Compositions and methods for analysis of nucleic acids

• US 7,270,958 B2
• Filed: 07/13/2004
• Issued: 09/18/2007
• Est. Priority Date: 09/10/1998
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Makarov, Vladimir L., Langmore, John P.
• Primary Examiner(s): Kim; Young J.
• Application Number:
  US10/890,483
• Publication Number:
  US 20050053986A1
• Time in Patent Office: 1,162 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

Disclosed are a number of methods that can be used in a variety of embodiments, including, creation of a nucleic acid terminated at one or more selected bases, sequence analysis of nucleic acids, mapping of sequence motifs within a nucleic acid, positional mapping of nucleic acid clones, and analysis of telomeric regions. The methods utilize double-stranded templates, and in most aspects involve a strand replacement reaction initiated at one or more random or specific locations created in a nucleic acid molecule, and in certain aspects utilizing an oligonucleotide primer.

Claims

1. A method for preparing a DNA molecule comprising the steps of:

a) obtaining a sample of DNA that includes double-stranded DNA molecules that have been fragmented enzymatically;

b) conditioning DNA fragments of the sample to provide a 3′

hydroxyl group thereon through the use of a mixture comprising DNA polymerase and exonuclease III to provide conditioned DNA fragments.

2. The method of claim 1, wherein the DNA molecules have been fragmented using an endonuclease.

3. The method of claim 2, wherein the DNA molecules have been fragmented through the use of a restriction endonuclease.

4. The method of claim 3, wherein the DNA molecules have been fragmented through the use of a restriction endonuclease having a two base recognition sequence.

5. The method of claim 3, wherein the DNA molecules have been fragmented through the use of a restriction endonuclease having a four base recognition sequence.

6. The method of claim 3, wherein the restriction endonuclease has introduced random double strand breaks into DNA molecules.

7. The method of claim 2, wherein the endonuclease introduced a blunt end.

8. The method of claim 1, wherein the DNA polymerase possesses a 3′

to 5′

exonuclease activity.

9. The method of claim 1, further comprising attaching an oligonucleotide adaptor to the conditioned DNA fragments.

10. The method of claim 9, wherein the oligonucleotide adaptor is a double-stranded oligonucleotide adaptor.

11. The method of claim 10, wherein the double-stranded oligonucleotide adaptor is attached to the conditioned DNA by only one of its two strands.

12. The method of claim 11, wherein the double stranded adaptor is attached to the conditioned DNA by means of a 5′

terminus of the adaptor.

13. The method of claim 12, wherein the double-stranded oligonucleotide adaptor is blocked at at least one of its 3′

termini.

14. The method of claim 13, wherein the double-stranded oligonucleotide adaptor is blocked at both of its 3′

termini.

15. The method of claim 1, wherein the conditioned DNA fragments are amplified.

16. The method of claim 15, wherein DNA fragments are amplified through a PCR reaction.

17. The method of claim 16, wherein the DNA fragments are amplified through a PCR reaction through the use of double-stranded adaptors that have been attached to the conditioned DNA fragments.

18. The method of claim 1, further defined as comprising the steps of:

a) obtaining a sample of DNA wherein the sample includes DNA fragments that do not include a 3′

hydroxyl group, wherein the sample has been subjected to enzymatic fragmentation;

b) conditioning DNA fragments of the sample that lack a 3′

hydroxyl by incorporating a 3′

hydroxyl group thereon through the use of a mixture comprising a DNA polymerase and exonuclease III;

c) attaching adaptors to DNA fragments of the sample; and

d) amplifying DNA fragments of the sample through the use of the adaptors.