Uniform fragmentation of DNA using binding proteins

• US 8,202,691 B2
• Filed: 01/22/2009
• Issued: 06/19/2012
• Est. Priority Date: 01/25/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Steemers, Frank, Boutell, Jonathan Mark
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/357,995
• Publication Number:
  US 20090191563A1
• Time in Patent Office: 1,244 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The invention provides a method for preparing and analysing a population of fragmented polynucleotide sequences having a substantially uniform size. The method can include steps of (a) binding at least one protection molecule to at least one polynucleotide sequence; (b) cleaving the at least one polynucleotide sequence to generate a plurality of polynucleotide fragment sequences of substantially uniform size; (c) amplifying the polynucleotide fragments; and (d) determining a sequence characteristic of a plurality of the polynucleotide fragments.

Claims

1. A method for preparing and analysing a population of fragmented polynucleotide sequences having a substantially uniform size comprising:

(a) binding at least one protection molecule to at least one polynucleotide sequence;

(b) cleaving the at least one polynucleotide sequence to generate a plurality of polynucleotide fragment sequences of substantially uniform size;

(c) amplifying the polynucleotide fragments, wherein the amplification is performed on a solid support, wherein the amplification produces an array of locations, each location comprising multiple copies amplified from a single template; and

(d) sequencing the polynucleotide fragments, wherein a strand of each of the templates is sequenced, the strand of each of the templates is converted into a double stranded template using strand resynthesis, and wherein the resynthesized template is used for a second round of sequencing, thereby analysing a population of fragmented polynucleotide sequences having a substantially uniform size.

2. The method according to claim 1 wherein both ends of the fragment are analysed in a paired end sequencing procedure.

3. The method according to claim 1 wherein the at least one protection molecule is a histone or a DNA binding protein.

4. The method according to claim 3 wherein in step (b) the at least one polynucleotide sequence is cleaved by mechanical, chemical or enzymatic means.

5. The method according to claim 4 wherein the cleaving is by enzymatic means.

6. The method according to claim 5 wherein the enzymatic means is micrococcal nuclease.

7. The method according to claim 1, wherein the solid support is a pool of beads or microspheres.

8. The method according to claim 1, wherein the solid support is an planar surface.

9. The method according to claim 1, wherein the strand resynthesis is performed using multiple cycles of hybridisation, extension and denaturation.

10. The method according to claim 1, wherein the strand resynthesis is performed isothermally.

11. The method according to claim 1, further comprising removing the protection molecule from the polynucleotide fragment sequences after the cleaving.

12. The method according to claim 1, further comprising attaching universal adapter sequences to the ends of each polynucleotide fragment.

13. The method according to claim 12, wherein the amplifying of the polynucleotide fragments comprises using the universal adapter sequences as sites for primer hybridization.

14. The method according to claim 12, wherein the multiple copies produced by the amplifying are double stranded copies, and wherein the method further comprises making each of the double stranded copies into the single stranded template by removing at least a portion of one of the strands from the surface.

15. The method according to claim 1, wherein the plurality of polynucleotide fragment sequences comprises a sequence representation that includes the entire sequence complement of a genome from a particular organism.

16. The method according to claim 15, wherein the organism is a human.

17. The method according to claim 7, wherein the amplifying comprises emulsion PCR.

18. The method according to claim 1, wherein the multiple copies produced by the amplifying are double stranded copies, and wherein both strands of the double stranded copies are attached to the solid support.

19. The method according to claim 1, wherein greater than 100 of the polynucleotide fragments are sequenced in parallel on the solid support.