This invention relates to DNA separation systems and methods suitable for effecting a size-based (base pair length) separation of DNA. The invention concerns an improved separation column for increasing the range of base pair length of the DNA fragments that can be separated by Matched Ion Polynucleotide Chromatography (MIPC) and for improving the separation of heteroduplex and homoduplex DNA using MIPC under partially denaturing conditions.
Separations of polynucleotides such as DNA have been traditionally performed using slab gel electrophoresis or capillary electrophoresis. However, liquid chromatographic separations of polynucleotides are becoming more important because of the ability to automate the analysis and to collect fractions after they have been separated. Therefore, columns for polynucleotide separation by liquid chromatography (LC) are becoming more important.
DNA molecules are polymers comprising sub-units called deoxynucleotides. The four deoxynucleotides found in DNA comprise a common cyclic sugar, deoxyribose, which is covalently bonded to any of the four bases, adenine (a purine), guanine (a purine), cytosine (a pyrimidine), and thymine (a pyrimidine), referred to herein as A, G, C, and T respectively. A phosphate group links a 3xe2x80x2-hydroxyl of one deoxynucleotide with the 5xe2x80x2-hydroxyl of another deoxynucleotide to form a polymeric chain. In double stranded DNA, two strands are held together in a helical structure by hydrogen bonds between what are called complimentary bases. The complimentarity of bases is determined by their chemical structures. In double stranded DNA, each A pairs with a T and each G pairs with a C, i.e., a purine pairs with a pyrimidine. Ideally, DNA is replicated in exact copies by DNA polymerases during cell division in the human body or in other living organisms. DNA strands can also be replicated in vitro by means of the Polymerase Chain Reaction (PCR). Sometimes, exact replication fails and an incorrect base pairing occurs. Further replication of the new strand produces double stranded DNA offspring containing a heritable difference in the base sequence from that of the parent. Such heritable changes in base pair sequence are called mutations.
As used herein, double stranded DNA is referred to as a duplex. When a base sequence of one strand is entirely complimentary to a base sequence of the other strand, the duplex is called a homoduplex. When a duplex contains at least one base pair which is not complimentary, the duplex is called a heteroduplex. A heteroduplex is formed during DNA replication when an error is made by a DNA polymerase enzyme and a non-complimentary base is added to a polynucleotide chain being replicated. Further replications of a heteroduplex will, ideally, produce homoduplexes which are heterozygous, i.e., these homoduplexes will have an altered sequence compared to the original parent DNA strand. When the parent DNA has a sequence which predominates in a naturally occurring population, the sequence is generally referred to as a xe2x80x9cwild typexe2x80x9d.
Many different types of DNA mutations are known. Examples of DNA mutations include, but are not limited to, xe2x80x9cpoint mutationxe2x80x9d or xe2x80x9csingle base pair mutationsxe2x80x9d in which an incorrect base pairing occurs. The most common point mutations comprise xe2x80x9ctransitionsxe2x80x9d in which one purine or pyrimidine base is replaced for another and xe2x80x9ctransversionsxe2x80x9d wherein a purine is substituted for a pyrimidine (and visa versa). Point mutations also comprise mutations in which a base is added or deleted from a DNA chain. Such xe2x80x9cinsertionsxe2x80x9d or xe2x80x9cdeletionsxe2x80x9d are also known as xe2x80x9cframeshift mutationsxe2x80x9d. Although they occur with less frequency than point mutations, larger mutations affecting multiple base pairs can also occur and may be important. A more detailed discussion of mutations can be found in U.S. Pat. No. 5,459,039 to Modrich (1995), and U.S. Pat. No. 5,698,400 to Cotton (1997).
The sequence of base pairs in DNA is a code for the production of proteins. In particular, a DNA sequence in the exon portion of a DNA chain codes for a corresponding amino acid sequence in a protein. Therefore, a mutation in a DNA sequence may result in an alteration in the amino acid sequence of a protein. Such an alteration in the amino acid sequence may be completely benign or may inactivate a protein or alter its function to be life threatening or fatal. On the other hand, mutations in an intron portion of a DNA chain would not be expected to have a biological effect since an intron section does not contain code for protein production. Nevertheless, mutation detection in an intron section may be important, for example, in a forensic investigation.
Detection of mutations is therefore of great importance in diagnosing diseases, understanding the origins of disease, and the development of potential treatments. Detection of mutations and identification of similarities or differences in DNA samples is also of critical importance in increasing the world food supply by developing diseases resistant and/or higher yielding crop strains, in forensic science, in the study of evolution and populations, and in scientific research in general (Guyer, et al., Proc. Natl. Acad. Sci. USA 92:10841 (1995); Cotton, TIG 13:43 (1997)).
Alterations in a DNA sequence which are benign or have no negative consequences are sometimes called xe2x80x9cpolymorphismsxe2x80x9d. For the purposes of this application, all alterations in the DNA sequence, whether they have negative consequences or not, are defined herein as xe2x80x9cmutationsxe2x80x9d. For the sake of simplicity, the term xe2x80x9cmutationxe2x80x9d is used herein to mean an alteration in the base sequence of a DNA strand compared to a reference strand (generally, but not necessarily, a wild type). As used herein, the term xe2x80x9cmutationxe2x80x9d includes the term xe2x80x9cpolymorphismxe2x80x9d or any other similar or equivalent term of art.
Separation of double-stranded deoxyribonucleic acids (dsDNA) fragments and detection of DNA mutations is of great importance in medicine, in the physical and social sciences, and in forensic investigations. The Human Genome Project is providing an enormous amount of genetic information and yielding new information for evaluating the links between mutations and human disorders (Guyer, et al., Proc. Natl. Acad. Sci. USA 92:10841 (1995)). For example, the ultimate source of disease is described by genetic code that differs from the wild type (Cotton, TIG 13:43 (1997)). Understanding the genetic basis of disease can be the starting point for a cure. Similarly, determination of differences in genetic code can provide powerful and perhaps definitive insights into the study of evolution and populations (Cooper, et. al., Human Genetics vol. 69:201 (1985)). Understanding these and other issues related to genetic coding requires the ability to identify anomalies, i.e., mutations, in a DNA fragment relative to the wild type.
Traditional chromatography is a separation process based on partitioning of mixture components between a xe2x80x9cstationary phasexe2x80x9d and a xe2x80x9cmobile phasexe2x80x9d. The stationary phase is provided by the surface of solid materials which can comprise many different materials in the form of particles or passageway surfaces of cellulose, silica gel, coated silica gel, polymer beads, polysaccharides, and the like. These materials can be supported on solid surfaces such as on glass plates or packed in a column. The mobile phase can be a liquid or a gas in gas chromatography. This invention relates to liquid mobile phases.
The separation principles are generally the same regardless of the materials used, the form of the materials, or the apparatus used. The different components of a mixture have different respective degrees of solubility in the stationary phase and in the mobile phase. Therefore, as the mobile phase flows over the stationary phase, there is an equilibrium in which the sample components are partitioned between the stationary phase and the mobile phase. As the mobile phase passes through the column, the equilibrium is constantly shifted in favor of the mobile phase. This occurs because the equilibrium mixture, at any time, sees fresh mobile phase and partitions into the fresh mobile phase. As the mobile phase is carried down the column, the mobile phase sees fresh stationary phase and partitions into the stationary phase. Eventually, at the end of the column, there is no more stationary phase and the sample simply leaves the column in the mobile phase.
A separation of mixture components occurs because the mixture components have slightly different affinities for the stationary phase and/or solubilities in the mobile phase, and therefore have different partition equilibrium values. Therefore, the mixture components pass down the column at different rates.
In traditional liquid chromatography, a glass column is packed with stationary phase particles and mobile phase passes through the column, pulled only by gravity. However, when smaller stationary phase particles are used in the column, the pull of gravity alone is insufficient to cause the mobile phase to flow through the column. Instead, pressure must be applied. However, glass columns can only withstand about 200 psi. Passing a mobile phase through a column packed with 5 micron particles requires a pressure of about 2000 psi or more to be applied to the column. 5 to 10 micron particles are standard today. Particles smaller than 5 microns are used for especially difficult separations or certain special cases). This process is denoted by the term xe2x80x9chigh pressure liquid chromatographyxe2x80x9d or HPLC.
HPLC has enabled the use of a far greater variety of types of particles used to separate a greater variety of chemical structures than was possible with large particle gravity columns. The separation principle, however, is still the same.
An HPLC-based ion pairing chromatographic method was recently introduced to effectively separate mixtures of double stranded polynucleotides in general, and DNA in particular, wherein the separations are based on base pair length (U.S. Pat. No. 5,585,236 to Bonn (1996); Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993)). These references and the references contained therein are incorporated herein in their entireties. The term xe2x80x9cMatched Ion Polynucleotide Chromatographyxe2x80x9d (MIPC) is defined herein and applied to this method because the mechanism of separation was found to be based on binding and release of the DNA from the separation surfaces rather than traditional partitioning. MIPC separates DNA fragments on the basis of base pair length and is not limited by the deficiencies associated with gel based separation methods.
Matched Ion Polynucleotide Chromatography, as used herein, is defined as a process for separating single and double stranded polynucleotides using non-polar separation media, wherein the process uses a counter-ion agent, and an organic solvent to release the polynucleotides from the separation media. MIPC separations can be complete in less than 10 minutes, and frequently in less than 5 minutes.
The MIPC separation process differs from the traditional HPLC separation processes in that the separation is not achieved by a series of equilibrium separations between the mobile phase and the stationary phase as the liquids pass through the column. Instead, the sample is fed into the column using a solvent strength which permits the sample dsDNA to bind to the separation media surface. Strands of a specific base pair length are removed from the stationary phase surface and are carried down the column by a specific solvent concentration. By passing an increasing gradient of solvent through the sample, successively larger base pair lengths are removed in succession and passed through the column.
Descriptions of the use of MIPC, such as U.S. Pat. No. 5,585,236 to Bonn; U.S. Pat. No. 5,795,976 to Oefner; and U.S. patent application Ser. Nos. 09/183,123 to Gjerde filed October 30, 1998; and 09/183,450 to Gjerde filed Oct. 30, 1998, disclose separations of dsDNA having lengths less than about 1000-2000 base pairs. The limitation in the upper range of DNA length amenable to the technique has impeded the use of MIPC in the purification of fragments larger than 2000 bp, such as those routinely used in cloning procedures, for example.
A reliable way to detect mutations is by hybridization of the putative mutant strand in a sample with the wild type strand (Lerman, et al., Meth. Enzymol., 155:482 (1987)). If a mutant strand is present, then two homoduplexes and two heteroduplexes will be formed as a result of the hybridization process. Hence separation of heteroduplexes from homoduplexes provides a direct method of confirming the presence or absence of mutant DNA segments in a sample.
As the use and understanding of MIPC developed, it was discovered that when MIPC analyses were carried out at a partially denaturing temperature, i.e., a temperature sufficient to denature a heteroduplex at the site of base pair mismatch, homoduplexes could be separated from heteroduplexes having the same base pair length (U.S. Pat. No. 5,795,976; Hayward-Lester, et al., Genome Research 5:494 (1995); Underhill, et al., Proc. Natl. Acad. Sci. USA 93:193 (1996); Doris, et al., DHPLC Workshop, Stanford University, (1997)). These references and the references contained therein are incorporated herein in their entireties. Thus, the use of Denaturing HPLC (DHPLC) was applied to mutation detection (Underhill, et al., Genome Research 7:996 (1997); Liu, et al., Nucleic Acid Res., 26; 1396 (1998)).
The application of the Matched Ion Polynucleotide Chromatography (MIPC) under the partially denaturing conditions used for separating heteroduplexes from homoduplexes in mutation detection is hereafter referred to as DMIPC. In DMIPC, precise temperature control is required for maintaining both mobile and stationary phases at a partially denaturing temperature, that is, a temperature at which mismatched DNA present at the mutation site of a heteroduplex strand will denature but at which the matched DNA will remain bound into the double strand.
The hybridization process creates two homoduplexes and two heteroduplexes. Ideally, at an optimal temperature, the appearance of four distinct peaks is observed upon DMIPC analysis. DMIPC can separate heteroduplexes that differ by as little as one base pair. However, in some cases separations of homoduplexes and heteroduplexes are poorly resolved (e.g., as described by Liu et al. Nucleic Acids Res. 26: 1396-1400 (1998)). The presence of mutations may even be missed entirely. In some mutation analyses, only two peaks or partially resolved peak(s) are observed in DMIPC analysis. The two homoduplex peaks may appear as one peak or a partially resolved peak and the two heteroduplex peaks may appear as one peak or a partially resolved peak. In some cases, only a broadening of the initial peak is observed under partially denaturing conditions.
There is a need for improving the resolution of DNA fragments separated by MIPC, and for extending the range of base pairs which can be separated by this method. There is also a need for improving the resolution of heteroduplex and homoduplex DNA fragments using DMIPC.
It is an object of the present invention to provide a MIPC separation column which gives improved separation of polynucleotides; which allows separation of polynucleotides having lengths greater than about 1000; and which gives improved separation of homoduplex and heteroduplex DNA under partially denaturing conditions.
In one aspect, the invention concerns an improved separation column for separating a mixture of double stranded DNA fragments by Matched Ion Polynucleotide Chromatography (MIPC). The mixture contains fragments having lengths exceeding about 1000 base pairs. The column includes a cylinder having an ID greater than about 5 mm and containing polymer beads. The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA. In one embodiment, the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
In another aspect, the invention concerns an improved method for separating a mixture of double stranded DNA fragments by MIPC in which the mixture includes fragments having lengths exceeding about 1000 base pairs. The method includes a first step (a) of applying a solution of the DNA fragments fragments and counterion reagent to separation beads. The beads are retained within a separation column. The column has an ID greater than about 5 mm. The beads have an average diameter of 1 to 100 microns, and are composed of unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA. Step (b) involves eluting the fragments with a gradient of eluting solvent of increasing organic component concentration containing a counterion agent. During the elution, surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not trap or release multivalent metal cations therefrom. The eluting is carried out under conditions effective to at least partially denature the heteroduplexes and where the eluting results in the separation of the heteroduplexes from the homoduplexes. In one embodiment of this aspect, the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
In yet another aspect, the invention concerns an improved method for separating heteroduplex and homoduplex DNA molecules in a mixture. The method includes a first step (a) of applying a solution of the fragments and counterion reagent to separation beads, said beads retained within a separation column having an ID greater than about 5 mm. The beads have an average diameter of 1 to 100 microns. The beads are unsubstituted polymer beads or polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA, said column having an ID greater than about 5 mm. In step (b), fragments are eluted with a gradient eluting solvent of increasing organic component concentration containing a counterion agent. During the elution, the surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not trap or release multivalent metal cations therefrom. The eluting is carried out under conditions effective to at least partially denature the heteroduplexes and where the eluting results in the separation of the heteroduplexes from the homoduplexes. In one embodiment of this aspect, the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.
In still another aspect, the invention concerns an improved separation column for separating heteroduplex and homoduplex DNA molecules in a mixture, by Denaturing Matched Ion Polynucleotide Chromatography (DMIPC). The DNA molecules in the mixture consist of fragments having equal lengths. The column includes a cylinder having an ID greater than about 5 mm and containing polymer beads. The beads have an average diameter of 1 to 100 microns and are unsubstituted polymer beads or are polymer beads substituted with a hydrocarbon moiety having from 1 to 1,000,000 carbons. The preferred beads are characterized by being substantially free from multivalent cations which are free to bind with DNA. In one embodiment, the column ID is greater than about 7 mm. In another embodiment, the column ID is greater than about 10 mm. In yet another embodiment, the column ID is greater than about 50 mm. In still another embodiment the column ID is in the range of about 5 mm to about 1 m.