Method for analyzing the nucleotide sequence of nucleic acids

• US 20040115655A1
• Filed: 12/16/2002
• Published: 06/17/2004
• Est. Priority Date: 12/16/2002
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Ilsley, Diane D.
• Application Number:
  US10/320,197
• Publication Number:
  US 20040115655A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

Provided are methods for deciphering the sequence of a nucleic acid by synthesizing fragments with pol α-primase and analyzing the fragments. The fragments are preferably short Xmers that are analyzed by mass spectrometry.

Claims

1. A method for analyzing sequence of a target nucleic acid comprising the steps of:

a) synthesizing a fragment on the target nucleic acid with pol α

-primase, wherein the fragment includes an RNA primer synthesized by pol α

-primase;

b) separating the fragment from the target nucleic acid; and

c) analyzing the fragment to ascertain the sequence of the target nucleic acid.

2. The method of claim 1, wherein the synthesizing step involves synthesis of fragments at random locations on the target nucleic acid.

3. The method of claim 2, wherein the fragments are substantially from about 6 to about 12 nucleotides in length.

4. The method of claim 3, wherein the fragments are substantially from about 8 to about 11 nucleotides in length.

5. The method of claim 3, wherein the synthesizing a fragment step is terminated by using a ddNTP.

6. The method of claim 5, wherein the fragment is an RNA primer that is terminated by a single ddNTP in the absence of dNTPs.

7. The method of claim 3, wherein the analyzing step is carried out by a mass spectrometer.

8. The method of claim 1, further comprising a step of removing the RNA portion of the fragment before the analyzing step.

9. A method for analyzing a sequence of a target DNA comprising the steps of:

a) synthesizing complimentary fragments on the target DNA with pol α

-primase in a reaction mixture containing NTPs and at least one of four ddNTPs, in the optional presence of dNTPs;

b) separating the fragments from the target DNA;

c) measuring the mass of the fragments; and

d) ascertaining the sequence of the fragments from the mass.

10. The method of claim 9, wherein the ddNTP is mass tagged.

11. The method of claim 9, wherein the fragments are substantially from about 6 to about 12 nucleotides in length.

12. The method of claim 11, wherein the fragments are substantially from about 8 to about 11 nucleotides in length.

13. The method of claim 9, wherein the fragment are RNA fragments having a single ddNTP at 3′

end.

14. A method for preparing fragments for sequence analysis by mass spectrometry comprising the step of synthesizing with pol α

-primase fragments that are substantially from about 6 to about 12 nucleotides long at random locations on a target nucleic acid.

15. The method of claim 14, wherein the fragments are substantially from about to about 11 nucleotides in length.

16. The method of claim 14, wherein the fragments are RNA fragments having a single ddNTP at 3′

end.