WO2002028501A1 - Genotypage par chromatographie en phase liquide de fragments d'acides nucleiques courts - Google Patents

Genotypage par chromatographie en phase liquide de fragments d'acides nucleiques courts Download PDF

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WO2002028501A1
WO2002028501A1 PCT/US2001/030828 US0130828W WO0228501A1 WO 2002028501 A1 WO2002028501 A1 WO 2002028501A1 US 0130828 W US0130828 W US 0130828W WO 0228501 A1 WO0228501 A1 WO 0228501A1
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nucleic acid
acid molecules
buffer
nucleotides
column
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PCT/US2001/030828
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English (en)
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Jeffrey Van Ness
David J. Galas
Lori K. Garrison
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Keck Graduate Institute
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Priority to AU2001296491A priority Critical patent/AU2001296491A1/en
Priority to US10/398,005 priority patent/US20050089850A1/en
Publication of WO2002028501A1 publication Critical patent/WO2002028501A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • the invention is in the field of molecular biology, and is more specifically directed to genotyping methods and compositions useful therein.
  • the chromosomal mapping and nucleic acid sequencing of each of the 80,000 to 100,000 human genes, achieved through the Human Genome Project, provides an opportunity for a comprehensive approach to the identification of nucleotide loci responsible for genetic disease.
  • Many of the 150-200 common genetic diseases and -600-800 of the rarer genetic diseases are associated with one or more defective genes. Of these, more than 200 human diseases are known to be caused by a defect in a single gene, often resulting in a change of a single amino acid residue. (Olsen, "Biotechnology: An Industry Comes of Age” (National Academic Press, 1986)).
  • Mutations occurring in somatic cells may induce disease if the mutations affect genes involved in cellular division control, resulting in, for example, tumor formation.
  • loss-of-function mutations in many genes can give rise to a detectable phenotype in humans.
  • the number of cell generations in the germline, from one gamete to a gamete in an offspring, may be around 20-fold greater in the male germline than in the female.
  • an egg is formed after a second meiotic division and lasts for 40 years. Therefore the incidence of different types of germline mutations and chromosomal aberrations depends on the parent of origin.
  • cytogenetics remains the preferred technique to investigate these important genetic mechanisms.
  • the remaining normal allele may be replaced by a second copy of the mutant allele in one cell per 10 3 -10 4 .
  • Mechanisms causing this replacement include chromosomal nondisjunction, mitotic recombination, and gene conversion.
  • independent mutations destroying the function of the remaining gene copy are estimated to occur in one cell out of 10 .
  • Sensitive mutation detection techniques offer extraordinary possibilities for mutation screening. For example, analyses may be performed even before the implantation of a fertilized egg. (Holding et al., Landcet 5:532 (1989)). Increasingly efficient genetic tests may also permit screening for oncogenic mutations in cells exfoliated from the respiratory tract or the bladder in connection with health checkups. (Sidransky et al., Science 252:106, 1991). Alternatively, when an unknown gene causes a genetic disease, methods to monitor DNA sequence variants are useful to study the inheritance of the disease through genetic linkage analysis. Notwithstanding these unique applications for the detection of mutations in individual genes, the existing methodology for achieving such applications continues to pose technological and economic challenges.
  • allele-specific PCR methods can rapidly and preferentially amplify mutant alleles.
  • multiple mismatch primers have been used to detect H-ras mutations at a sensitivity of one mutant in 10 5 wild-type alleles and sensitivity as high as one mutant in 10 wild-type alleles have been reported.
  • Haliassos et al. Nucleic Acids Res. 77:8093-8099 (1989); and Chen et al., Anal. Biochem. 244:191-194 (1997).
  • the discriminating mismatch on the 3' primer end i.e., G:T or C:A mismatch
  • G:T or C:A mismatch the discriminating mismatch on the 3' primer end
  • RFLP detection methods are limited by the requirement that the location of the mutations must coincide with restriction endonuclease recognition sequences.
  • primers that introduce a restriction site have been employed in "primer-mediated RFLP.”
  • “primer-mediated RFLP” Jacobson et al., PCR Methods Applicant. 1:299 (1992); Chen et al., Anal. Biochem. 195:51-56 (1991); Di Giuseppe et al., Am. J. Pathol. 144:889-895 (1994); Kahn et al., Oncogene :1079-1083 (1991); Levi et al., Cancer Res.
  • nucleotide analogs that are designed to base pair with more than one of the four natural bases are termed "convertides.” Base incorporation opposite different convertides has been tested. (Hoops et al., Nucleic Acids Res. 25:4866-4871 (1997)). For each analog, PCR products were generated using Taq DNA polymerase and primers containing an internal nucleotide analog. The products generated showed a characteristic distribution of the four bases incorporated opposite the analogs.
  • the present invention fulfills this and other related needs by providing methods for the detection of mutations at defined nucleotide loci in target nucleic acids that, inter alia, display increased speed, convenience and specificity.
  • methods according to the present invention are based on digestion of target nucleic acids with restriction endonucleases that cleave outside their recognition sites, thus producing short nucleic acid fragments, and subsequently the successful use of liquid chromatography in characterizing such short fragments.
  • the present invention provides methods for identifying one or more nucleotide(s) at a defined location in a double-stranded target nucleic acid, comprising the following ' steps:
  • each of the first and the second ODNPs comprises a 5 ' end and a 3' end, a first portion of each ODNP at the 5' end and a second portion of each ODNP at the 3 ' end are at least substantially complementary to a first portion and a second portion ofthe target nucleic acid, and 4-8 nucleotides between the first portion and the second portion of the ODNP comprise a recognition site for a restriction endonucleotide (RE) that cleaves outside its recognition site, and wherein each ODNP is complementary to an opposite strand of the target nucleic acid, the first and the second ODNPs are complementary to two noncontiguous regions of the target nucleic acid, and the defined position is between the two non-contiguous regions;
  • RE restriction endonucleotide
  • step (b) amplifying the target nucleic acid using the first and the second ODNPs; (c) digesting the amplification product of step (b) with the restriction endonuclease(s) that recognize the recognition sequences in the first and the second
  • step (d) characterizing a short digestion product of step (c) with liquid chromatography.
  • the first portion of each ODNP at the 5 ' end and the second portion of each ODNP at the 3 ' end are exactly complementary to the first portion and the second portion ofthe target nucleic acid.
  • the first portion of each ODNP is at least 6, 8,
  • the second portion of each ODNP is at least 1, 3, 5 or 7 nucleotides in length.
  • the second portion of each ODNP is at least 1, 3, 5 or 7 nucleotides in length.
  • ODNP is at most 16, 18, 20, 22 or 24 nucleotides in length.
  • the distance between the first and the second portions in the target nucleic acid is up to 10 nucleotides in length. Preferably, the distance is 4-8 nucleotides in length.
  • the target nucleic acid does not comprise a recognition site between the first and the second portions that is recognizable by a restriction endonuclease cleaving outside its recognition site.
  • the recognition sequences in the first and the second ODNPs are the same. In other embodiments, the recognition sequences in the first and the second ODNPs are different.
  • the RE(s) that recognizes the recognition sequences in the first and the second ODNPs are Type IIS RE(s).
  • the RE is Bpm I.
  • the distance between the two non-contiguous regions in the target nucleic acid is 1 to 30, preferably 1 to 20, more preferably 1-10 nucleotides in length.
  • the target nucleic acid is genomic DNA or cDNA.
  • the nucleotide(s) at the defined location is associated with a disease. In other embodiments, the nucleotide(s) at the defined location is associated with drug resistance of a pathogenic microorganism.
  • the invention provides a method of performing chromatography in order to characterize and identify nucleic acid molecules, particularly nucleic acid molecules formed from less than 20 nucleotides.
  • the present invention provides a method of performing liquid chromatography comprising: applying two nucleic acid molecules to a liquid chromatography column, where the two nucleic acid molecules have an identical number of nucleotide bases, but have different nucleotide sequences; and eluting the two nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules have different elution times; the elution buffer being formed from Buffer A and Buffer B, where elution buffer of incrementally increasing organic solvent concentration is applied to the column, where the elution buffer comprises an ammonium salt and the ammonium salt comprises a secondary or tertiary amine complexed with an organic or inorganic acid.
  • the two nucleic acid molecules are composed of identical nucleotides, but the order of the nucleotides in the two nucleic acid molecules is non-identical.
  • the two nucleic acid molecules each have the sequence 5'-n-X-m-3', where n and m each represent a sequence of from 0-1 nucleotides, and X represents a single nucleotide, and the two nucleic acid molecules each have the same sequences n and m, but differ in the identify ofthe nucleotide at location X.
  • the present invention provides a method method of performing liquid chromatography comprising: applying two nucleic acid molecules to a liquid chromatography column, where the two nucleic acid molecules have an identical number of nucleotide bases within the range of 2-10, but have different nucleotide sequences, and the liquid chromatography column is a reverse phase chromatography column; and eluting the two nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules have different elution times; the elution buffer being formed from Buffer A and Buffer B, where elution buffer of incrementally increasing organic solvent concentration is applied to the column, where the elution buffer comprises an ammonium salt and the ammonium salt comprises a secondary or tertiary amine complexed with an organic or inorganic acid.
  • the present invention also provides a method of performing liquid chromatography comprising: applying two nucleic acid molecules to a liquid chromatography column, where the two nucleic acid molecules have an identical number of nucleotide bases, but have different nucleotide sequences; and eluting the two nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules have different elution times; the elution buffer being formed from Buffer A and Buffer B, where Buffer A comprises water and an ammonium salt that is formed from a secondary or tertiary amine complexed with an organic or inorganic acid; and Buffer B comprises water, organic solvent, and an ammonium salt that is formed from a secondary or tertiary amine complexed with an organic or inorganic acid; where elution buffer of incrementally increasing organic solvent concentration is applied to the column.
  • the present invention provides a method of performing liquid chromatography and mass spectrometric analysis comprising: applying a plurality of pairs of nucleic acid molecules to a liquid chromatography column, where each pair of nucleic acid molecules is formed from two nucleic acid molecules that have an identical number of nucleotide bases, but have different nucleotide sequences; eluting the plurality of pairs of nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules that form each pair have different elution times; and characterizing each nucleic acid molecule by mass spectroscopy; the elution buffer being formed from Buffer A and Buffer B, where elution buffer of incrementally increasing organic solvent concentration is applied to the column, where the elution buffer comprises an ammonium salt and the ammonium salt comprises a secondary or tertiary amine complexed with an organic or inorganic acid.
  • the present invention also provides a composition referred to herein as Buffer B comprising water, the reaction product of secondary or tertiary amine with organic or inorganic acid, and organic solvent.
  • Buffer B comprising water, the reaction product of secondary or tertiary amine with organic or inorganic acid, and organic solvent.
  • the present invention provides a composition comprising water, the reaction product of secondary or tertiary amine with organic or inorganic acid, organic solvent, and two nucleic acid molecules, where the two nucleic acid molecules have an identical number of nucleotide bases, but have different nucleotide sequences.
  • the present invention also provides a kit for chromatographic analysis comprising: (a) a container holding components comprising water and the reaction product of secondary or tertiary amine with organic or inorganic acid (Buffer A); and
  • Figure 1 is a diagram of a double-stranded target nucleic acid.
  • Figure 2 is a diagram showing the relationship between the first ODNP and the top strand ofthe target nucleic acid.
  • Figure 3 is a diagram showing the situation where the first and second portions of a first ODNP are complementary to bases of the top strand of a target nucleic acid so that there are more nucleotides separating the first and second portions than there are nucleotides separating the corresponding complementary regions in the top strand.
  • Figure 4 is a diagram of major steps in one aspect ofthe present method for identifying a nucleotide at a defined position in a target nucleic acid using an ODNP pair and an exemplary restriction endonuclease recognition sequence for Fok I.
  • Figure 5 is a high pressure liquid chromatogram (HPLC) of a set of 4, 6, 8 and 10 nucleotide ODNP.
  • Figures 6A and 6B show HPLC separation of three 8-mers (Figure 6A) and three 10-mers ( Figure 6B).
  • Figures 7A and 7B show the HPLC separation of one 4-mer, one 6-mer, three 8-mers and three 10-mers (Figure 7A) and the elution of two 6-mers ( Figure 7B).
  • Figure 8 is a control HPLC chromatogram.
  • Figure 9 shows HPLC fractionation and detection of short fragments generated by the Fok I double digestion.
  • the present invention provides methods of obtaining genetic information such as genotype analysis to identify nucleic acid variations.
  • the invention prepares short nucleic acid segments by amplifying a target nucleic acid using specially designed oligonucleotide primers (ODNPs).
  • ODNPs oligonucleotide primers
  • the amplification product is subsequently digested with restriction endonuclease(s) that recognize restriction endonuclease recognition sequence(s) that have been incorporated into the product via the specially designed ODNPs.
  • the digestion product includes one or more short polynucleotide segments that can be analyzed by liquid chromatography to identify whether nucleic acid variation is present in the target nucleic acid.
  • the present method is simple to implement (e.g., no purification of digestion products required before liquid chromatographic analysis), accurate, and more economical than genotyping methods using mass spectrometric analysis (Laken et al., published PCT application No. WO 00/31300).
  • the present method can be used in a variety of applications such as genetic analysis for hereditary diseases, tumor diagnosis, disease predisposition, forensics or paternity, crop cultivation, animal breeding, expression profiling of cell function and/or' disease marker genes, and identification and/or characterization of infectious organisms that cause infectious diseases in plants or animals and/or that are related to food safety.
  • the target nucleic acid can be any polynucleotide that contains an (one or more) unknown nucleotide at a defined location.
  • the target nucleic acid is formed by an organism, and has been at least partially, preferably completely, isolated from the organism. Methods by which the target may be isolated from the organism are well known and often practiced in the art.
  • the subject organism can be any organism, for example, a human or other animal, a plant, a fungus, or a microorganism such as a bacterium or a virus.
  • the target nucleic acid is synthetic in origin, i.e., has been made according to human intervention or design. For instance, many companies are now in the business of making "genes" or other long polynucleic acids, and these materials may be the target nucleic acid of the present invention.
  • the target nucleic acid may be represented by the generalized formula shown in Figure 1. It is composed of two strands, each strand being a nucleic acid molecule, which are arbitrarily identified and distinguished by the names "top strand” and "bottom strand.” In general, these two strands may be given other convenient names, where first/second, coding/non-coding, and 375' are other conventions used in the art to distinguish the two strands that form the double-stranded target.
  • the top and bottom strands are each either an oligonucleotide (having up to 100 nucleotides) or a polynucleotide (having more than 100 nucleotides).
  • Each strand is identified as having a 3' end and a 5' end, which is conventional naming used in the nucleic acid art for oligonucleotides and polynucleotides.
  • each of the numbers represents a base (adenine (A), guanine (G), cytosine (C) or thymine (T)) connected to a neighboring base by a sugar and a phosphate group .represented by two straight lines that together form a right angle, e.g., ⁇ , I, I , and — I.
  • Either the base designated by "0" or the base designated by "0*” is the base at the "defined position" in the method of the present invention.
  • This position is "defined” in that the investigator knows the base sequence in the 3' direction from the defined position, i.e., the sequence 1 3 5 7 9 etc., and also knows the base sequence in the 5' direction as measured from the defined position, i.e., the sequence 2, 4, 6, 8, etc. However, the investigator does not know the identity of the base at the defined position.
  • the present invention provides a method by which the investigator may identify the base at the defined position.
  • the target nucleic acid contains twelve nucleotides in each of the top and bottom strands.
  • the upper strand contains VaQ + '/-.(P + 1) + 1 nucleotides
  • the bottom strand contains V2Q* + ⁇ (P* + 1) + 1 nucleotides.
  • the upper and lower strands may, but need not contain the same number of nucleotides. That is, P does not necessarily equal P*, and Q does not necessarily equal Q*.
  • each number and its complement e.g., 0 and 0*, 1 and 1*, 2 and 2*, etc., represent a base pair selected from A and T, and G and C.
  • the dashed line between a number and its complement represents hydrogen bonding between the two bases.
  • the present invention forms a mixture that includes, among other possible components, a target nucleic acid as described above, a first oligonucleotide primer (first ODNP) and a second oligonucleotide primer (second ODNP).
  • first ODNP first oligonucleotide primer
  • second ODNP second oligonucleotide primer
  • the first ODNP must be able to (1) prime an extension and/or amplification reaction in combination with the target nucleic acid; (2) be able to hybridize to the top strand of the target nucleic acid at a location 3 ' from the defined position in the target nucleic acid and not overlapping the defined position, and (3) contain a sequence that will be recognized by a restriction endonuclease.
  • the first ODNP contains a nucleotide sequence such that it will hybridize to a region ofthe top strand that is located in the 3' direction from the defined location of the target nucleic acid.
  • Figure 2 shows that complements 4/4*, 6/6*, 8/8*, 18/18*, 20/20* and 22/22* are present between the first ODNP and the top strand of the target nucleic acid, where each of these complements is selected from A/T, T/A, G/C, and C/G. These complements provide thermal stability when the first ODNP hybridizes to the top strand.
  • an ODNP is "exactly complementary" to its template sequence if every nucleotide of the ODNP is complementary to every corresponding nucleotide of the template sequence wherein A is complementary to T and G is complementary to C.
  • mismatching of bases can occur according to the present invention even between the bases in the first and second portions of the first ODNP and the complimentary locations in the top strand.
  • the duplex cannot have too many mismatches or else the duplex will either not form at all or will not be sufficiently stable under the extension/amplification conditions to effectively allow the extension/amplification reaction to occur.
  • the first ODNP and the top strand are substantially complimentary.
  • an ODNP is substantially complementary to a target nucleic acid when it is at least 90% identical to the complement ofthe target over the length ofthe ODNP as determined by the Smith- Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molelcular) using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
  • R 1 through R 4 there is a contiguous sequence of bases within the first ODNP, denoted R 1 through R 4 , that do not completely hybridize to the top strand of the target nucleic acid.
  • This sequence R -R is selected, according to the present invention, to be a recognition sequence for a restriction endonuclease.
  • One or more of the nucleotides R ! -R may, coincidentally, hybridize to the corresponding base in the top strand of the target nucleic acid. However, it is unnecessary that any of the bases R ! -R 4 hybridize to any base in the top strand of the target nucleic acid. Should such hybridization occur, it is not detrimental to the present invention, and may provide additional, desirable stability for the duplex formed between the first ODNP and the top strand ofthe target nucleic acid.
  • an ODNP of the present invention there is a sequence of nucleotides located between the recognition sequence and the 5' end of the ODNP that will be referred to herein as the first portion of the ODNP.
  • the second portion of the ODNP there is a sequence of nucleotides located between the recognition sequence and the 3 ' end of the ODNP that will be referred to herein as the second portion of the ODNP. Referring to Figure 2, which is illustrative only, the first portion consists of 18*, 20* and 22*, while the second portion consists of 8*, 6* and 4*.
  • first and/or second portions are the regions of the ODNP that are designed to be sufficiently complementary to the top strand ofthe target nucleic acid such that the first ODNP will hybridize to the top strand to a sufficient extent that the first ODNP can prime an polymerase catalyzed extension reaction whereby the first ODNP is extended in the 3 ' direction to provide a partial complement ofthe top strand ofthe target nucleic acid.
  • this extension reaction copies into the extended first ODNP a complement ofthe nucleotide at the defined position in the target nucleic acid. In this way, the base information present at the defined position of the top strand of the target nucleic acid is transferred into the extended first ODNP.
  • those bases of the top strand of the target nucleic acid that are exactly complementary or substantially complementary to the first portion of the first ODNP will be referred to herein as the first portion of the top strand of the target nucleic acid.
  • those bases of the top strand of the target nucleic acid that are exactly complementary or substantially complementary to the second portion of the first ODNP will be referred to herein as the second portion of the top strand of the target nucleic acid.
  • An ODNP of the present invention must have either a first portion or a second portion, and preferably has both a first and second portion. If the ODNP does not contain a second portion, then the polymerase catalyzed extension reaction will generally be quite slow to occur because the polymerase requires a double-stranded end in order to begin the extension reaction. In the absence of this second portion, the necessary double-stranded end will only occur occasionally, that is, when the terminal base ofthe recognition sequence in the primer (R 4 in Figure 2) happens hybridize to the base opposite itself (10 in Figure 2) in the top strand ofthe target nucleic acid sequence.
  • the first ODNP contain a second portion, where that second portion is substantially or, preferably, exactly complementary to the top strand ofthe target nucleic acid, and as an additional preferred embodiment, the second portion contains at least 1, or at least 3, or at least 5, or at least 7, or at least 9 nucleotides. Preferably, the second portion is at most 16, 18, 20, 22, or 24 nucleotides in length.
  • the first ODNP does not contain a first portion, then it is necessary that the first ODNP contain a second portion that is exactly or substantially complementary to the first portion of the top strand of the target.
  • the first ODNP contains both a first and second portion, where together these two portions stably hold the first ODNP in a hybridized arrangement with the top strand ofthe target nucleic acid, so that the polymerized extension reaction will readily proceed.
  • the size of the first portion this is generally in the range of 2-20 nucleotides.
  • the first portion may contain at least 6, 8, 10, 12, or 14 nucleotides.
  • the first portion need not be too long, because the second portion alone will be able to hold the first ODNP in a hybridized state with the top strand of the target nucleic acid.
  • the second portion is relatively short, on the order of 2-4 nucleotides, then the first portion must be more substantial, on the order of 8-20 nucleotides.
  • the total number of nucleotides in the first and second portions depends on the bases present in the first and second portions. As is well known in the art, it is empirically observed that a G/C base pair provides more stability to a hybrid than does an A/T base pair, the reason probably being that G/C base pairs are formed from three hydrogen bonds while an A/T base pair is formed from only two hydrogen bonds.
  • the first ODNP has a second portion that contains 4-8 nucleotides where those nucleotides are substantially complementary to the second portion of the target nucleic acid.
  • first and second portions of a first ODNP Another factor to consider when designing a first and second portion of a first ODNP is the location of the restriction site.
  • the first and second portions are designed such that the restriction site is located across from an equal number of nucleotides in the top strand of the target nucleic acid.
  • Figure 3 illustrates the situation where the first and second portions of the first ODNP are complementary to bases of the top strand of the target nucleic acid such that there are more nucleotides separating the first and second portions than there are nucleotides separating the corresponding complementary regions in the top strand.
  • the restriction enzyme recognition sequence will need to form a "bubble" as identified in this Figure.
  • the bubble will contain nucleotides that are not part of the restriction enzyme recognition sequence.
  • nucleotides present in the restriction enzyme recognition sequence will be able to hybridize to the top strand ofthe target nucleic acid, and accordingly even when a bubble is formed, some ofthe nucleotides that form the restriction enzyme recognition sequence may not be part ofthe bubble.
  • the nucleotides interposed between the first and second portions of the first ODNP need not all be part of a recognition sequence for a restriction enzyme.
  • the first and second portions of the first ODNP are separated by 0-16, preferably 4-8 nucleotides, where these nucleotides contain a restriction enzyme recognition sequence.
  • the first ODNP need not hybridize to the nucleotide adjacent to the defined position in the target nucleic acid.
  • Figure 2 where the first ODNP actually hybridizes to the top strand of the target nucleic acid such that there is a gap of a single nucleotide (denoted "2") between the second portion ofthe top strand ofthe target nucleic acid and the defined position ofthe target nucleic acid.
  • this "gap" ranges in length from 0 nucleotide (i.e., no gap is present) to not more than 20, preferably not more than 10, nucleotides.
  • the invention may be practiced with a gap greater than 20 nucleotides, this is typically not advantageous because the inventive method then affords a relatively large digestion product when restriction enzyme acts on amplification product of first and second ODNPs and the target nucleic acid. Typically, greater discrimination is afforded according to the present invention when the digestion product is shorter, and thus introducing a larger gap when designing first (and second) ODNPs is not typically advantageous. Thus, in one preferred aspect ofthe present invention, there is no gap between the first portion of the ODNP, while in another aspect the gap is only a single nucleotide.
  • the guidelines provided above for designing a first ODNP to hybridize to the top strand ofthe target nucleic acid are the same as the guidelines that should be used in designing a second ODNP to hybridize to the bottom strand ofthe target nucleic acid. Accordingly, less specific discussion will be provided regarding the design ofthe second ODNP.
  • the parameters that were chosen for the first ODNP e.g., the number of nucleotides in the first portion of the first ODNP, the number of nucleotides in the second portion of the first ODNP, the number of bases separating the first and second portions of the first ODNP, whether the second portion of the first ODNP is exactly complementary to the second portion of the top strand of the target nucleic acid, or is merely substantially complementary, whether hybridization between the first ODNP and the top strand of the target nucleic acid produces a bubble in the top strand of the target nucleic acid or in the first ODNP, etc. need not be the same between the first and second ODNPs.
  • the first ODNP may have a first portion containing 7 nucleotides
  • the second ODNP may have a first portion containing 5 nucleotides.
  • the second ODNP is designed so that it will hybridize to the bottom strand of the target nucleic acid, at a location 3 ' of the defined position of the bottom strand, i.e., in the 3' direction from the nucleotide defined as 0*.
  • the second ODNP will hybridize to the bottom strand by way of first and/or second portions, just as the first ODNP hybridized to the top strand ofthe target nucleic acid by way of first and second portions.
  • the second ODNP is designed so that the hybridization product between it and the bottom strand ofthe target nucleic acid may, or may not, have a gap between the double-stranded hybridization product and the defined position of the bottom strand.
  • the second ODNP will contain a restriction enzyme recognition sequence
  • the enzyme that recognizes the sequence in the first ODNP need not be the same as the enzyme that recognizes the sequence in the second ODNP.
  • the restriction enzyme recognition sequence of the first ODNP need not be identical to the restriction enzyme recognition sequence of the second ODNP.
  • the first and second ODNPs share the same restriction enzyme recognition sequence. Either the first ODNP, or the second ODNP, or both may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 or 70 nucleotides.
  • the first and second ODNPs are at least partially complementary to two non-contiguous regions of opposing strands of a double-stranded target nucleic acid. These two regions may be separated from each other by 1 to about 10, 15, 20, 25, 30, 35, or 40 base pairs, and preferably by 1 to 20 base pairs. The region that separates these two regions contains the defined position at which a nucleotide of interest resides.
  • the present invention uses restriction endonuclease(s) that cleave nucleic acid molecules outside their recognition sites.
  • a “restriction endonuclease” or “restriction enzyme” refers to the class of nucleases that bind to unique double-stranded nucleic acid sequences referred to herein as a recognition site, and generate a cleavage in the double-stranded nucleic acid that results in either blunt, double-stranded ends, or single-stranded ends with either a 5' or a 3' overhang.
  • a single- stranded nucleic acid molecule may be described herein as comprising a recognition sequence for a restriction enzyme.
  • the actual recognition site for the endonuclease consists of the recognition sequence and the complement thereof.
  • a restriction endonuclease will bind to a recognition site and then cleave each strand that forms the recognitions site.
  • the position at which the endonuclease cuts the double- stranded nucleic acid molecule is referred to as the "cleavage site.”
  • the position of the cleavage site is relative to the recognition site and is a characteristic of the endonuclease.
  • the restriction endonuclease can be, for example, a Type IIS restriction endonuclease such as Bpml, Bsgl, Eco57 I, or Fok I.
  • Type IIS restriction endonucleases have asymmetric recognition sites and cleave at a specific distance of up to about 20 bp from their recognition site.
  • restriction endonuclease that cleaves outside the recognition site is advantageous because the product of endonuclease digestion can be a nucleic acid fragment smaller than that if the endonuclease cleaved within the recognition site, thereby generating a fragment that is particularly suitable for liquid chromatographic analysis.
  • the restriction endonuclease should have a cleavage site distal from its recognition site by at least 3, 4, 5, 6, 8, 10, 12, or 15 nucleotides, and preferably by at least 8 nucleotides.
  • ODNPs according to the invention can be synthesized by any method known in the art for oligonucleotide synthesis. For instance, solid phase oligonucleotide synthesis can be performed by sequentially linking 5' blocked nucleotides to a nascent oligonucleotide attached to a resin, followed by oxidizing and unblocking to form phosphate diester linkages. ODNPs of the present invention may then be isolated.
  • isolated refers to a molecule that is substantially free of undesired contaminants, such as molecules having other sequences.
  • ODNPs are used to amplify a segment from a sample of a target nucleic acid.
  • amplification refers to any process using a pair of ODNPs described above that produces a target nucleic acid fragment between, and including, the portion complementary to the 5' ends ofthe pair of ODNPs. Any amplification method known in the art may be used such as PCR methods disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159.
  • PCR methods are also described in several books, e.g., Gelfand et al., "PCR Protocols: A Guide to Methods and Application” (1990); Burke (ed), “PCR: Essential Techniques”; McPherson et al. "PCR (Basic: From Background to Bench).”
  • a DNA polymerase e.g., Taq or Pfu polymerase
  • the ODNPs will bind to the target and the polymerase will cause the ODNPs to be extended along the target nucleic acid sequence by the addition of nucleotides.
  • the extended ODNPs will dissociate from the target to form reaction products, excess ODNPs will bind to the target and to the reaction product, and the process is repeated until the sequence of interest is amplified.
  • Exemplary PCR conditions include, but are not limited to, the following: 100 ⁇ l PCR reactions comprise 100 ng target nucleic acid; 0.5 ⁇ M of each first ODNP and second ODNP; 10 mM Tris, pH 8.3; 50 mM KC1; 1.5 mM MgCl 2; 200 ⁇ M each dNTP; 4 units TaqTM DNA Polymerase (Boehringer Mannheim; Indianapolis, IN), and (optionally) 880 ng TaqStartTM Antibody (Clontech, Palo Alto, CA).
  • Exemplary thermocycling conditions may be as follows: 94°C for 5 minutes to achieve initial denaturation; 45 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute; final extension at 72°C for 5 minutes.
  • Exemplary nucleic acid polymerases may include one of the thermostable DNA polymerases that are readily available in the art such as, e.g., TaqTM , VentTM or PFUTM. Depending on the particular application contemplated, it may be preferred to employ one ofthe nucleic acid polymerases having a defective 3' to 5' exonuclease activity.
  • the amplified product is cleaved (digested) using a restriction endonuclease whose recognition site comprises a recognition sequence present in an ODNP.
  • a restriction endonuclease whose recognition site comprises a recognition sequence present in an ODNP.
  • Digestion of a double-stranded nucleic acid molecule with a restriction endonuclease refers to the process of allowing the endonuclease to bind to its recognition site, cleave at its cleavage site, and release the cleavage products (segments or fragments, these terms being used synonymously).
  • each member of an ODNP pair of this invention contains a recognition sequence for the same restriction endonuclease, so that digestion of the amplified product with the endonuclease will result in cleavage at two sites and consequently the release of a defined fragment ofthe product.
  • the two ODNPs of an ODNP pair contain recognition sequences for different restriction endonucleases.
  • the first ODNP has a recognition sequence that differs from that of the second ODNP.
  • the amplification products described above are digested with two restriction endonucleases that recognize the recognition sites generated by the first and the second ODNPs.
  • the product of the restriction endonuclease digestion will be a short fragment of double-stranded DNA, whose length can be from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 14, 16, 18, 20, 22, 24, 26, 30, 35, or 40 bp, including every combination therein.
  • the short fragment of double- stranded nucleic acid can be denatured, and the resulting single-stranded segments can be subjected to liquid chromatography to provide a retention time upon identification in the eluent using, e.g., UV absorbance.
  • nucleic acid-containing eluent from liquid chromatographic analysis may be further characterized by techniques known in the art that are applicable to short oligonucleotide fragments, such as mass spectrometry. This further analysis may be used to validate the conclusion reached after liquid chromatographic analysis.
  • a surprising discovery of the present invention is that liquid chromatography can be used to identify small nucleic acid molecules and/or distinguish small nucleic acid molecules from one another.
  • two nucleic acid molecules may be of the same length, i.e., contain the same number of nucleotides, and differ from each other only in their sequences at one nucleotide position, and yet these two molecules can be distinguished from one another based on retention time obtained from liquid chromatographic analysis performed according to the present invention.
  • nucleic acid molecules that are of the same length and of the same nucleotide composition, and differ from each other only in the order in which the nucleotides are arranged may be distinguish from one another based on retention time obtained from liquid chromatographic analysis performed according to the present invention.
  • the order of two nucleotides in a nucleic acid molecule can be switched to provide a variant nucleic acid molecule, and the original and variant nucleic acid molecules may be distinguished by their different retention times as observed by liquid chromatographic analysis (see, e.g., 8merB and 8merC in Example 1).
  • an important factors in being able to use retention time to distinguish two similar nucleic acid molecules is the selection of the mobile phase for the chromatographic analysis.
  • the mobile phase is a gradient formed from two buffer solutions (Buffer A and Buffer B). That is, after the nucleic acid molecules have been applied to the column, Buffer A, or an elution buffer that is largely Buffer A, is used to elute the molecules. During the course of the chromatography, Buffer B is gradually (incrementally) added to Buffer A so that the eluting buffer gradually becomes enriched in the components specific to Buffer B.
  • Buffer A is an aqueous solution of an (one or more) ammonium salt
  • Buffer B is an organic solution of an (one or more) ammonium salt.
  • an aqueous solution necessarily contains water, but may also contain non-aqueous components including organic components, e.g., organic solvent(s).
  • an organic solution necessarily contains organic material, but may and typically will also contain non-organic components including inorganic components, e.g., water as a solvent.
  • the water used in Buffers A and/or B is preferably HPLC grade water, where this quality of water is well known in the art.
  • the ammonium salt(s) present in Buffer A need not be identical to the ammonium salt(s) present in Buffer A, however, in one aspect ofthe invention Buffer A and Buffer B contain the same ammonium salts.
  • Buffer A or Buffer B may, independently of the other, contain more than one ammonium salt, however, in one aspect of the invention Buffer A and Buffer B each contain a single ammonium salt structure. In one aspect of the invention, each of Buffer A and Buffer B contain a single ammonium salt and furthermore these two buffers contain the same ammonium salt.
  • water is the only solvent present in this buffer.
  • water constitutes at least 99%, at least 97%, at least 95%, at least 90%, at least 85%, or at least 80% of the total volume of solvent present in Buffer A.
  • this buffer contains both organic solvent and water.
  • water is necessarily present in Buffer B in order to solubilize the ammonium salt in the buffer.
  • the organic solvent constitutes up to 75%, up to 60%, up to 45%), up to 30%, or up to 15% of the total volume of solvents present in Buffer B, with water contributing the residual volume of solvent.
  • the volume ratio of organic sol vent: water in Buffer B is 5-75:95-25, or 10-50:90-50; or 15-40:85-60; or 20-35:80-65 with the total of the organic solvent and the water equaling 100.
  • the organic solvent this is preferably a solvent that is very miscible with water.
  • the organic solvent is miscible with water to an extent of at least about 5 vol% based on the total volume of water and organic solvent, and in various aspects is miscible to an extent of 10 vol%, 20 vol%, 30 vol%, 40 vol%, or 50 vol% (i.e., an equal volume of water and organic solvent forms a homogeneous solution).
  • a solvent is "organic" so long as it contains at least one carbon.
  • the organic solvent is preferably a liquid at room (ambient, standard) temperature and pressure. Such solvents are very well known in the art.
  • the organic solvent is an alcohol, i.e., an organic liquid at room temperature than contains at least one hydroxyl (OH) group.
  • exemplary alcohols include, without limitation, methanol, ethanol, ethylene glycol, wo-propanol, r ⁇ -propanol, propylene glycol, and glycerol.
  • the organic solvent is acetonitrile.
  • the organic solvent is dimethylsulfoxide (DMSO).
  • ammonium salt(s) these are selected from primary (RNH ), secondary (R NH) or tertiary (R 3 N) amines that are complexed with protic acid (H-A).
  • the ammonium salt is a primary amine
  • the ammonium salt is a secondary amine
  • the ammonium salt is a tertiary amine
  • the ammonium salt is selected from secondary and tertiary amine salts.
  • the ammonium salt should be soluble in the solvent(s) that form the buffer into which the ammonium salt is to be added.
  • the ammonium salt is water soluble at a concentration up to 1,000 mM, or up to 800 mM, or up to 600 mM, or up to 400 mM, or up to 200 mM, or up to 100 mM. Particularly if the liquid chromatographic analysis will be followed by mass spectroscopic analysis, it is desirable to minimize the amount of ammonium salt in the fractions that are obtained from liquid chromatography. High concentrations of ammonium salt can impair the ionization efficiency ofthe mass spectrometer, particularly when the mass spectrometer operates by electrospray ionization. Ammonium salts typically display water solubility so long as the R groups that form the ammonium salts are not, as a whole, too hydrophobic. In various aspect the ammonium salt is selected so as to provide an aqueous solution with a pH in the range of from 5.0 to 9.0, or 5.5 to 8.5, or 6.0 to 8.0, or 6.5 to 7.5.
  • an R group is, independently at each occurrence within an ammonium molecule, and for each of primary, secondary and tertiary ammonium salts, hydrocarbon, i.e., the R group is formed entirely of hydrogen and carbon; hydrocarbons with 1-10 carbons; hydrocarbons with 1-6 carbons; hydrocarbons with 5-8 carbons; selected from alkyl and cycloalkyl groups; selected from methyl, ethyl, propyl (including geometric isomers thereof), butyl (including geometric isomers thereof), pentyl (including geometric isomers thereof), hexyl (including geometric isomers thereof), cyclohexyl, and cycopentyl (including methyl-substituted cyclopentyl); selected from organic groups having an atomic mass of 15-250, which may or may not include atoms other than carbon and hydrogen; halide substituted in one occurrence; and/or halide substituted in two occurrences; where an R group may according to the present invention
  • alkyl and cylcoalkyl groups may contain unsaturation, e.g., a double bond, however cycloalkyl does not include aromatic rings. Nevertheless, in one aspect the R group is an aromatic ring selected from phenyl and C ⁇ -C 6 alkyl substituted phenyl. In another aspect, the R group is an alkyl group having aryl substitution, e.g., benzyl. In one aspect, the ammonium salt is a secondary or tertiary ammonium salt having alkyl or cycloalkyl groups with 5-8 carbons.
  • the ammonium salt contains a cationic, i.e., protonated form of triethylamine, diallylamine, diisopropylamine, N,N- dimethyl-N-cyclohexylamine, or N,N-dimethyl-N-isopropylamine.
  • the ammonium salt is the salt form of N-N-dimethylaminobutane.
  • the ammonium salt is the salt form of N,N-dimethylcyclohexylamine.
  • the ammonium salt is the salt form of triethylamine.
  • the selection ofthe amino component ofthe ammonium salt can have an effect on the signal generated by the nucleic acid molecule using liquid chromatography followed by mass spectroscopy. For instance, while dimethylaminobutane and dimethylcyclohexylamme provide similar responses, triethylamine decreases response as observed by mass spectrometry following liquid chromatography, by about 25% compared to dimethylaminobutane.
  • the protic acid of the ammonium salt is of the formula Ra-COOH so that the anionic counterion to the ammonium group has the formula Ra-COO .
  • Ra is hydrogen (i.e., the counterion is derived from formic acid) methyl, ethyl, propyl, selected from methyl and ethyl, or selected from methyl, ethyl and propyl, and mono- and poly-halogenated versions thereof.
  • the protic acid used to form the ammonium salt is a protonated form of the carbonate family of anions, i.e., bicarbonate (HCO ) and/or carbonate (CO 3 ) is the anionic counterion to the ammonium group.
  • the protic acid is an inorganic acid, e.g., HC1 and HBr.
  • the protic acid of the ammonium salt is acetic acid, formic acid, carbonic acid (so as to provide bicarbonate anion), or hydrogen chloride.
  • the ammonium group is a tertiary ammonium group having R groups as defined above, and the counterion is acetate (not including halogenated acetate), carbonate/ bicarbonate, or is selected from acetate (not including halogenated acetate) and carbonate/ bicarbonate.
  • exemplary ammonium salts include, without limitation, triethylamine acetate, dimethylbutamine acetate, dimethylisopropylamine acetate, dimethylhexylamine acetate, dimethylcyclohexylamme acetate, and disopropylamine acetate.
  • the conditions under which the liquid chromatography is operated are important in being able to resolve, or distinguish, two nucleic acid structures of identical length but of somewhat different nucleotide base sequence.
  • the liquid chromatographic analysis of the present invention is carried out at either room temperature, i.e., about 25°C, or at elevated temperature. Typically, elevated temperatures are less than 75°C.
  • the chromatography column is maintained within the following temperature ranges during the chromatography: 20°C-80°C, 25°C-70°C, 25°C-65°C, 25°C-60°C, 30°C- 80°C, 30°C-70°C, 30°C-65°C, 30°C-60°C, 30°C-55°C, 30°C-50°C, or 30°C-45°C.
  • Elevated temperature is typically desirable because it may provide a chromatogram wherein the peaks are higher, narrower, and display greater resolution. However, as the temperature exceeds about 70°C, bubble formation in the eluent is sometimes observed and this may cause a loss in resolution.
  • the pH of the elution buffer is also an important factor in the successful chromatographic analysis.
  • the elution buffer maintains a pH ranging from about 5.0 to 9.0, as discussed above.
  • retention time is unaffected so long as the pH of the elution buffer is maintained within this pH range.
  • the chromatography is typically run under pressure, i.e., pressure is used to push the elution buffer through the column. As the pressure is increased there is typically an increase in the flow rate of the buffer through the column.
  • a pressure ranging from about 200-1600 bars, and a flow rate ranging from about 10 ⁇ L to 2000 ⁇ L per minute are typically suitable conditions for operating the column chromatography.
  • the length and stationary phase of the liquid chromatography column are two other parameters that must be selected.
  • the column is 18-500 mm in length.
  • the column is 18, 25, 50, 100, 250, or 500 mm in length and can be at a micro-, or macro-scale.
  • the stationary phase is selected so as to provide a reverse phase column, i.e., a column having a hydrophobic phase surrounding the solid phase.
  • a suitable reverse phase column is the MICROSORBTM C18 column from Varian Inc. (Palo Alto, CA; www. varianinc . com) .
  • This column is a monomeric silica column, with 5 micron spherical particles, 300A pores, C18 stationary phase, 12% carbon load and is endcapped.
  • a substantially equivalent column is the JUPITERTM C18 column made by Phenomenex U.S.A. (Torrance, CA; www.phenomenex.com).
  • a similar column is the XTERRATM column from Waters (Milford, MA: www.waters.com) which contains a hybrid particle made of silica and polymer to extend pH stability with a pore size of 120A and particle size from 2.5 micron to 5 micron. Columns having a completely polymeric solid support may also be used.
  • the reverse phase column has a C18 stationary phase and a pore size of at least 120 A. Endcapping of silica columns is desirable in order to minimize tailing and improve peak shape, and accordingly a preferred column has this feature.
  • the carbon load of the column is important to ensure sufficient retention (carbon load does not apply to polymeric columns), and is preferably in the range of 5-20%.
  • the particle size typically varies from about 2 microns up to about 10 microns, where these are typical sizes for columns that are currently commercially available. Smaller particle size generally result in improved chromatography, so particle sizes smaller than 5 ⁇ M are preferred. Column dimensions are not critical, but may be chosen based on scale and type of analysis.
  • a small column is preferred, such as the 2.1 x 15 mm (diameter x length) XTERRATM column with 2.5 ⁇ M particles allowing a complete run in four minutes at a flow rate of 250 ⁇ L/min.
  • a larger column where larger columns are about 4.6 mm in diameter and about 250 mm in length.
  • Columns having dimensions from 0.3 mm to 4.6 mm in internal diameter and from 10 mm to 250 mm long are available from many commercial suppliers (e.g., Water) and are suitably used in the present invention.
  • a typical LCMS column will be 1 mm x 50 mm.
  • the flow rate will be dependent on the column dimensions and will vary from a few microliters per minute for a 0.3 mm ID column up to about 1500-2000 microliters per minute for a 4.6 mm ID column.
  • the mobile phase is preferably formed by incrementally combining two different solutions (Buffers A and B).
  • the salt is preferably present in the solution at a concentration of 1 mM to 200 mM, more preferably at a concentration of 1 mM to 100 mM, still more preferably at a concentration of 5 mM to 50 mM. Particularly when the liquid chromatographic analysis is followed by mass spectroscopic analysis, lower salt concentration is preferred, and in such a situation a salt concentration of about 5 mM is preferred.
  • Buffer B preferably contains 10-90% (volume/volume) polar organic molecules (e.g., acetonitrile, methanol, or isopropanol) in Buffer A.
  • Buffers A and/or B may contain optional components, and in one aspect an optional component present in Buffer A is also present in Buffer B.
  • a suitable optional component is EDTA, where EDTA may be used at a concentration of about 0.1 mM in the buffers.
  • liquid chromatography according to the present invention may be performed as follows. First, a shallow gradient of acetonitrile or other suitable solvent may be used to elute the nucleic acid molecules and provide for sample clean up. For instance, when using acetonitrile, the gradient can start at, or about at, 5% and increase to, or about to, 20%, where these percent values refer to the volume percent of organic solvent in water as the elution buffer.
  • Methanol can be used in place of acetonitrile, but the use of methanol may require increasing the final gradient composition to 50% or even 75% methanol.
  • the fraction of solvent required depends partly on the column used, and also on the length range of nucleic acid molecules being analyzed. Generally a strong solvent wash is applied to the column at the end ofthe run to elute any large, hydrophobic components.
  • the analysis portion of the gradient starts at 5% acetonitrile and increases to 15% over about 90 seconds, where this is followed by a wash which quickly pushes a "plug" of 45% acetonitrile onto the column for just a few seconds followed by a return to starting conditions of 5% acetonitrile.
  • a preferred buffer system incorporates 5 mM N,N- dimethylaminobutyl acetate, and operates at pH 7. Concentrations of ammonium salts from 1 mM up to 50 mM are observed to have equivalent response in a mass spectrometer (e.g., there is no evidence of ion suppression within this range) but variation of ammonium salt concentration within this range may have slight effects on the retention times of the nucleic acid molecules. Variation in retention times may be compensated for by adjusting the solvent composition during the LC run. As mentioned above, the pH range is flexible, where a range from pH 6 to pH 8 can be used with little or no noticeable change in outcome, i.e., little or no effect on nucleic acid retention times.
  • the present invention provides kits.
  • the kit includes a container holding ammonium salt and a separate container holding organic solvent.
  • the container of ammonium salt and/or the container of organic solvent may include optional ingredients, e.g., EDTA and/or water.
  • the container of ammonium salt and the separate container of organic solvent each contain water so that the ammonium salt is present in a dissolved form in the kit. In another aspect, the water is held in a separate container.
  • kits in another aspect includes a container that holds all the ingredients necessary to make Buffer A, except that water is not present in the container, and also separately includes a container that holds all the ingredients necessary to make Buffer B, except that water is not present in the container.
  • Water may, or may not, be included in a separate container that is found within the kit.
  • the water is preferably HPLC grade water.
  • kits of the present invention contain one, each combination of two, each combination of three, each combination of four, or all five of the following components:
  • a container holding components comprising ammonium salt dissolved in water, where the water should preferably be HPLC grade water.
  • the ammonium salt may be any of the one or more ammonium salts identified above.
  • the ammonium salt is N,N-dimethyl-N-butylammonium with a counterion, where, in a further aspect, the counterion is acetate.
  • the ammonium salt is present in the water at a concentration of about 1 - 200 mM, preferably about 1-100 mM, preferably about 5-50 mM, and more preferably about 5 mM.
  • the container may hold ammonium salt at a concentration greater than 100 mM, but in such case the components in this container will probably need to be diluted with water in order to form an effective buffer.
  • This solution of ammonium salt and water preferably has a pH of about 7.0-7.5, and is typically about 7.2, and is referred to herein as Buffer A. In one aspect, this container holds only ammonium salt dissolved in water.
  • a container holding components comprising ammonium salt, water and organic solvent comprising Buffer A and organic solvent, where the mixture is preferably homogeneous, i.e., the organic solvent dissolves or is miscible in Buffer A.
  • the organic solvent is acetonitrile.
  • the organic solvent is methanol.
  • the organic solvent constitutes, on a volume percent basis, based on the total volume of the components in the container, 15%-20%, 20%-25%, 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%- 50% or 50%-55% or 55-60% or 65-70% or 75-80%.
  • the organic solvent constitutes 25% acetonitrile.
  • the organic solvent constitutes 50% acetonitrile.
  • a preferred component is a solution of 75 vol% Buffer A and 25 vol% acetonitrile, while another preferred component is a solution of 50 vol% Buffer A and 50 vol% acetonitrile.
  • a chromatography column preferably a reverse phase chromatography column as described above, may be included in the kit.
  • a preferred reverse phase chromatography is a C18 reverse phase chromatography column.
  • a preferred C18 reverse phase column has a pore size of at least 120A.
  • a preferred C18 reverse phase column has a particle size of 2 microns to 10 microns.
  • a preferred column size is 0.3 mm to 4.6 mm in inner diameter and from 10 mm to 250 mm in length.
  • the instructions booklet will provide useful information for running the column chromatographic analysis. Useful information may include a description of how to program a chromatogram using the two Buffers ofthe present invention.
  • a container holding water is preferably HPLC grade water.
  • the kit may optionally include one or more components useful in preparing the short nucleic acid molecules that will be characterized by the chromatographic technique.
  • the kit may include primers as described herein, restriction enzymes as described herein, and/or polymerase and other components necessary for amplification of nucleic acid.
  • two nucleic acid molecules are separated by liquid chromatography, where these two molecules each have 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18 nucleotides and, furthermore, these two nucleic acid molecules either have the same base sequence of nucleotides with one exception, or they are made from the same nucleotides but these nucleotides are arranged in a different sequence.
  • higher temperature and/or higher organic solvent concentration are generally preferred when the nucleic acid molecule contains much more than about 8-10 nucleotides.
  • the two nucleic acid molecules may have the sequences 5'- A-G-C-T-A-3' and 5'-A-G-A-T-A-3', where this is an example of two nucleic acid molecules each having 5 nucleotides where the two nucleic acid molecule have the same base sequence of nucleotides with one exception (C vs. A in the third position from the 5' end).
  • the two nucleic acid molecules may have the sequences 5'-A-C-G-T-A-3' and 5'-A-G-C-T-A-3', where this is an example of two nucleic acid molecules each having 5 nucleotides where the two nucleic acid molecules are made from the same nucleotides but these nucleotides are arranged in a different sequence.
  • the two nucleic acid molecules are composed of identical nucleotides, but the order of the nucleotides in the two nucleic acid molecules is non-identical.
  • the two nucleic acid molecules each have the sequence 5'-n-X-m-3', where n and m each represent a sequence of from 0-10 nucleotides, and X represents a single nucleotide, and the two nucleic acid molecules each have the same sequences n and m, but differ in the identify of the nucleotide at location X.
  • the present invention provides a method of performing liquid chromatography comprising applying two nucleic acid molecules to a liquid chromatography column, where the two nucleic acid molecules have an identical number of nucleotide bases, but have different nucleotide sequences; and eluting the two nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules have different elution times; the elution buffer being formed from Buffer A and Buffer B, where elution buffer of incrementally increasing organic solvent concentration is applied to the column, where the elution buffer comprises an ammonium salt and the ammonium salt comprises a secondary or tertiary amine complexed with an organic or inorganic acid.
  • nucleic acid molecules may be applied to a liquid chromatography column.
  • four nucleic acid molecules may be applied to the column, where those four nucleic acid molecules consist of two pairs of nucleic acid molecules, each pair consisting of two nucleic acid molecules that have the same number of nucleotide bases, but have different nucleotide sequences.
  • SNP single nucleotide polymorphism
  • the present invention may also be used in genotyping genetic variations in a defined location of a wild type sequence resulting from insertions, deletions or substitutions involving more than one nucleotide.
  • the insertions, deletions or substitutions involve up to 20 nucleotides, and typically involve 1, 2, 3, 4 or 5 nucleotides.
  • single nucleotide polymorphism refers to any nucleotide sequence variation that involves a single nucleotide, preferably one that is common in a population of organisms and is inherited in a Medelian fashion. Typically, an SNP is either of two possible nucleotides, and there is no possibility of finding a third or fourth nucleotide identity at an SNP site.
  • a "wild type” sequence refers to a nucleotide sequence that is most popular in a selected population of an organism from which the sequence may be isolated.
  • the genetic variation may be characterized first.
  • the sequence containing the genetic variation may be determined first.
  • (1) a nucleic acid fragment from the individual, (2) a wild type sequence, and (3) a sequence having the pre-determined genetic variation are then individually used as a template for amplification using specifically designed ODNPs as described above.
  • the resulting products from the three separate amplification reactions are then separately digested with restriction endonuclease(s) that recognize the recognition site(s) created from the recognition sequences in the ODNPs.
  • the short digestion product from each digestion reaction is subsequently subjected to liquid chromatographic analysis.
  • the result of this analysis using the nucleic acid fragment from the individual as the amplification template ("Result 1") is compared with that using a wild type sequence ("Result 2”) or a sequence containing the genetic variation
  • Result 3 as the template. If Result 1 is the same as Result 2, the individual has a wild type genotype with respect to that defined position. If Result 1 is the same as
  • the individual has the predetermined genetic variation at the defined position.
  • the present method may also be used to determine whether a subject organism (e.g., a person) is homozygous or heterozygous as to a particular genomic fragment or gene. If a person is homozygous with respect to that genomic fragment or gene, there will be two peaks corresponding to the two single-stranded DNA fragments resulting from the denaturation of a short double-stranded digestion fragment detected by liquid chromatography. If the person is heterozygous with respect to that genomic fragment or gene, there will be four peaks detected corresponding to four single- stranded DNA fragments resulting from the denaturation of two short double-stranded digestion fragments: one corresponding to one allele from one parent, the other from the other parent.
  • a subject organism e.g., a person
  • target nucleic acids as double- stranded nucleic acids
  • present invention is applicable to single-stranded target nucleic acids as well.
  • a single-stranded target nucleic acid may be used as a template for synthesizing its complementary strand and thus becomes double-stranded nucleic acid before forming a mixture with an ODNP pair of the present invention.
  • a single-stranded target nucleic acid may be directly mixed with the ODNPs of the present invention and its complementary strand is produced using one ofthe ODNPs as a primer during the amplification reaction for producing a nucleic acid containing the nucleotide(s) at a defined location.
  • the present invention provides, in one aspect, a method for identifying one or more nucleotide bases at a defined position of a polynucleotide, where the position is defined relative to known nucleotide base sequences at both a 3 ' and 5 ' direction from the defined position, comprising:
  • nucleotide base sequence that is a recognition sequence for a restriction endonuclease, the restriction endonuclease having a cleavage site outside the recognition sequence
  • nucleotide base sequence that is a recognition sequence for a restriction endonuclease, the restriction endonuclease having a cleavage site outside the recognition sequence
  • the above method may also be applied to oligonucleotides, preferably oligonucleotides having more than 50 bases.
  • the present invention provides a method for identifying one or more nucleotide bases at a defined position of a polynucleotide, where the polynucleotide is preferably DNA, and more preferably a cDNA or genomic DNA, and where the position is defined relative to known nucleotide base sequences at both a 3 ' and 5 ' direction from the defined position, comprising: amplifying the polynucleotide from a subject using a pair of primers (as described above, or as describe below) to thereby form amplified polynucleotide; digesting the amplified polynucleotide with a restriction endonuclease to form a short nucleic acid molecule; and characterizing the short nucleic acid molecule by liquid chromatography, so as to identify the one or more nucleotide bases at the defined position.
  • a pair of primers as described above, or as describe below
  • each primer comprises a linear oligonucleotide comprising a 5' and a 3' end, said oligonucleotide consisting of at least 35 nucleotides, wherein a first portion of said oligonucleotide of at least 13 nucleotides at the 5' end of said oligonucleotide and a second portion of the oligonucleotide of from 5 to 22 nucleotides at the 3' end ofthe oligonucleotide are at least substantially complementary to a first portion and a second portion of the polynucleotide, wherein 4-8 nucleotides between the first and second portions of each oligonucleotide comprise a recognition sequence for a restriction enzyme that cleaves at least 5 nucleotides from its recognition site, wherein the segment ofthe polynucleotide (e.g., cDNA or genomic
  • the present invention provides the following:
  • a method of performing liquid chromatography comprising: applying two nucleic acid molecules to a liquid chromatography column, where the two nucleic acid molecules have an identical number of nucleotide bases, but have different nucleotide sequences; and eluting the two nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules have different elution times; the elution buffer being formed from Buffer A and Buffer B, where elution buffer of incrementally increasing organic solvent concentration is applied to the column, where the elution buffer comprises an ammonium salt and the ammonium salt comprises a secondary or tertiary amine complexed with an organic or inorganic acid.
  • the ammonium salt includes the protonated form of a secondary amine of the formula R NH and R at each occurrence is independently selected from C]-C] 0 hydrocarbon groups, and the hydrocarbon is optionally an alkyl or cycloalkyl group;
  • the ammonium salt is includes the protonated form of a secondary amine ofthe formula R NH and R at each occurrence is independently selected from C ⁇ -C 6 hydrocarbon groups, where the hydrocarbon of the amine group is optionally selected from alkyl and cycloalkyl groups;
  • the ammonium salt is the protonated form of a secondary amine ofthe formula R 2 NH, and R at each occurrence is independently selected from Cs- hydrocarbon groups, and the hydrocarbon is optionally selected from alkyl and cycloalkyl groups;
  • the amine component of the ammonium salt is a secondary amine selected from diallylamine and diis
  • a method of performing liquid chromatography comprising: applying two nucleic acid molecules to a liquid chromatography column, where the two nucleic acid molecules have an identical number of nucleotide bases within the range of 2-10, but have different nucleotide sequences, and the liquid chromatography column is a reverse phase chromatography column; and eluting the two nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules have different elution times; the elution buffer being formed from Buffer A and Buffer B, where elution buffer of incrementally increasing organic solvent concentration is applied to the column, where the elution buffer comprises an ammonium salt and the ammonium salt comprises a secondary or tertiary amine complexed with, i.e., is the reaction product of, an organic or inorganic acid.
  • a method of performing liquid chromatography comprising: applying two nucleic acid molecules to a liquid chromatography column, where the two nucleic acid molecules have an identical number of nucleotide bases, but have different nucleotide sequences; and eluting the two nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules have different elution times; the elution buffer being formed from Buffer A and Buffer B, where Buffer A comprises water and an ammonium salt that is formed from a secondary or tertiary amine complexed with an organic or inorganic acid; and Buffer B comprises water, organic solvent, and an ammonium salt that is formed from a secondary or tertiary amine complexed with an organic or inorganic acid; where elution buffer of incrementally increasing organic solvent concentration is applied to the column.
  • the two nucleic acid molecules are composed of identical nucleotides, but the order of the nucleotides in the two nucleic acid molecules is non-identical; the two nucleic acid molecules each have the sequence 5'-n-X-m-3', where n and m each represent a sequence of from 0-10 nucleotides, and X represents a single nucleotide, and the two nucleic acid molecules each have the same sequences n and m, but differ in the identify of the nucleotide at location X; and the two nucleic acid molecules each have 2-10 nucleotides.
  • a method of performing liquid chromatography and mass spectrometric analysis comprising: applying a plurality of pairs of nucleic acid molecules to a liquid chromatography column, where each pair of nucleic acid molecules is formed from two nucleic acid molecules that have an identical number of nucleotide bases, but have different nucleotide sequences; eluting the plurality of pairs of nucleic acid molecules from the column with an elution buffer so that the two nucleic acid molecules that form each pair have different elution times; and characterizing each nucleic acid molecule by mass spectroscopy; the elution buffer being formed from Buffer A and Buffer B, where elution buffer of incrementally increasing organic solvent concentration is applied to the column, where the elution buffer comprises an ammonium salt and the ammonium salt comprises a secondary or tertiary amine complexed with an organic or inorganic acid.
  • each member of a pair of two nucleic acid molecules is composed of identical nucleotides, but the order of the nucleotides in the two nucleic acid molecules is non-identical; each member of a pair of two nucleic acid molecules has the sequence 5'-n-X-m-3', where n and m each represent a sequence of from 0-10 nucleotides, and X represents a single nucleotide, and the two nucleic acid molecules each have the same sequences n and m, but differ in the identify of the nucleotide at location X; the liquid chromatography column is a reverse phase chromatography column; the liquid chromatography column is a C18 reverse phase chromatography column; the liquid chromatography column is a C18 reverse phase chromatography column having a pore size of at least 120 A; the liquid chromatography column is a Cl 8 reverse phase
  • a composition referred to as Buffer B comprising water, the reaction product of secondary or tertiary amine with organic or inorganic acid, and organic solvent.
  • the reaction product of secondary or tertiary amine with organic or inorganic acid is selected from an acetate salt of an amine selected from the group consisting of triethylamine, diallylamine, diisopropylamine, N,N-dimethyI-N- cyclohexylamine, N,N-dimethyl-N-isopropylamine, and N,N-dimethyl-N-butylamine.
  • this composition has a pH of 6-8.
  • the organic solvent is selected from methanol and acetonitrile.
  • the composition has a water:organic solvent ratio of 95-25:5-75.
  • the reaction product is present in Buffer B at a concentration of 1-100 mM.
  • a composition comprising water, the reaction product of secondary or tertiary amine with organic or inorganic acid, organic solvent, and two nucleic acid molecules, where the two nucleic acid molecules have an identical number of nucleotide bases, but have different nucleotide sequences.
  • the two nucleic acid molecules are composed of identical nucleotides, but the order ofthe nucleotides in the two nucleic acid molecules is non-identical; optionally, the two nucleic acid molecules each have the sequence 5'-n-X-m-3', where n and m each represent a sequence of from 0-10 nucleotides, and X represents a single nucleotide, and the two nucleic acid molecules each have the same sequences n and m, but differ in the identify of the nucleotide at location X; optionally the two nucleic acid molecules have 4-10 nucleotides; optionally the reaction product of secondary or tertiary amine with organic or inorganic acid is selected from an acetate salt of an amine selected from the group consisting of triethylamine, diallylamine, diisopropylamine, N,N-dimethyl-N- cyclohexylamine, N,N-dimethyl-N-isopropylamine
  • the composition comprises four nucleic acid molecules, the four nucleic acid molecules being two pairs of nucleic acid molecules, each pair of nucleic acid molecules being formed from two nucleic acid molecules that have an identical number of nucleotide bases, but have different nucleotide sequences.
  • a kit for chromatographic analysis comprising: (a) a container holding components comprising water and the reaction product of, i.e., the salt of, secondary or tertiary amine with organic or inorganic acid (Buffer A); and (b) a container holding components comprising the components of (a) and organic solvent (Buffer B).
  • the reaction product of (salt of) secondary or tertiary amine with organic or inorganic acid is selected from an acetate salt of an amine selected from the group consisting of triethylamine, diallylamine, diisopropylamine, N,N-dimethyl-N- cyclohexylamine, N,N-dimethyl-N-isopropylamine, and N,N-dimethyl-N-butylamine; optionally the reaction product of secondary or tertiary amine with organic or inorganic acid is present in the water at a concentration of 1-200 mM; optionally the reaction product of secondary or tertiary amine with organic or inorganic acid is present in the water at a concentration of about 5 mM; optionally the organic solvent is selected from methanol and acetonitrile; optionally the water is HPLC grade water; optionally the kit further includes a (i.e., one or more) chromatography column; optionally the chromatography column is
  • the chromatography system is from Varian (Walnut Creek, CA) and is a ProStar Helix System (catalog # HelixsysOl) that is composed of two pumps, a column oven, a UV detector, a degasser, a mixer and an autoinjector.
  • the column is a Varian Microsorb MV (catalog number R0086203F5), C18 packing with 5 uM particle size, with 300 Angstroms pore size, 4.6 mm x 50 mm.
  • the column was run at 30°C to 40°C with a gradient of acetonitrile in 100 mM triethylamine acetate (TEAA) and 0.1 mM EDTA. The type of gradient is described in the text.
  • 10-merB 5'-GAATATCCAC -3' (SEQ ID NO. 2)
  • 10-merC 5'-GAACATCCAT -3' (SEQ ID NO. 3)
  • the polymorphisms in the 8-mers and 10-mers are underlined.
  • the 8- mers B and C differ from 8-mer A by only a single base.
  • the 10-mers B and C differ from 10-mer A by only a single base.
  • Buffer A is 100 mM TEAA with 0.1 mM EDTA
  • Buffer B is 100 mM TEAA with 0.1 mM EDTA and 25% (V/V) acetonitrile
  • the column was run at 40°C by adjusting the column oven to 40°C. The flow rate was 1.5 ml per minute.
  • the injection volume was 10 microliters and 200 nanogram of fragment was injected per 10 microliter volume.
  • Different combinations of the 4-mer, 6-mer, 8-mer and 10-mer were injected to determine the chromatographic behavior.
  • the first result is shown in Figure 5.
  • Trace 1 in Figure 5 all 8 fragments composed of the 4-mer, 6-mer, 8-mer and 10-mer were separated. All three 8-mers and all three 10-mers were separated even though they differed by only a single base. The fragments are single-stranded.
  • the order of elution in Trace 1 is (from left to right): 4-mer, 6-mer, 8-merB, 8-merA, 10-merA, 8-merC, 10-merB, 10-merC.
  • Trace 2 the 6-mer and 10-merC were coinjected and the elution times of the 6-mer and 10- merC were the same as seen in Trace 1.
  • Trace 3 the three 10-mers were co-injected and separated. The elution times ofthe three 10-mers were the same as seen in Trace 1.
  • Trace 4 the three 8-mers were co-injected and separated. The elution times of the three 8-mers were the same as seen in Trace 1.
  • Trace 5 shows a single peak of 8-merA and Trace 6 shows a single Trace of 8-merB. Genotypes can be directly inferred from the retention times during the cliromatography, even from fragments that differ by only a single base.
  • Figure 6 shows HPLC fractionation and detection of three 8-mers (Fig. 6A) and three 10-mers (Fig. 6B).
  • panel A the "T” allele at position 2 of the 1 st 8- mer is discriminated from the "C” allele at position 2 ofthe 2 nd 8-mer and the "T” allele at position 5 ofthe 2 nd 8-mer from the "C” allele at position 5 ofthe 3 rd 8-mer.
  • panel B the "T” allele at position 4 of the 3 rd 10-mer is discriminated from the "C” allele at position 4 ofthe 2 nd 10-mer and the "C” allele at position 10 ofthe 2 nd 10-mer from the "T” allele at position 10 ofthe 1 st 10-mer.
  • Genotypes can be directly inferred from the retention times during the chromatography, even from fragments that differ by only a single base.
  • Figure 7 in Panel A, one 4-mer (4-merA), one 6-mer (6-merA), three 8-mers (8-merA, 8-merB and 8-merC) and three 10-mers (10-merA, 10-merB, and 10- merC) are separated, and in Panel B, two 6-mers are shown eluting between 2 and 3 minutes. The 6-mers were generated by double Fok I digestion of a 41-mer which contained the forward and reverse Fok I recognition site which was separated by 6 nucleotides.
  • FOK I RESTRICTION ENDONUCLEASE The following example describes the amplification of a specific sequence from the lambda genome in which the primers contain the Fok I recognition sequence (both the forward and reverse primers contain this sequence).
  • the resulting amplicon contains a "double-Fok I" cutting site which liberates a small oligonucleotide fragment, which is then subjected to a chromatography step and identified by UV absorbance.
  • Two sets of primers were designed to generate two different amplicons from two different regions of the lambda genome.
  • the Fok I recognition sequences of the primers are in bold face.
  • RE5P01F 5'- GAAGTGATGGGGATGCGGAAAGAG-3' (SEQ ID NO. 4)
  • RE5P02R 5'- GTAAGCCACAGGATGAGGAACGGG-3'
  • RE5P03F 5'-AAAGCTGGCAGGATGACCGGCAGA -3'
  • RE5P04R 5'-AGCGTCTGTTGGATGTCGTGGCGG -3'
  • RE5P01F and RE5P02R are primer set one and RE5P03F and RE5P04R are primer set 2.
  • All oligonucleotides were synthesized by Midland Reagent CO. of Midland TX.
  • the templates for primer set one and two are as follows:
  • PCR reaction mixture was used in 25 ⁇ l volumes: The 25 ⁇ l PCR reactions were composed of 25 ng genomic DNA, 0.5 ⁇ M forward and reverse primers, 10 M Tris pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 200 ⁇ M each dNTP, 4 Units Taq DNA Polymerase (Boehringer Mannheim, Indianapolis, IN), and 880 ng TaqStart Antibody (Clontech, Palo Alto, CA).
  • Thermocycling conditions were as follows: 94°C for 5 minutes initial denaturation; 45 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute; final extension at 72°C for 5 minutes.
  • a MJ Research 9600 thermocycler (MJ Research, Watertown, MA) was used for all PCR reactions.
  • Buffer A is 100 mM TEAA with 0.1 mM EDTA
  • Buffer B is 100 mM TEAA with 0.1 mM EDTA and 25% (V/V) acetonitrile
  • B was re-equilibrate the column.
  • the column was run at 40°C by adjusting the column oven to 40°C.
  • the flow rate was 1.5 ml per minute.
  • the injection volume was 10 to 30 microliters.
  • the controls for the chromatograms are shown.
  • the no-template control is shown in which the unincorporated primers (primer set 1) can be seen eluting after the 4 minute mark.
  • the "no-template” control is shown in which the unincorporated primers (primer set 2) can be seen eluting after the 4 minute mark.
  • the "plus-template” control is seen prior to cutting with Fok I. The large amplicon is seen eluting at 4.6 minutes.
  • a peak is seen at 1.24 minutes which is due to the Fok I buffer (the no-template control was mixed with the Fok I buffer components as a control). The large peak at 0.5 to 1 minute is due to the PCR components.
  • Figure 9 is shown the short fragments generated by the Fok I enzyme double digest. 6-mers (5 '-TTATTA-3 ' and its complement) and 8-mers (5'- CATTATTC-3 ' and its complement) were expected. For primer set one, peaks are seen at 2.0 and 3.2 minutes and for primers set two, peaks are seen at 2.4 and 3.3 minutes. Therefore, SNP fragments can be easily generated and detected by the double-Fok I amplification and cutting.

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Abstract

L'invention concerne le génotypage par chromatographie en phase liquide de fragments d'acides nucléiques courts qui sont des produits d'amplification. Ces produits mettent en oeuvre des oligonucléotides de conception spécifique servant d'amorces, d'une part, et des acides nucléiques cibles présentant les nucléotides recherchés et servant de matrices, d'autre part. Les oligonucléotides contiennent des séquences de reconnaissance spécifiques aux endonucléases de restriction, moyennant quoi lesdites endonucléases réalisent le clivage en dehors de ces séquences de reconnaissance. Il est possible d'effectuer une analyse rapide et fiable des fragments d'acides nucléiques courts par le biais de la chromatographie en phase liquide, éventuellement suivie d'une analyse en spectrométrie de masse, et d'identifier ainsi les nucléotides recherchés.
PCT/US2001/030828 2000-10-02 2001-10-01 Genotypage par chromatographie en phase liquide de fragments d'acides nucleiques courts WO2002028501A1 (fr)

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AU2001296491A AU2001296491A1 (en) 2000-10-02 2001-10-01 Genotyping by liquid chromatographic analysis of short nucleic acid fragments
US10/398,005 US20050089850A1 (en) 2000-10-02 2001-10-01 Genotyping by liquid chromatographic analysis of short nucleic acid fragments

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US23740900P 2000-10-02 2000-10-02
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US24717200P 2000-11-10 2000-11-10
US24716700P 2000-11-10 2000-11-10
US24727500P 2000-11-10 2000-11-10
US60/247,172 2000-11-10
US60/247,173 2000-11-10
US60/247,166 2000-11-10
US60/247,275 2000-11-10
US60/247,167 2000-11-10
US26397101P 2001-01-24 2001-01-24
US60/263,971 2001-01-24
US26924401P 2001-02-15 2001-02-15
US60/269,244 2001-02-15
US30035001P 2001-06-21 2001-06-21
US30031901P 2001-06-21 2001-06-21
US60/300,350 2001-06-21
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PCT/US2001/030743 WO2002040126A2 (fr) 2000-10-02 2001-10-01 Procedes d'identification de nucleotides dans des positions definies dans des acides nucleiques cibles au moyen de la polarisation de fluorescence
PCT/US2001/042432 WO2002029006A2 (fr) 2000-10-02 2001-10-01 Procédé pour le mesurage parallèle des variations génétiques
PCT/US2001/030742 WO2002046447A2 (fr) 2000-10-02 2001-10-01 Procede d'identification de nucleotides a des positions determines dans des acides nucleiques cibles

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PCT/US2001/042432 WO2002029006A2 (fr) 2000-10-02 2001-10-01 Procédé pour le mesurage parallèle des variations génétiques
PCT/US2001/030742 WO2002046447A2 (fr) 2000-10-02 2001-10-01 Procede d'identification de nucleotides a des positions determines dans des acides nucleiques cibles

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008642A2 (fr) * 2001-07-15 2003-01-30 Keck Graduate Institute Amplification de fragments d'acide nucleique au moyen d'agents de coupure
WO2004053159A2 (fr) * 2002-12-07 2004-06-24 Guoliang Fu Analyse d'expression genique dirigee a l'aide d'oligonucleotides

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7033762B2 (en) * 2001-08-29 2006-04-25 Amersham Biosciences Corp Single nucleotide amplification and detection by polymerase
US7052839B2 (en) 2001-08-29 2006-05-30 Amersham Biosciences Corp Terminal-phosphate-labeled nucleotides and methods of use
US7223541B2 (en) 2001-08-29 2007-05-29 Ge Healthcare Bio-Sciences Corp. Terminal-phosphate-labeled nucleotides and methods of use
DK1421213T3 (da) 2001-08-29 2010-06-07 Ge Healthcare Bio Sciences Mærkede nukleosidpolyphosphater
US7560254B2 (en) 2001-08-29 2009-07-14 Ge Healthcare Bio-Sciences Corp. Allele specific primer extension
US7256019B2 (en) 2001-08-29 2007-08-14 Ge Healthcare Bio-Sciences Corp. Terminal phosphate blocked nucleoside polyphosphates
WO2004083432A1 (fr) 2003-03-21 2004-09-30 Academisch Ziekenhuis Leiden Modulation de la reconnaissance d'exons dans le pre-arnm par interference avec la structure d'arn secondaire
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USRE48468E1 (en) 2007-10-26 2021-03-16 Biomarin Technologies B.V. Means and methods for counteracting muscle disorders
EP2119783A1 (fr) 2008-05-14 2009-11-18 Prosensa Technologies B.V. Procédé pour l'omission efficace de l'exon (44) dans la dystrophie musculaire de Duchenne et moyens connexes
JP2012524540A (ja) 2009-04-24 2012-10-18 プロセンサ テクノロジーズ ビー．ブイ． Ｄｍｄを処置するためのイノシンを含むオリゴヌクレオチド
CN112251436A (zh) 2012-01-27 2021-01-22 比奥马林技术公司 治疗杜兴型肌营养不良症和贝克型肌营养不良症的具有改善特性的rna调节性寡核苷酸

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5284758A (en) * 1992-08-14 1994-02-08 Merck & Co., Inc. Process for forming cholesterol lowering compound using pseudodiplodia sp.
US5556768A (en) * 1993-06-29 1996-09-17 Osaka Gas Company Limited Method for preparing peptides and proteins utilizing ribosome
US5837536A (en) * 1992-05-22 1998-11-17 Genetic Therapy, Inc. Expression of human multidrug resistance genes and improved selection of cells transduced with such genes
US6027898A (en) * 1997-08-18 2000-02-22 Transgenomic, Inc. Chromatographic method for mutation detection using mutation site specifically acting enzymes and chemicals

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040166A (en) * 1985-03-28 2000-03-21 Roche Molecular Systems, Inc. Kits for amplifying and detecting nucleic acid sequences, including a probe
DE69333650T2 (de) * 1992-02-19 2006-01-12 The Public Health Research Institute Of The City Of New York, Inc. Neue anordnungn von oligonukleotiden und ihr nutzen zum sortieren, isolieren, sequenzieren und manipulieren von nukleinsäuren
US6110709A (en) * 1994-03-18 2000-08-29 The General Hospital Corporation Cleaved amplified modified polymorphic sequence detection methods
US6180408B1 (en) * 1998-08-21 2001-01-30 Washington University Fluorescence polarization in nucleic acid analysis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5837536A (en) * 1992-05-22 1998-11-17 Genetic Therapy, Inc. Expression of human multidrug resistance genes and improved selection of cells transduced with such genes
US5284758A (en) * 1992-08-14 1994-02-08 Merck & Co., Inc. Process for forming cholesterol lowering compound using pseudodiplodia sp.
US5556768A (en) * 1993-06-29 1996-09-17 Osaka Gas Company Limited Method for preparing peptides and proteins utilizing ribosome
US6027898A (en) * 1997-08-18 2000-02-22 Transgenomic, Inc. Chromatographic method for mutation detection using mutation site specifically acting enzymes and chemicals

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003008642A2 (fr) * 2001-07-15 2003-01-30 Keck Graduate Institute Amplification de fragments d'acide nucleique au moyen d'agents de coupure
WO2003008642A3 (fr) * 2001-07-15 2003-07-31 Keck Graduate Inst Amplification de fragments d'acide nucleique au moyen d'agents de coupure
WO2004053159A2 (fr) * 2002-12-07 2004-06-24 Guoliang Fu Analyse d'expression genique dirigee a l'aide d'oligonucleotides
WO2004053159A3 (fr) * 2002-12-07 2005-01-20 Guoliang Fu Analyse d'expression genique dirigee a l'aide d'oligonucleotides

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WO2002040126A2 (fr) 2002-05-23
AU2002239228A8 (en) 2006-11-02
WO2002029006A3 (fr) 2002-08-29
WO2002046447A3 (fr) 2003-12-24
AU3922802A (en) 2002-05-27
WO2002046447A2 (fr) 2002-06-13
AU2001296491A1 (en) 2002-04-15
WO2002029006A2 (fr) 2002-04-11
AU1183902A (en) 2002-04-15
AU4146002A (en) 2002-06-18

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