Methods for analyzing nucleic acids using a type IIs restriction endonuclease
First Claim
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1. A method for identifying differences between a first and second nucleic acid sample, comprising:
- fragmenting the samples with a first type IIs endonuclease;
(a) ligating to the fragments a first adapter sequence comprising a second type-IIs endonuclease recognition site;
(b) fragmenting the samples with a third endonuclease and ligating a second adapter sequence to the fragments;
(c) amplifying the fragments;
(d) marking the amplified fragments from the first sample;
(e) mixing the first and second samples under conditions that will allow heteroduplex formation between sequences in the first and second samples;
(f) selectively degrading one strand from heteroduplexes which contain a mismatch and isolating the remaining single-stranded fragments;
(g) creating double-stranded DNA from the single-stranded fragments and amplifying the double-stranded DNA;
(h) cleaving the DNA with the second type-IIs endonuclease; and
determining the sequence of polynucleotides between the first and second type-IIs endonuclease recognition sites.
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Abstract
The present invention relates to novel methods for sequencing and mapping genetic markers in polynucleotide sequences using Type-IIs restriction endonucleases. The methods herein described result in the “capturing” and determination of specific oligonucleotide sequences located adjacent to Type-IIs restriction sites. The resulting sequences are useful as effective markers for use in genetic mapping, screening and manipulation.
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Citations
18 Claims
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1. A method for identifying differences between a first and second nucleic acid sample, comprising:
- fragmenting the samples with a first type IIs endonuclease;
(a) ligating to the fragments a first adapter sequence comprising a second type-IIs endonuclease recognition site;
(b) fragmenting the samples with a third endonuclease and ligating a second adapter sequence to the fragments;
(c) amplifying the fragments;
(d) marking the amplified fragments from the first sample;
(e) mixing the first and second samples under conditions that will allow heteroduplex formation between sequences in the first and second samples;
(f) selectively degrading one strand from heteroduplexes which contain a mismatch and isolating the remaining single-stranded fragments;
(g) creating double-stranded DNA from the single-stranded fragments and amplifying the double-stranded DNA;
(h) cleaving the DNA with the second type-IIs endonuclease; and
determining the sequence of polynucleotides between the first and second type-IIs endonuclease recognition sites. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
the second type-IIs endonuclease is selected from the group consisting of HgaI, BbvI, BspMI, BsmFI and FokI; and
the third endonuclease cleaves more frequently than the first type-IIs endonuclease.
- fragmenting the samples with a first type IIs endonuclease;
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5. The method of claim 4, wherein the first type IIs endonuclease is BseRI;
- the second type-IIs endonuclease is Fok; and
the third endonuclease is HaeIII.
- the second type-IIs endonuclease is Fok; and
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6. The method of claim 1, wherein prior to said determining step a third adapter sequence comprising a primer is ligated to the cleavage product of step (i) and the sequence of nucleotides in the polynucleotide between the first and third adapter sequences is amplified.
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7. The method of claim 1, wherein the sequence of nucleotides between the first and second type-IIs endonuclease recognition sites is determined by hybridization to an oligonucleotide probe.
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8. The method of claim 7, wherein said oligonucleotide probe is a positionally distinct probe on an oligonucleotide array, a position of the probe being indicative of the sequence of the probe.
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9. The method of claim 1, wherein the mixture of step (f) is treated with a mismatch repair enzyme and an exonuclease.
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10. The method of claim 9, wherein the mismatch repair enzyme is MutLSH and the exonuclease is ExoIII.
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11. A method of reducing the complexity of a nucleic acid sample comprising:
- fragmenting the sample using a first type-IIs restriction enzyme to produce fragments;
ligating a first adapter sequence to at least some of the fragments;
fragmenting the samples with an endonuclease;
ligating a second adapter sequence to at least some of the fragments; and
amplifying at least some of the fragments. - View Dependent Claims (12, 13, 14, 15)
- fragmenting the sample using a first type-IIs restriction enzyme to produce fragments;
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16. A method of reducing the complexity of a nucleic acid sample comprising:
- fragmenting the sample using a type-IIs restriction enzyme to produce fragments;
ligating an adapter sequence to at least some of the fragments; and
amplifying at least some of the fragments. - View Dependent Claims (17, 18)
- fragmenting the sample using a type-IIs restriction enzyme to produce fragments;
Specification