COMPOSITION AND METHOD FOR SEQUENCING NUCLEIC ACID

• US 20150191774A1
• Filed: 12/15/2014
• Published: 07/09/2015
• Est. Priority Date: 02/04/2008
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: LEE, Linda G.
• Application Number:
  US14/570,839
• Publication Number:
  US 20150191774A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

A composition for sequencing DNA is provided and comprises a nuclease and a nuclease-resistant sequencing primer. A method of preparing DNA for sequencing and a method of sequencing DNA are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition under conditions in which excess amplification primer is degraded by the nuclease and the nuclease-resistant sequencing primer is essentially non-degraded.

Claims

1. A method comprising the steps of:

amplifying a DNA sequence under conditions to produce an amplification reaction product, the amplification reaction product comprising an excess amplification primer; and

contacting the amplification reaction product with a reaction mixture comprising an exonuclease I and a sequencing primer configured to be resistant to degradation by the exonuclease I, whereby, the excess amplification primer is degraded by the exonuclease I and the nuclease-resistant sequencing primer is essentially non-degraded.

2. The method of claim 1, wherein the amplification reaction products further comprise a target amplicon.

3. The method of claim 1, further comprising inactivating the nuclease after the excess amplification primer is degraded by the nuclease.

4. The method of claim 3, wherein inactivating the nuclease comprises heating.

6. The method of claim 1, wherein the nuclease-resistant sequencing primer comprises at least one phosphorothioate linkage.

7. The method of claim 1, wherein the nuclease-resistant sequencing primer comprises a phosphorothioate linkage at a terminal 3′

end of the sequencing primer.

5. (canceled)

8. A method comprising the steps of:

amplifying DNA in a first reaction mixture comprising nuclease-sensitive amplification primers to form amplified DNA;

contacting the first reaction mixture of the amplifying step with a second reaction mixture comprising an exonuclease I and a sequencing primer configured to be resistant to degradation by the exonuclease I, whereby the nuclease sensitive amplification primers are degraded by the exonuclease I;

inactivating the exonuclease I; and

causing the amplified DNA to react in a sequencing reaction under conditions in which the nuclease-resistant sequencing primer primes the sequencing reaction.

9. The method of claim 8, further comprising:

obtaining results based on the sequencing reaction; and

determining a nucleotide base sequence of the amplified DNA based on the results.

10. The method of claim 8, wherein amplifying DNA comprises polymerase chain reaction amplification.

12. The method of claim 8, wherein the nuclease-resistant sequencing primer comprises at least one phosphorothioate linkage.

13. The method of claim 12, wherein the nuclease-resistant sequencing primer comprises a phosphorothioate linkage at a terminal 3′

end.

14. The method of claim 8, wherein inactivating the nuclease comprises heating.

15. The method of claim 8, wherein the sequencing reaction comprises cycle sequencing.

16. The method of claim 8, wherein the second reaction mixture further comprises deoxynucleotide triphosphates, dye-labeled dideoxynucleotide triphosphates, and DNA polymerase.

11. (canceled)

17. A composition for sequencing nucleic acid, comprising a polymerase, an exonuclease, a nuclease-resistant sequencing primer, deoxynucleotide triphosphates, and dideoxynucleotide triphosphates.

18. The composition of claim 17, further comprising PCR amplification reaction product that comprises non-nuclease-resistant amplification primer.

20. The composition of claim 17, wherein the polymerase is Taq polymerase.

22. The composition of claim 17, wherein the nuclease-resistant primer comprises at least one phosphorothioate linkage.

23. The composition of claim 22, wherein the nuclease-resistant primer comprises a phosphorothioate linkage at a terminal 3′

end of the primer.

24. The composition of claim 17, wherein the nuclease-resistant primer comprises an exonuclease I resistant primer.

19. (canceled)

21. (canceled)

25. (canceled)

26. (canceled)