Methods and compositions for large-scale analysis of nucleic acids using DNA deletions

• US 9,334,490 B2
• Filed: 11/09/2007
• Issued: 05/10/2016
• Est. Priority Date: 11/09/2006
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje T.
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US11/938,096
• Publication Number:
  US 20080213771A1
• Time in Patent Office: 3,105 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are constructs that include pairs of target sequences which are separated by a known distance in the polynucleotide from which they are derived.

Claims

1. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first linear construct, wherein the linear construct comprises a first adaptor interposed between a first target polynucleotide fragment and a second target polynucleotide fragment, and wherein the first target polynucleotide fragment and the second target polynucleotide fragment comprise contiguous nucleic acid sequences within a genome or target polynucleotide;

(b) ligating a deletion adaptor to the first linear construct to form a second linear construct, wherein the deletion adaptor comprises a recognition site for a restriction endonuclease, and wherein the restriction endonuclease cleaves at a known distance from said recognition site;

(c) cleaving the second linear construct with the restriction endonuclease to form a third linear construct comprising the first adaptor;

(d) circularizing the third linear construct to form a first circularized construct comprising the first adaptor,

thereby forming the polynucleotide comprising a deletion mate pair, wherein the deletion mate pair comprises a first target sequence and a second target sequence that are separated by Y bases within the genome or target polynucleotide from which the first and second target sequences are derived, but which are contiguous in the first circularized construct, wherein Y is less than 100 bases.

2. The method of claim 1, further comprising prior to the circularizing step (d):

ligating a second adaptor to the third linear construct.

3. The method of claim 2, the method further comprising:

(a) cleaving the first circularized construct to form a fourth linear construct; and

(b) ligating a third adaptor to the fourth linear construct.

4. The method of claim 3, the method further comprising circularizing the fourth linear construct.

5. The method of claim 1, wherein the cleaving step (c) comprises deleting a known number of bases from the second target polynucleotide fragment.

6. The method of claim 1, wherein the first adaptor, the deletion adaptor, or both the first adaptor and the deletion adaptor comprise at least one recognition site for the restriction endonuclease on each end.

7. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first circular construct, wherein the construct comprises a first adaptor and a target polynucleotide, wherein the first adaptor comprises a recognition site for a first restriction endonuclease that cleaves at a known distance from the recognition site and a recognition site for a second restriction endonuclease that cleaves within the first adaptor;

(b) cleaving the first circular construct with the first restriction endonuclease to form a first linear construct;

(c) cleaving the first linear construct with the second restriction endonuclease to form a second linear construct comprising a fragment of the first adaptor; and

(d) circularizing the second linear construct to create a second circular construct comprising the fragment of the first adaptor;

thereby forming the polynucleotide comprising a deletion mate pair, wherein the deletion mate pair comprises a first target sequence and a second target sequence that are separated by Y bases within the genome or target polynucleotide from which the first and second target sequences are derived, but which would be contiguous in the second circular construct if the fragment of the first adaptor were removed, wherein Y is less than 300.

8. The method of claim 7, further comprising repeating steps (b) through (d) on the second circular construct, thereby forming a third circular construct comprising a deletion mate pair.

9. The method of claim 8, wherein steps (b) through (d) are repeated at least three times to create a series of constructs.

10. The method of claim 8, wherein the first restriction endonuclease is different than the second restriction endonuclease.

11. The method of claim 8, wherein the first restriction endonuclease is the same as the second restriction endonuclease.

12. A method for forming a polynucleotide comprising a deletion mate pair, the method comprising:

(a) providing a first linear construct comprising a target polynucleotide and a first adaptor, wherein the first adaptor is attached to one end of the target polynucleotide;

(b) ligating a deletion adaptor to the end of the first linear construct opposite the first adaptor, wherein the deletion adaptor comprises a recognition site for a restriction endonuclease that cleaves at a known distance from the recognition site; and

(c) cleaving the first linear construct with the restriction endonuclease to form a second linear construct;

(d) circularizing the second linear construct to form a first circularized construct,

thereby forming the polynucleotide comprising a deletion mate pair, wherein the deletion mate pair comprises a first target sequence and a second target sequence separated by Y bases within the genome or polynucleotide molecule from which the first and second target sequence are derived, wherein Y is less than 100.

13. The method of claim 12, wherein prior to the circularizing step (d), the method further comprises:

ligating a second adaptor to the end of the second linear construct to the end that is opposite of the first adaptor.

14. The method of claim 12, wherein prior to the circularizing step (d), steps (b) and (c) are repeated on the second linear construct.

15. The method of claim 14, wherein steps (b) and (c) are repeated three or more times.