desired large size substance or substances from the separation medium. The exact concentration of the driving solvent is determined from a standard table or curve or calculated using values determined in a calibration of the medium performed with a double stranded DNA (dsDNA) ladder or standardized enzymatic digestion products. can do.

7) Vacuum valve 218 until liquid from the mixture contained in each chamber collects in chamber 222.
A vacuum is applied to the vacuum chamber 208 by opening. Disassemble the vacuum device and chamber 2
Take out the contents of 22. Remove large size material from chamber and collect.

The concentration of the driving solvent in the third solution can be selected to remove a single fragment size or a series of fragment sizes. This method can be elaborated by selecting a driving solvent concentration in a third aqueous solution that removes all substances below the target size and then repeatedly repeating the driving solvent concentration to remove substances of a specific size of the target size. it can.

Obviously, the plate 220 can be changed between steps or washed between steps to prevent contamination of the product fraction or fractions.

These steps can be repeated with successive higher concentrations of solvent to remove a series of successively increasing fragment sizes.

It will be readily apparent to those skilled in the art that other variations can be adapted to remove a series of purified fractions in much the same way as shown above and illustrated in the examples and figures of the present application.

In MIPC, fragments are foreign components (ie, fluorescent dyes, biotin, etc.).
It is unrivaledly suitable for the method of purifying nucleic acids, whether labeled with or without. Adapting MIPC conditions to isolate fragments based on size, sequence or both gives the highest possible purity for nucleic acids. This level of performance is obtained by tight control over chemical / eluent conditions, as well as by using appropriate matrix materials. Separations performed using the apparatus and methods of the present invention are more suitable for cloning than those obtainable by gel electrophoresis.
NA fragments can be generated, eg, Hecker, Karl
H. et al, "Optimization of cloning eff"
icacy by pre-cloning DNA fragment an
alysis ", Biotechniques, 26 (6): 216-222 (
(February 1999).

Since MIPC is a chemical method, nucleic acid purification can be performed at any pressure. This is particularly useful for size-based purification of PCR products and restriction digests at ambient pressure as shown in Examples 3 and 4 presented below and Figures 22-27. It is based on the size of the PCR product at high pressure before sequencing as shown in Example 1 and Figures 12-21 below (Marino, et a.
1, Electrophoresis, 1998, 19, 108-118) and for sequence-based purification as well. It can also be applied to size-based purification of plasmid restriction digests prior to cloning. US Provisional Application No. 60/1, filed 4/23/99, incorporated herein in its entirety
See No. 30,700.

The general method for obtaining purified nucleic acids does not depend on pressure conditions. Whether operating at high or low pressure, purification features the following steps: Capturing the nucleic acid (s) of interest on a DNA chromatography matrix under appropriate DNA chromatography conditions.

2. Exposing the captured nucleic acid (s) to chemical and / or thermal conditions that quantitatively liberate components selected for removal.

[0169]   3. Collecting the removed components of interest for later use (optional).

[0170]   4. Detecting removed components (optional).

5. Exposing the remaining captured nucleic acid (s) to chemical and / or thermal conditions that quantitatively liberate the remaining nucleic acid (s).

[0172]   6. Collecting the liberated nucleic acid (s) of interest (optional).

[0173]   7. Detecting the liberated nucleic acid (s) of interest (optional).

The following are examples of the application of DNA chromatography to nucleic acid purification: It is known that the current technology used for PCR purification has serious problems. One of the single biggest problems with this application of current technology is the incomplete removal of primers, primer dimers or other non-specific amplification products. These "background" components confuse many subsequent molecular biology tasks (such as sequencing and cloning, to name but a few) by competition. As one example, these problems are particularly exacerbated in sequencing when using the industry standard method of dye-labeled terminators in cycle sequencing. When using dye-labeled terminators, purification of PCR-generated templates must give exceptionally high template recovery and template purity. This is due to potentially interfering contaminants (amplification primers and primer dimers, non-specific amplification products, dNTPs, Taq) that do not always occur with current silica-based technologies.
) Must be completely removed.

Since it can lead to the cloning of unwanted fragments,
Other later problems also occur when cloning. These cloning problems are described in Hecker, Karl H. et al. et al, outlined above, the entire contents of which are incorporated by reference.

The purification method utilized by the major producers of PCR removal technology is based on the adsorption of nucleic acids (ssDNA, dsDNA, RNA) on silica gel. Once silica gel is treated with a high salt solution, nucleic acids adsorb to the silica gel by an exchange mechanism. The nucleic acid is then desorbed by adding a low salt eluent. This works very well with clean DNA, but has problems with other components. The chemical processes that occur on silica gel surfaces are not tightly controlled and therefore rely on radical changes occurring uniformly across the particle surface. It is well known that there is no efficient primer, primer dimer and removal of non-specific amplification products. Example 5 illustrates the removal of PCR byproducts. In addition, the (typically) porous surface of silica gel is able to capture the contaminants most in need of removal.

The materials currently used for the MIPC column matrix of the present invention, as well as other materials suitable for MIPC, such as larger polymer particle size non-porous reverse phase materials, are exceptional for long nucleic acids. It is known to have high capacity and selectivity. Quantitative analysis of short and long nucleic acids of various lengths by using the appropriate counterion ion and then changing nothing but the acetonitrile concentration (or any other suitable solvent such as alcohol). Adsorption / desorption essentially starts and stops. In addition, since the matrix is made of a non-porous polymeric material, infiltrant (dNTPs)
, Primers, primer dimers, non-specific amplification products) and there is no chance of later becoming a problem. In essence, our matrix material (either non-alkylated or alkylated, non-porous polystyrene-divinylbenzene) is one of the predominant PCR products for most demanding molecular biology applications. Perfectly suitable for purification. Also suitable are non-porous polymeric or modified silica materials that have been manufactured or purified to yield a contaminant-free surface. These can be in the form of beads, monoliths, grooves, capillaries or planes. The polymer surface can be provided by non-alkylated and alkylated materials including polystyrene, divinylbenzene, hydroxyethylmethacrylate and other nonionic polymers. Polymers with a negative charge can also be used if the charged groups are protonated to give a neutral surface, ie a carboxylic acid. This example is illustrated by Example 1. When attempting size-based cloning from a pool of fragments (cDNA fragment pool, fragment pool from restriction digests of plasmids / artificial chromosomes / genomic DNA, etc.), the efficiency of fragment ligation into the vector (and thus cloning efficiency) is Biased for smaller fragments. As an extension of the same principles applied to the purification of PCR products, fragment pool fractionation can be performed on the kit. The fragment pool was smeared and expanded on the gel by adding increasing concentrations of acetonitrile and analyzing the collected fractions with an appropriate 260/280 detector and a safe razor blade to reach the desired size range. It provides a very fast method that effectively replaces thin sectioning. This general method is
It has been shown for high pressure sizing of the fragment prior to cloning and is directly in the low pressure form.

Clones (this particularly applies to minipreps of < 20 μg of plasmid)
Of many silica-based and anion exchange-based purification kits are known to have difficulties in purification from TB (rich medium) due to the high copy number of plasmids present (relative to LB medium). Is. We recognize that the DNA chromatography matrix has a higher capacity than typical silica gel based preparations (ie QiaGen minipreps). Moreover, because our separation mechanism is homologous to anion exchange, the purity obtained by DNA chromatography is comparable to that of the competition's finest (and significantly more expensive) "ultra pure" anion exchange products. It is highly likely that it will be equal to or more than, and thus represents a significant cost savings at the same time. The following is the protocol for this method.

First, plasmid DNA is released from E. Coli (or ssDNA from M13 phage). Alkaline lysis of Escherichia coli is a typical first step in the isolation of plasmids from Escherichia coli. To do this, the cells are centrifuged and they are supernatant / growth medium (Luria-
Separate from either Bertani (also known as LB) or rich medium (also known as TB). Once pelleted, cells are resuspended (usually with glucose / EDTA / Tris, aka GET) and then lysed with NaOH / SDS. This avoids the introduction of smaller fragments of sheared chromosomal DNA,
Must be done gently. The resulting solution should be fairly clear and viscous. The solution is neutralized with a suitable buffer (usually potassium acetate), thus producing a white precipitate containing cell debris, SDS, chromosomal DNA and some proteins. The plasmid DNA remains in solution (along with various soluble lipids, various proteins, carbohydrates, salts, RNA). The lysate solution is typically clarified by centrifugation. Plasmid or ss
This clarified lysate containing DNA is then introduced into a DNA chromatography matrix.

Once introduced into the matrix, the washing method is performed. This usually means that T with a higher concentration of acetonitrile (ie 15% acetonitrile)
It is an EAA solution. This should even remove most of the hydrophobic contaminants and leave the plasmid intact on the resin. Once washed with this solution, the plasmid is eluted with a minimal amount of elution buffer (25% acetonitrile, higher for larger plasmids). In the case of ssDNA from M13 phage, it may be necessary to have different alkylammonium ion pairing reagents to maintain size-based separation. The collected plasmid / M13 phage (optionally solvent can be removed by evaporation) is then available for any subsequent use.

These are very large DNA constructs (typically> 100 kb) that often have to be purified for a number of reasons (sequencing, subcloning, etc.).
Is. Note that there are a vast number of methods available to release these constructs from their respective media (ie bacteria, yeast, other cells). In any case, once the constructs were present in the clarified lysate medium (much the same as the plasmids and M13 phages described above), appropriate amounts of ion-pairing reagents were added so that they could be captured on the DNA chromatography matrix. You have to add to them. Once captured, a wash solution with an appropriate concentration of organic solvent (acetonitrile, alcohol, etc.) to remove contaminating species is added, leaving the larger DNA construct on the matrix. Since these constructs are fairly large, they often require fairly high concentrations of acetonitrile (or other suitable solvent) to release them from the matrix. Once they are assembled (optionally the solvent can be removed by evaporation), the construct is available for any subsequent use.

MIPC is well suited for the purification of oligonucleotides and longer probes, both of which are ssDNA constructs. Oligonucleotides and probes (either labeled or unlabeled) are ordered prior to use, directly after in-house synthesis and from external suppliers (and are believed to have undergone these particular purification criteria). In some cases, quality control and / or purification is required. As detailed in Examples 1 and 6, these purifications can be carried out in high pressure form.

It is likewise possible to carry out these analyzes in low-pressure form. As with the nucleic acids above, the ssDNA construct is combined with an appropriate ion-pairing reagent at an appropriate concentration to provide discrimination based on DNA size.

The standard means of genotyping involves the use of primer extension. Anneal the oligonucleotides adjacent to the 5'region of interest on the template. Nucleotides are added in standard proportions except that one of them is a terminating dideoxynucleotide (ddNTP). A suitable high fidelity polymerase such as Pfu is added and the nucleotides are added onto the template, effectively extending the primer to the point where ddNTP is incorporated and thus stops the primer extension step. Therefore, the length of the obtained extension product indicates the identity of the extension-terminated base. This extension can occur for only one base or can continue until a base complementary to the terminator is encountered. In either case, the extended primer can then be denatured from the template, and then the extended primer can be sized to determine the correct genotype of the template.

In the context of DNA chromatography, it is possible to use temperature (> 75 ° C.) to completely denature the extended primer from its template. When introduced into a preheated matrix, the (ion-paired) template and (ion-paired) extended primer selectively desorb only the (significantly short) primer and primer extension product (probably for collection), The first template remains adsorbed on the surface until the appropriate concentration of organic solvent (acetonitrile, alcohol, etc.) is left on the matrix. This is an example where the smaller product is collected first and the larger (unwanted) product is left behind.

Once the primer extension products have been selectively released, they can be sized directly on the DNA chromatography matrix (H
oooooooooooooo. , Et al "Genotyping single
nucleotide polymorphisms by primer
extension and high performance liqui
d chromatography ", Human Genetics, 199.
9, Vol. 104, No. 1, pp. 89-93). When collecting purified primer extension products, they can also be sized by other means such as gel electrophoresis or mass spectrometry.

When applying the above purification method, it is possible to detect the purified product by either UV absorbance or fluorescence intensity. In the case of high pressure or medium pressure purification, which is the usual liquid chromatographic form, this detection is on-line with a suitable detector (which is described in detail elsewhere). If these steps are in low pressure form, it is also possible to detect the purified product in online form. However, a simple means of detecting the purified product is the off-line form (either in a cuvette-based system or by a well-designed well-plate reader).

Regardless of the detection means described above, an alternative to gel-based purification has been provided where nucleic acids are detected separately after purification. As an illustration of this concept, an example is given below for the detection of (low pressure) purified PCR products prior to dye-terminator sequencing.

Once the PCR product has been purified and collected in appropriate well-plates, it requires spectroscopic analysis. The reason for this is twofold: successful DNA sequencing (especially the dye terminator cycle sequencing method) requires considerable control over the amount of DNA introduced for sequencing; ensuring that there are no competing enzymatic reactions. For this, the amount of protein impurities must be measured. In order to make these measurements, purified DNA must be measured by absorbance at 260 nm (for DNA measurements) and 280 nm (for protein measurements). This can be done by collecting the eluted DNA in UV transparent well-plates. Must ensure that there is no photometric "interference" between wells,
And the path length is "tunable" to reflect different eluent volumes. The spectrophotometric requirements at each wavelength are very important. When operating at maximum DNA volume, 10 μg DNA / 50 μL = 200 ng / μL; when operating under typical conditions: ˜1 μg / 50 μL = 40 ng / μL. 0.2 cm (polyfi
A nominal path length of 50 μL on the ltronics' UVMAX plate can be assumed. Absorbance of 1.0 (A2 at 260 nm with a path length of 1.0 cm).
60 = 1) ≡ 50 ng / μL DNA. 50ng at ∴0.2cm path length
A260 = 0.2 for μL DNA. If the above conditions are true, the following absorbance criteria (background subtracted) is A260 at a path length of 0.20 cm.
It must be achievable in the measurement: Maximum (10 μg DNA / 50 μL = 200 ng / μL): A260 = 0.
8 typical (1 μg DNA / 50 μL = 20 ng / μL): A260 = 0.
08 Low limit (50 ng DNA / 50 μL = 1 ng / μL): A260 = 0.
For the purposes of quantifying protein "quantification" and DNA purity, it is necessary to make measurements at 280 nm. The ratio of A260 / A280 measurements is used for this work. The above absorbance standard as a standard, and A260 for a typical nucleotide sequencing reaction
With the knowledge that the / A280 ratio must be above 1.8, the following must be achievable at 280 nm.

Maximum (200 ng / μL): A280 = 0.440 Typical (20 ng / μL): A280 = 0.044 Low limit (1 ng / μL): A280 = 0.022 DNA or RNA test strand (or There are numerous methods within molecular biology that require complementary (or near complementary) hybridization of a probe (or probes) to multiple strands. Once the probe (or probes) have hybridized, the hybridization event is then detected by a number of means. In order to detect the hybridization event, it is possible to examine the signal from the reporter component bound to the probe (radioactive probe, fluorescent probe, etc.), the mobility change in the gel electrophoresis experiment, and the like.

It is also possible to detect hybridization events directly by using DNA chromatography. Once the probe probe event occurs and any other steps are followed (ie extension of the annealed primer,
Denaturing the resulting single-stranded structure (which is smaller than the template it is examining) directly from the template, and removing it online (ie, by removing the probe for analysis).
UV absorbance, fluorescence, mass spectrometry) or off-line method (ie MALDI)
-TOF-MS). The following are examples of these types of operations and analyses. Note that all the methods described herein are chemical methods and can be carried out in a high pressure or low pressure environment. Furthermore, all of these methods can be performed on any scale, including micro or nano scales.

A standard means of performing genotyping involves the use of primer extension. Mold (DN
Anneal the oligonucleotides adjacent to the 5'region of interest on either A or RNA). Nucleotides are added in standard proportions, except that one of them is a terminating dideoxynucleotide (ddNTP). Appropriate high fidelity polymerase (ie Pfu) is added and nucleotides are added one by one onto the template, effectively extending the primer to the point where ddNTP is incorporated and thus terminates the primer extension step. Therefore, the length of the obtained extension product indicates the identity of the extension-terminated base. This extension can occur for only one base or can continue until a base complementary to the terminator is encountered. In either case, the extended primer can then be denatured from the template, and then the extended primer can be sized to determine the correct genotype of the template.

In the context of DNA chromatography, it is possible to use temperature (ie> 75 ° C.) or other means to completely denature the extended primer from its corresponding template (Hecker, Karl H). , Et al, supra).
For example, when introduced into a preheated matrix, the (ion-paired) template and the (ion-paired) extended primer selectively desorb only the (significantly short) primer and the primer extension product, and thus the initial template. It remains adsorbed on the surface until an appropriate concentration of organic solvent (acetonitrile, alcohol, etc.) that remains on the matrix is introduced. This is the smaller product first purified and collected,
And an example of leaving behind a larger (unwanted) product.

It is also possible to analyze these extension products directly online by DNA chromatography. By applying the appropriate chemical conditions for the correct sizing of the ssDNA, the extension product is eluted so as to give information about where the extension step was terminated and thus indicate the identity of the terminated nucleotide. For example, 18
The mer primer is annealed (underlined) 5'of the mutant site (assuming the bold mutant is either C or T) and the terminating nucleotide added is ddGTP. Possible variants are given below as examples.

Variant 1: GTCATCGAATCCCATGCTA CAC Variant 2: GTCATCGAATCCCATGCTA TAC Once primer extension occurs, the product is 19 bases long (for C mutants) or 21 bases long (for T mutants: The extension stops when the next C nucleotide is encountered).

If the subject is homozygous for variant 1, only one peak (at 19 mer) is detected. If they are homozygous for variant 2, only one peak (of 21 mer) is detected. If they are heterogeneous, two peaks (19mer and 21mer) are detected. In practice, by applying denaturing conditions and appropriate ssDNA sizing procedures, all of these determinations can be made to be automated. Moreover, since DNA chromatography is a quantitative method, this makes it directly to genotyping so that "allelic counts" can easily be found when studying population genetics.

A further description of these is given in Table A, where (C) represents the single nucleotide polymorphism (SNP) to be detected (C rather than A).

[0198]

[Table 1]

It is also possible to analyze these extension products directly by a suitable “real-time” mass spectrometric detection scheme, which detects these species as they emerge from the DNA chromatography system. This is applicable if all of the possible terminating nucleotides (ddATP, ddCTP, ddGTP, ddTTP) are added at once, thus giving a mass difference between the eluted extension products. However, this allows the mutant nucleotides to be identified directly in one step. Using the example above, if the subject is heterozygous (mutant 1 and mutant 2 are present), two 19mer primer extension products will result. However, it

[0200]

[Outer 1]

Mass spectrometer is "offline" (MALDI-TOF) or "online"
The ability to size fragments prior to mass spectrometric analysis, whether (electrospray), is very important, especially when trying to perform more than one primer extension at a time (ie multiplex). Useful for. A large number of primers (and their respective extension products) were analyzed by mass spectrometry, and if the target product requiring determination was a 20mer, the 18mer to 22mer overlap with the theoretical mass of the 20mer product. It is theoretically possible to have a mass. Therefore, separating the primer extension products based on size prior to their analysis by mass spectrometry results in a real-time analysis of the data, thus further simplifying interpretation.

[0202]

[Table 2]

The probe may be used to probe a site within the template (DNA or RNA), and this probing event is monitored by a number of means.
This is typically done by sizing the fragments on a gel and then examining the resulting separation with a radioactive probe, "Southern" blotting for DNA and Northern blotting for RNA.

There are other probes available that can be analyzed once removed from the original template. By way of example, some of these probes carry an exogenous "mass label" which helps identify the probe once it has been cleaved off (usually by photolysis).

However, in order to detect this “mass label” efficiently, the probe to which it is bound must first be released from the template (PCT application No. WO).
No. 9905322, WO 9905321, WO 9905320, WO 9905319, WO 9727327, WO 9727325, WO 9905308, WO 9904896, WO 972733.
No. 1, all contents above are incorporated by reference). This can be done in much the same way as shown above to release the primer extension product. Amplification products are denatured and probed with "mass labeled" probes. These duplexes were introduced into the DNA chromatography matrix under highly denaturing conditions (ie> 75 ° C.), so that the probe and the corresponding template were separated from each other and the top of the DNA chromatography matrix. Held in. Under appropriate sizing conditions for ssDNA, the probe is then selectively eluted from the DNA chromatography matrix. In this case, instead of directing the probe directly to the mass spectrometer (as in the case of primer extension products), the "mass label" is cleaved from the probe and then for detection (and thus detection of the probe probe event) To the mass spectrometer. This is done by giving another dimension that can simultaneously give selectivity, namely the dimension of the probe length.
The detection of "mass labels" can be further simplified.

The present invention is further illustrated by the following specific but non-limiting examples, wherein
The past tense description of the method represents a completed scientific experiment. The tense description is not currently being carried out in the laboratory, and is construed here for interpretation to be practiced according to the application of the present application.

Example 1 Size Fractionation of DNA Restriction Fragments in the MIPC DNA Fragment Analysis System One application of the WAVE ^™ system is the accurate and rapid sizing of DNA fragments. This feature can be used to isolate DNA fragments.
Described herein are methods and buffer gradients used for size fractionation and isolation of restriction fragments generated by digestion of pUC18 with HaeIII. The nine isolated fragments range in size from 80 to 587 base pairs.

Liquid chromatography (LC) is a powerful technique for nucleic acid separation due to its high resolution, short analysis time, and ease of recovery of DNA fragments for later studies. The WAVE ^™ system combines the accuracy of ion-pair reversed phase LC with automatic sampling, data acquisition and reporting functions. Fragments can be easily assembled and used in subsequent experiments such as subcloning, amplification and sequencing.

[0209]

[Table 3]

Table D shows octadecylalkyl substituted styrene-divinylbenzene copolymer beads (DNASep ^R column, Transgenomic, Inc., O.
3 shows fragment sizing of pUC18 plasmid cut with HaeIII using the protocol in Table C on a 4.6 × 50 mm column containing maha, NE). A total of 9.96 μg of digested DNA was injected in a volume of 20 μl. Table D shows the amount of DNA per fragment based on an injection of 9.96 μg. Fractions were collected manually.

[0211]

[Table 4]

Peaks corresponding to the 80, 102, 174, 298 and 587 bp fragments were collected in a volume of 200 μl. The 257/267 fragments that differ only by 10 bp elute within less than 20 seconds of each other, as do the 434/458 bp fragment. To achieve separation of these peaks, the fraction size should be 1
It was lowered to 00 μl. Fractions were dry concentrated after collection and resuspended in 30 μl TE buffer (10 mM Tris, 1 mM EDTA), 25 μl of which were reinjected for analysis using the protocol in Table C. The fragment sizes and the corresponding retention times are listed in Table E.

[0213]

[Table 5]

The results obtained are shown in Figures 12 to 21, where the drawing numbers and base pair lengths are shown in Table F.

[0215]

[Table 6]

DNA fragments ranging in size from 80 to 587 bp were isolated and recovered as shown in FIGS. 80, 102, 174, 257, 298, 4
The 34 and 587 bp DNA fragments were completely purified without contamination with any other fragments of the restriction digest. 267 bp fragment and 4
Only the isolation of the 58 bp fragment showed minor contamination with the corresponding shorter fragments, preceded by the fragments in the 257 and 434 bp digestion products, respectively.

Example 2 Preparation of Spin Columns 50 mg of resin is added to each spin column (see schematic diagram of column in FIG. 7) while applying vacuum on the column. The side of the column was tapped to remove the resin from the wall. The polyethylene filter was placed on top of the resin in each vial, followed by the retaining ring, while gently tapping with a hammer to firmly position the retaining ring against the filter. 50% acetonitrile (ACN) and 0
． The spin column was washed with an aqueous solution containing 1M triethylammonium acetate (TEAA). The vial was then washed with an aqueous solution of 25% ACN and 0.1M TEAA, followed by an aqueous solution of 0.1M TEAA.

Example 3 Separation of pUC18 MspI on a Spin Column A sample solution was prepared by diluting 35 ml of stock pUC18 MspI to 1 ml with 0.1 M TEAA (1 ml total volume). A 400 ml aliquot (corresponding to 6.6 mg loaded on the column) was selected. The WAVE separation system (Transgenomic, Inc., Omaha) described in FIGS.
, NE) was used to perform the base pair length separation of the solution, and the chromatograms obtained on two of the columns are shown in FIG.

Pipet aliquots of the sample solution into separate spin columns and
Let stand for a minute. Each vial was centrifuged at 5000 rpm for 5 minutes. Next, 9
． Pipet 400 μl of freshly prepared aqueous solution containing 5% ACN and 0.1 M TEAA into each spin column, allow each vial to sit for 5 minutes, centrifuge each vial at 5000 rpm for 5 minutes, and WAVE separation system. Was used to analyze the filtrate. The chromatogram obtained for the eluent is shown in Figure 23 for the pUC18 MspI standard. The treatment method was repeated.

The above method was repeated by replacing the 38% B solution with 100 μl of 100% B solution. A chromatogram from the analysis of the eluent on the WAVE separation system is shown in Figure 24 for the pUC18 MspI standard.

This example demonstrates the removal of small size fragments from a column while retaining the large size fragments on the column, and the subsequent removal of large size fragments from the column. This is particularly useful for purifying large size fragments or multiple fragments from small size contaminants.

Example 4 Separation of pBR322 HaeIII on a Spin Column 18 ml of stock pBR322 HaeIII digested product was added to 0.1M TEAA.
A sample solution was prepared by diluting to 1 ml with (1 ml total volume). Four
An aliquot of 00 ml (corresponding to 6.6 mg loaded on the column) was selected.
The WAVE separation system (Transgenomic, Inc. described in FIGS.
． , Omaha, NE) and base-length separation of the solution was performed, and the chromatograms obtained on two of the columns are shown in FIG.
Shown in.

Pipet an aliquot of the sample solution into a separate spin column and
Let stand for a minute. Each vial was centrifuged at 5000 rpm for 5 minutes. Then 3
Containing 8% B (B is 25% ACN in water, 0.1M TEAA) 4
00 μl of freshly prepared aqueous solution was pipetted into each spin column, each vial was allowed to sit for 5 minutes, each vial was centrifuged at 5000 rpm for 5 minutes, and the filtrate was analyzed using the WAVE separation system. The chromatogram obtained for the eluent is shown in Figure 26 for the pBR322 HaeIII standard. The treatment method was repeated.

The above method was repeated substituting 100 μl of 100% B solution for the 38% B solution. The chromatogram obtained by analyzing the eluent with the WAVE separation system is
FIG. 27 shows the 322 HaeIII standard.

This example demonstrates the removal of small size fragments from a column while retaining the large size fragments on the column, and the subsequent removal of large size fragments from the column. This is particularly useful for purifying large size fragments or multiple fragments from small size contaminants.

Example 5 Purification of PCR Products on Spin Columns 100 μl 0.2M TEAA is pipetted onto the spin column and 100 μl PCR amplified 200 bp fragment (p53 exon 6 genomic DNA) is pipetted on. To prepare a sample solution. Figure 1-
WAVE separation system (Transgenomic, Inc., O.
Separation of the solution was carried out using (Maha, NE) and is shown in FIG.

Pipet an aliquot of the sample solution into a separate spin column and
Let stand for a minute. Each vial was centrifuged at 5000 rpm for 5 minutes. Then 3
Containing 8% B (B is 25% ACN in water, 0.1M TEAA) 4
00 μl of freshly prepared aqueous solution was pipetted into each spin column, each vial was allowed to sit for 2 minutes, each vial was centrifuged at 5000 rpm for 5 minutes, and the filtrate was analyzed using the WAVE separation system. The treatment method was repeated.

The above method was repeated substituting 100 μl of 100% B solution for the 38% B solution. 200 chromatograms obtained by analyzing the eluent with a WAVE separation system
The bp fragment is shown in FIG. FIG. 29 shows 97.9% purified P
Recovery of CR products and removal of greater than 99.2% byproducts are shown.

This example shows that this method can elute PCR products with high recovery and remove PCR byproducts almost completely.

Example 6 Separation of Labeled Oligonucleotides of Same Length by MIPC A 200 bp fragment (plitm) using one biotinylated primer.
was injected onto a MIPC column and eluted under gradient conditions to produce a chromatogram as shown in FIG. The WAVE separation system (T
transgenomic, Inc. , Omaha, NE). Mobile phase is component A, 0.1M triethylammonium acetate (TEAA)
And component B, 0.1 M TEAA, 25% acetonitrile. A linear gradient was used for the separation, starting with 30% component B and reaching 70% component B in 12 minutes at 75 ° C. The column is DNASep ^™ (Transgenomic, Inc. S).
an Jose, CA).

In FIG. 30, biotinylated oligonucleotides are eluted later than non-biotinylated oligonucleotides. The greater retention time is due to the hydrophobicity of the biotin component. Thus, under denaturing conditions, 200 nucleotide ssDNA species can be separated from biotinylated ssDNA species of the same length.

Example 7 Kinematic Separation of Homoduplex and Heteroduplex by MIPC A 209 bp fragment from the human Y chromosome locus DYS271 having an A → G mutation at position 168 is chromatographed by MIPC, and The results are substantially similar to those seen in Figure 31.

The MIPC analysis conditions using the WAVE ^™ DNA Fragment Analysis System are as follows: Component A: 0.1M TEAA, Component B: 0.1M TEAA, 25%.
Acetonitrile; Flow rate: 0.9 mL / min; Temperature: 56 ° C; Detection: UV, 254n
M. The gradient used is as follows:

[0234]

[Table 7]

[0235]   The heteroduplex elutes before the homoduplex.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. Therefore, within the scope of the appended claims, it should be understood that the invention can be practiced otherwise than as specifically described herein.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a high pressure system for carrying out the Matched Ion Pair Chromatography (MIPC) method of the present invention having a proportional valve system that provides a gradient of solvent concentration in the separation.

FIG. 2 is a partial schematic diagram corresponding to the representation of FIG. 1, but with a proportional pump system that provides a gradient of solvent concentration in the separation system with a proportional valve.

FIG. 3 is a cross-sectional view of a high pressure separation column suitable for use in the apparatus of FIGS.

FIG. 4 is a schematic representation of the hybridization of homomutant strands with wild-type DNA strands showing the production of two homoduplexes and two heteroduplexes.

[Figure 5]   5 is a DMIPC chromatogram showing the separation of the standard mixture of FIG.

FIG. 6 shows a DMIPC chromatogram showing mutation detection using the blind target compartment elution method.

[Figure 7]   1 is a cross-sectional view of a spin vial system for low pressure separation according to the present invention.

[Figure 8]   Figure 3 is a multi-well plate separation system of the present invention in combination with a vacuum accessory.

[Figure 9]   FIG. 9 is a top view of the multi-well plate of FIG. 8.

[Figure 10]   FIG. 9 is a sectional view of the separation tray of FIG. 8 taken along line AA.

FIG. 11   FIG. 11 is an enlarged view of a single separation cell of the multiwell plate of FIG. 10.

FIG. 12 is a chromatogram of an unseparated mixture of DNA fragments in the method of Example 1.

FIG. 13 is a chromatogram of the separated 80 bp fragment obtained by the method of Example 1.

14 is a chromatogram of the separated 102 bp fragment obtained by the method of Example 1. FIG.

FIG. 15 is a chromatogram of the separated 174 bp fragment obtained by the method of Example 1.

FIG. 16 is a chromatogram of the separated 257 bp fragment obtained by the method of Example 1.

FIG. 17 is a chromatogram of the separated 267 bp fragment obtained by the method of Example 1.

18 is a chromatogram of the separated 298 bp fragment obtained by the method of Example 1. FIG.

FIG. 19 is a chromatogram of the separated 434 bp fragment obtained by the method of Example 1.

20 is a chromatogram of the separated 458 bp fragment obtained by the method of Example 1. FIG.

21 is a chromatogram of the separated 587 bp fragment obtained in the method of Example 1. FIG.

22 is a chromatogram of the pUC18 MspI standard mixture of dsDNA fragments used in Example 3. FIG.

FIG. 23 is a chromatogram of the low molecular weight and small base pair long fraction eluent obtained in Example 3.

FIG. 24 is a chromatogram of the high base pair long fraction eluent obtained in Example 3 showing the efficacy of the spin column device for purifying the high base pair length components of a mixture of DNA fragments.

FIG. 25 is a chromatogram of a pBR322 standard mixture of dsDNA fragments used in Example 4.

FIG. 26 is a chromatogram of the low molecular weight and small base pair long fraction eluent obtained in Example 4.

FIG. 27   4 is a chromatogram of the high base pair long fraction eluent obtained in Example 4.

FIG. 28   5 is a chromatogram obtained by the method of Example 5.

FIG. 29   5 is a chromatogram obtained by the method of Example 5.

FIG. 30   9 is a chromatogram obtained by the method of Example 6.

FIG. 31 is a chromatogram of separated hybridized dsDNA mixtures obtained by elution with an isocratic mobile phase.

FIG. 32   6 illustrates the hysteresis effect obtained in kinematic separation.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, C N, CU, CZ, DE, DK, EE, ES, FI, GB , GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, L C, LK, LR, LS, LT, LU, LV, MD, MG , MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, T J, TM, TR, TT, UA, UG, UZ, VN, YU , ZA, ZW (72) Inventor Taylor, Paul Day             94301 Pa, California, United States             Roalto hawthorne avenue 248 (72) Inventor Resiender, Benjamin El, Jiyu             near             68123 Belbi, Nebraska, United States             You Apartment No.2 M Su             Carborau 1753 (72) Inventor Hefel, Robert Em             95008 Key, California California             Yanbell Reading Road 270 F term (reference) 4B024 AA11 AA20 CA01 CA09 HA12                 4B029 AA07 AA23 BB20 FA15                 4B063 QA12 QQ42 QR82 QS17 QS24                       QS25 QS34 QX01 [Continued summary] The lower eluent receiving chamber and the solids of the separation medium supported therein. A polynucleotide comprising a tube having a constant unit Polynucleotide fragments from a mixture of fragments Ambient or lower pressure equipment to separate I will describe. The separation medium reacts with the counterions and forms a surface of the separation medium. A polyvalent yang that produces an insoluble polar coating on a surface It has a non-polar separating surface without ions.

Claims (44)

[Claims] 1. A) subjecting a mixture of DNA fragments to a separation column containing a separation medium having a non-polar, non-porous surface, wherein the mixture of DNA fragments comprises a counterion and a DNA binding concentration of a driving solvent, In a first solution containing; and b) a counterion, and a concentration of the driving solvent that has been previously determined to remove the DNA fragment with the target fragment from the separation medium into a separate compartment of the eluent. The target DN from the medium by contacting the medium with the second solution
Removing the A fragment, and isolating a target DNA fragment having a predetermined base pair length from the mixture of DNA fragments comprising: 2. The method of claim 1 comprising the steps of: c) collecting separate compartments of the eluent, thereby recovering the target DNA fragment. 3. The method according to claim 2, wherein the recovered target DNA fragment is amplified. 4. The method according to claim 2, wherein the recovered target DNA fragment is cloned.
the method of. 5. d) subjecting the sample mixture to a separation column containing a separation medium having a non-polar, non-porous surface, the sample mixture comprising a counterion and a DNA binding concentration of the driving solvent in a co-solvent. In a first solvent mixture containing; and e) a second ion containing a counterion and a driving solvent concentration that has been previously determined to remove a DNA fragment having a particular base pair length from the separation medium. Removing any DNA in the sample having the specific base pair length by contacting a solvent solution with a separation medium, the presence of a DNA fragment having the specific base pair length in a sample mixture comprising How to determine. 6. The method of claim 5, wherein the second solvent solution formed in step (b) is collected. 7. The method of claim 6, wherein the second solvent solution product of step (b) is analyzed to determine if the DNA fragment having the base pair length is present in the sample mixture. 8. The method of claim 7, wherein any DNA in the second solvent solution product of step (b) is amplified. 9. A DNA fragment having the base pair length is present in a sample mixture at a concentration that is too low to be detected in an assay, and is amplified to confirm the presence of the DNA fragment having the base pair length in the sample mixture. 9. The method of claim 8, wherein the product is analyzed. 10. A) applying a mixture of homoduplex and heteroduplex DNA fragments to a separation column containing a separation medium having a non-polar, non-porous surface, homoduplex and heteroduplex. The mixture of strand DNA fragments is applied in a first solution containing a counterion and a DNA binding concentration of a driving solvent; and b) the medium and the temperature at which the heteroduplex fragment locally denatures at its mismatch sites. While maintaining the solution, a counterion and a concentration of a driving solvent that has been previously determined to separate the target homoduplex and / or heteroduplex DNA fragments from the separation medium in separate fractions. The desired homoduplex and / or heteroduplex DNA fragment is separated from the separation medium by contacting the second solution with the separation medium. Separating the target homoduplex from a mixture of homoduplex and heteroduplex DNA fragments having the same base pair length, wherein the heteroduplex fragment has at least one mismatch site. And / or a method for separating hetero double-stranded DNA fragments by DMIPC. 11. The desired fragment is a homoduplex fraction and the concentration of the driving solvent is selected to remove one or both homoduplex fractions from the homoduplex fraction as separate fractions. The method of claim 10. 12. The method of claim 11, comprising the step of collecting the homoduplex fraction. 13. The method of claim 12, comprising amplifying the pooled homoduplex fraction. 14. The desired fragment is a heteroduplex fraction and the concentration of the driving solvent is selected to remove one or both of the heteroduplex fractions as a separate fraction from the homoduplex fraction. The method of claim 10. 15. The method of claim 11, comprising the step of collecting the heteroduplex fraction. 16. The method of claim 12, comprising amplifying the pooled heteroduplex fraction.
the method of. 17. The method of claim 10, wherein the ratio of the amount of heteroduplex fragment to homoduplex fragment is less than 1: 1. 18. The method of claim 17, wherein the amount of heteroduplex in the distinct heteroduplex fraction is below the level of detection. 19. The method of claim 10, wherein the retention time used to separate the homoduplex or heteroduplex fractions was previously determined from a reference standard. 20. The method of claim 19 wherein the reference standard is obtained by separating a standard mixture of homoduplexes and heteroduplexes having the same base pair sequence as the sample by matched ion polynucleotide chromatography. 21. a) applying a mixture of homoduplex and heteroduplex DNA fragments to a separation column containing a separation medium having a non-polar, non-porous surface; homoduplex and heteroduplex The mixture of strand DNA fragments is applied in a first solution containing a counterion and a DNA binding concentration of a driving solvent; and b) a medium and a temperature at which the heteroduplex fragment locally denatures at its mismatch sites. While maintaining the solution, a counterion and a concentration of the driving solvent that has been previously determined to separate the target homoduplex and / or heteroduplex DNA fragments from the separation medium in separate fractions. The desired homoduplex and / or heteroduplex DNA fragment is separated from the separation medium by contacting the second solution with the separation medium. The step of separating, wherein the heteroduplex fragment has at least one mismatch site, the homoduplex in a mixture of homoduplex and heteroduplex DNA fragments having the same base pair length or A method for determining the presence of a heteroduplex fraction. 22. The method of claim 21, comprising the step of: c) collecting separate sections of the eluent, thereby recovering the target DNA fragment. 23. The method of claim 22, wherein the recovered target DNA fragment is amplified. 24. The method of claim 22, wherein the recovered target DNA fragment is cloned. 25. The method of claim 22, wherein the DNA sample contains a large amount of background wild type. 26. The method of claim 22, wherein the mutant DNA is below the detection limit. 27. The method of claim 22, wherein the DNA sequences of wild type DNA and mutant DNA are known. 28. The method of claim 21, wherein the mutant DNA differs from wild-type DNA by at least one base pair. 29. The method of claim 21, wherein the retention time used to separate the homoduplex or heteroduplex fractions has been previously determined from a reference standard. 30. The method of claim 29, wherein the reference standard was obtained by separating a standard mixture of homoduplexes and heteroduplexes having the same base pair sequence as the sample by matched ion polynucleotide chromatography. 31. A tube having an upper solution input chamber, a lower eluent receiving chamber, and a fixed unit of separation medium supported therein, the separation medium reacting with counterions on the surface of the separation medium. An ambient or lower pressure device for separating polynucleotide fragments from a mixture of polynucleotide fragments having a non-polar separating surface free of polyvalent cations that produces an insoluble polar coating. 32. The ambient or lower pressure device of claim 31, wherein the separation medium is a member selected from the group consisting of beads, capillaries and monolith structures. 33. The ambient or lower pressure apparatus of claim 32, wherein the separation medium fixation unit comprises a fixed bed of separation medium particles. 34. The ambient or lower pressure device of claim 33, wherein the separation medium particles are selected from the group consisting of organic polymers having non-polar surfaces and inorganic particles. 35. The ambient or lower pressure device of claim 31, wherein the lower chamber is closed. 36. The ambient or lower pressure device of claim 31, wherein the lower chamber has an open bottom portion. 37. The ambient or lower pressure device of claim 36 in combination with an eluent container adapted to receive said lower chamber. 38. The ambient or lower pressure device of claim 37, wherein the eluent chamber is a centrifuge vial. 39. The ambient pressure of claim 37, wherein the cylinder is a member of a series of cylinders, the eluent container is a member of a series of eluent containers, and the arrangement of cylinders and the arrangement of containers have a matching arrangement. Or a lower pressure device. 40. A multi-lumen separation plate having an outer sealed edge, a multi-well collection plate, and a separation plate sealing means for producing a sealing engagement with the outer sealed edge of the multi-well separated plate and the vacuum cavity receiving the multi-well collection plate. The multi-lumen separation plate comprises a series of tubes, each tube comprising a top solution input chamber, a bottom eluent receiving chamber having a bottom opening therein, and fixation of a separation medium supported therein. A unit, the separation medium having a non-polar separation surface without polyvalent cations which reacts with counterions to produce an insoluble polar coating on the surface of the separation medium;
A multi-well collection plate is an ambient or lower pressure separation system having collection wells arranged to receive liquid from the bottom opening of the lower eluent receiving chamber. 41. The ambient or lower pressure separation system of claim 40, wherein the separation medium is a member selected from the group consisting of beads, capillaries and monolith structures. 42. The ambient or lower pressure apparatus of claim 41, wherein the separation medium fixation unit comprises a fixed bed of separation medium particles. 43. The ambient or lower pressure device of claim 42, wherein the separation medium particles are selected from the group consisting of organic polymers having non-polar surfaces and inorganic particles. 44. a) applying a mixture of DNA fragments, which may contain target DNA fragments, to a separation column containing a medium having a non-polar, non-porous surface, the mixture of DNA fragments being counterions and auxiliary A first solvent mixture containing a DNA binding concentration of a driving solvent in a solvent; and b) a counterion and a previously determined aid for removing a DNA fragment having a target DNA fragment base pair length from the medium. Removing a target DNA fragment from the medium by contacting the medium with a second solvent solution containing the concentration of the driving solvent in the solvent, the upper solution input chamber, the lower eluent receiving The chamber comprises a chamber and a tube having a fixed unit of separation medium supported therein, the separation medium being a pair of tubes. Ambient or lower pressure for separating polynucleotide fragments from a mixture of polynucleotide fragments having a non-polar separating surface free from polyvalent cations that reacts with water to form an insoluble polar coating on the surface of the separation medium Apparatus for removing a target DNA fragment having a predetermined base pair length from a mixture of DNA fragments.