Complexity management of genomic DNA

• US 8,492,121 B2
• Filed: 06/29/2011
• Issued: 07/23/2013
• Est. Priority Date: 10/27/1999
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Dong, Shoulian
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/171,892
• Publication Number:
  US 20110287491A1
• Time in Patent Office: 755 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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 presence or absence of a polymorphism.

Claims

1. A method for selective amplification of a nucleic acid sample, the method comprising:

fragmenting the nucleic acid sample using a first restriction enzyme and a second restriction enzyme and producing a digested nucleic acid sample comprising fragments;

ligating a double stranded first adaptor and a double stranded second adaptor to the fragments and producing ligated fragments, wherein the 3′

end of a first strand of the first adaptor is blocked from ligating to the 5′

ends of the fragments such that only a second strand of the first adaptor can ligate to the fragments, and wherein the 5′

end of a first strand of the second adaptor is blocked from ligating to the 3′

ends of the fragments such that only a second strand of the second adaptor can ligate to the fragments, wherein a subset of the ligated fragments comprises ligated fragments having a single strand which is ligated to the first adaptor at its 3′

end and the second adaptor at its 5′

end; and

subjecting the ligated fragments to amplification conditions, and selectively and exponentially amplifying the ligated fragments having a single strand which is ligated to the first adaptor at its 3′

end and the second adaptor at its 5′

end, thereby selectively amplifying the nucleic acid sample.

2. The method of claim 1, wherein the fragments which can ligate to both the first adaptor and the second adaptor comprise at least 0.01% of the digested nucleic acid sample.

3. The method of claim 1, wherein the fragments which can ligate to both the first adaptor and the second adaptor comprise at least 0.5% of the digested nucleic acid sample.

4. The method of claim 1, wherein the fragments which can ligate to both the first adaptor and the second adaptor comprise at least 3% of the digested nucleic acid sample.

5. The method of claim 1, wherein the fragments which can ligate to both the first adaptor and the second adaptor comprise at least 12% of the digested nucleic acid sample.

6. The method of claim 1, wherein the fragments which can ligate to both the first adaptor and the second adaptor comprise at least 30% of the digested nucleic acid sample.

7. The method of claim 1, wherein the fragments which can ligate to both the first adaptor and the second adaptor comprise at least 50% of the digested nucleic acid sample.

8. 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.

9. 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.

10. The method of claim 1, wherein the ligated fragments having a single strand which is ligated to the first adaptor at its 3′

end and the second adaptor at its 5′

end are amplified by PCR, wherein at least a first primer and a second primer are utilized within the PCR, wherein the first primer is complementary to a first sequence of one strand of the first adaptor, and wherein the second primer is complementary to a second sequence of one strand of the second adaptor.

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

12. The method of claim 1, wherein the first restriction enzyme and the first adaptor are selected such that a first gap of at least one nucleotide is formed during the ligation step and blocks ligation of the first strand of the first adaptor to the fragments, and wherein the second restriction enzyme and the second adaptor are selected such that a second gap of at least one nucleotide is formed during the ligation step and blocks ligation of the first strand of the second adaptor to the fragments.

13. The method of claim 1, wherein the first strand of the second adaptor lacks a phosphate at its 5′

end, wherein lack of the phosphate at the 5′

end of the first strand of the second adaptor blocks ligation of the first strand of the second adaptor to the fragments.

14. The method of claim 1, wherein the first adaptor comprises a modified nucleotide at the 3′

end of the first strand and the second adaptor comprises a modified nucleotide at the 5′

end of the first strand, and wherein the modified nucleotides block ligation of the first strand of the first adaptor and the first strand of the second adaptor to the fragments.

15. The method of claim 1, wherein the first adaptor includes a terminal modification on the 3′

end of the first strand and the second adaptor includes a terminal modification on the 5′

end of the first strand, and wherein the terminal modifications block ligation of the first strand of the first adaptor and the first strand of the second adaptor to the fragments.

16. The method of claim 1 wherein the first adaptor comprises a 5′

overhang comprising a primer binding site, and wherein the second adaptor comprises a 3′

overhang comprising a primer binding site.

17. 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.

18. The method of claim 1, wherein the ligated fragments having a single strand which is ligated to the first adaptor at its 3′

end and the second adaptor at its 5′

end which are selectively and exponentially amplified are analyzed by a technique selected from the group consisting of sequencing, HPLC and hybridization analysis.

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

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