Complexity management of genomic DNA

Patent metadata

Field	Value
Publication number	US-9388460-B2
Application number	US-201313926510-A
Country	US
Kind code	B2
Filing date	Jun 25, 2013
Priority date	Oct 27, 1999
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

Abstract

The presently claimed invention provides for novel methods and kits for reducing the complexity of a nucleic acid sample by providing non-gel based methods for amplification of a subset of the sequences in a sample. In a preferred embodiment, amplification of a subset can be accomplished by digesting a sample with two or more restriction enzymes and ligating adaptors to the fragments so that only a subset of the fragments can be amplified. The invention further provides for analysis of the above amplified sample by hybridization to an array, which may be specifically designed to interrogate the desired fragments for particular characteristics, such as, for example, the to presence or absence of a polymorphism.

First claim

I claim:

1. A method of amplifying fragments of a nucleic acid sample comprising:
fragmenting the nucleic acid sample using a first restriction enzyme and a second restriction enzyme, thereby producing a fragmented nucleic acid sample including double stranded nucleic acid fragments having a first end cut by the first restriction enzyme and a second end cut by the second restriction enzyme;
ligating a first double-stranded adaptor to the first end of the double stranded nucleic acid fragments and a second double-stranded adaptor to the second end of the double stranded nucleic acid fragments, wherein one strand of the first adaptor comprises a 5′ overhang that provides a template for a primer binding site and one strand of the second adaptor comprises a 3′ overhang comprising a primer binding site, thereby producing ligated fragments comprising the 5′ overhang and the 3′ overhang, wherein the 5′ overhang and the 3′ overhang are present on the same strand of each of the ligated fragments comprising the 5′ overhang and the 3′ overhang; and
exponentially amplifying the ligated fragments comprising the 5′ overhang and the 3′ overhang.

2. The method of claim 1 , wherein the first restriction enzyme has a six base pair recognition sequence and the second restriction enzyme has a four base pair recognition sequence.

3. The method of claim 1 , wherein the first restriction enzyme has an eight base pair recognition sequence and the second restriction enzyme has a four base pair recognition sequence.

4. The method of claim 1 , wherein the ligated fragments comprising the 5′ overhang and the 3′ overhang are amplified by polymerase chain reaction (PCR), wherein at least a first primer and a second primer are utilized in the PCR, wherein the first primer is complementary to the 3′ overhang, and wherein the 5′ overhang is complementary to the second primer.

5. The method of claim 4 , wherein the first primer and/or the second primer comprises at least one nucleotide at their 3′ ends or its 3′ end that hybridizes to the variable region of the double stranded nucleic acid fragments.

6. The method of claim 1 , wherein the nucleic acid sample is genomic DNA, DNA, cDNA derived from RNA, cDNA derived from total RNA or cDNA derived from mRNA.

7. The method of claim 1 , wherein the ligation of one strand of each of the first adaptor and the second adaptor to the double stranded nucleic acid fragments is blocked.

8. The method of claim 7 , wherein the ligation of one strand of each of the first double-stranded adaptor and the second double-stranded adaptor to the double stranded nucleic acid fragments is blocked by a gap of at least one nucleotide between the 5′ end of one strand of the first adaptor and the 3′ end of one strand of the double stranded nucleic acid fragments and a gap of at least one nucleotide between the 3′ end of one strand of the second adaptor and the 5′ end of one strand of the double stranded nucleic acid fragments.

9. The method of claim 7 , wherein the ligation of one strand of the first double-stranded adaptor to the double stranded nucleic acid fragments is blocked due to the absence of a phosphate at the 5′ end of one strand of the first adaptor.

10. The method of claim 7 , wherein the 5′ end of one strand of the first adaptor comprises a modified nucleotide and/or the 3′ end of one strand of the second adaptor comprises a modified nucleotide, and wherein the modified nucleotides block ligation of the 5′ end of one strand of the first adaptor and the 3′ end of one strand of the second adaptor to the double stranded nucleic acid fragments.

11. The method of claim 7 , wherein the first adaptor includes a terminal modification at the 5′ end of one strand of the first adaptor and/or the second adaptor includes a terminal modification at the 3′ end of one strand of the second adaptor, and wherein the terminal modifications block ligation of the 5′ end of the one strand of the first adaptor and the 3′ end of the one strand of the second adaptor to the double stranded nucleic acid fragments.

12. The method of claim 7 , wherein the ligation of one strand of the first adaptor to the double stranded nucleic acid fragments is blocked by blocking the ligation of the 5′ end of the one strand of the first adaptor to the 3′ end of one strand of the double stranded nucleic acid fragments, and wherein the ligation of one strand of the second adaptor is blocked by blocking the ligation of 3′ end of the one strand of the second adaptor to the 5′ end of a strand of the double stranded nucleic acid fragments.

13. The method of claim 1 , wherein the first restriction enzyme recognizes a first recognition sequence, wherein the second restriction enzyme recognizes a second recognition sequence, and wherein the first recognition sequence occurs less frequently than the second recognition sequence within the nucleic acid sample.

14. The method of claim 1 , wherein the first adaptor and/or the second adaptor includes a blunt end and the ligating the first adaptor and/or the second adaptor to the double stranded nucleic acid fragments comprises blunt end ligation.

15. The method of claim 1 , wherein amplified products generated from the exponential amplifying step are analyzed by a technique selected from the group consisting of sequencing, HPLC and hybridization analysis.

16. The method of claim 1 , wherein the fragmenting step, ligating step and the exponential amplifying step are carried out in a single tube.

Assignees

• Affymetrix Inc

Inventors

• Dong Shoulian

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/683

  involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP] · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title