NUCLEIC ACID AMPLIFICATION

• US 20080128298A1
• Filed: 10/12/2007
• Published: 06/05/2008
• Est. Priority Date: 10/15/2001
• Status: Abandoned Application

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Lasken, Roger S., Bornarth, Carole, Hosono, Seiyu, Wisniewski, Michele, Raghunathan, Arumugham
• Application Number:
  US11/871,707
• Publication Number:
  US 20080128298A1
• Time in Patent Office: Days
• US Class Current: 206/223
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2523/119 Renaturing agents
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Disclosed are compositions and methods for amplification of nucleic acid sequences of interest. It has been discovered that amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed on an amount of nucleic acid at or over a threshold amount and/or on nucleic acids at or below a threshold concentration. The threshold amount and concentration can vary depending on the nature and source of the nucleic acids to be amplified and the type of amplification reaction employed. Disclosed is a method of determining the threshold amount and/or threshold concentration of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest. Because amplification reactions can produce high quality amplification products, such as low bias amplification products, below the threshold amount and/or concentration of nucleic acid, such below-threshold amounts and/or concentrations can be used in amplification reactions.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. A kit for amplifying a whole genome, the kit comprising

a composition for lysis,

a composition for neutralization,

a set of primers, and

a DNA polymerase.

7. The kit of claim 4 wherein the composition for lysis comprises potassium hydroxide.

8. The kit of claim 7 wherein the composition for lysis comprises 400 mM KOH.

9. The kit of claim 8 wherein the composition for lysis comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

10. The kit of claim 4 wherein the composition for lysis consists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

11. The kit of claim 26 wherein the composition for stabilization comprises Tris-HCl at pH 4.1.

12. The kit of claim 11 wherein the composition for stabilization comprises 800 mM Tris-HCl, pH 4.1.

13. The kit of claim 12 wherein the composition for stabilization consists of 800 mM Tris-HCl, pH 4.1.

14. The kit of claim 4 further comprising deoxynucleotide triphosphates.

15. The kit of claim 26 further comprisingdithiotheitol,

1×

Phosphase-Buffered Saline, pH 7.5, and

control DNA template,

wherein the composition for lysis comprises 400 mM KOH and 10 mM EDTA,

wherein the composition for stabilization comprises 800 mM Tris-HCl, pH 4,

wherein the set of primers comprises a reaction mix, wherein the reaction mix comprises 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4 mM deoxynucleotide triphosphates, and 0.2 mM random hexamer primers,

wherein the DNA polymerase is φ

29 DNA polymerase.

16. The kit of claim 4 further comprising one or more detection probes, wherein the detection probes each comprise a complementary portion, wherein the complementary portion is complementary to a nucleic acid sequence of interest.

17. The method of claim 16 wherein the kit is designed to detect nucleic acid sequences of interest in the genome.

18. The method of claim 17 wherein the kit is designed to assess a disease, condition or predisposition of an individual based on the nucleic acid sequences of interest.

24. The kit of claim 4, wherein the composition for neutralization is a composition for neutralization of a cell lysate.

25. The kit of claim 4, wherein the composition for neutralization is a composition for neutralizing lysed cells.

26. The kit of claim 4, further comprising a composition for stabilization.

5. (canceled)

6. (canceled)

19. A kit for amplifying a whole genome, the kit comprising

a composition for lysis, wherein the composition for lysis is alkaline,

a composition for neutralization,

a set of primers, wherein the primers are 6 nucleotides long, wherein the primers each contain at least one modified nucleotide such that the primers are nuclease resistant, and

a DNA polymerase, wherein the DNA polymerase is φ

29 DNA polymerase.

27. The kit of claim 19, wherein the composition for lysis is a composition for cell lysis, and wherein the composition for neutralization is a composition for neutralization of a cell lysate.

28. The kit of claim 19, wherein the composition for lysis is a composition for lysing cells, and wherein the composition for neutralization is a composition for neutralizing lysed cells.

20. (canceled)

21. (canceled)

22. A kit for amplifying a whole genome, the kit comprising

a composition for stabilization,

a reaction mix comprising a set of primers, and

a DNA polymerase mix comprising a DNA polymerase.

23. The kit of claim 22 further comprisingdithiotheitol,

1×

Phosphase-Buffered Saline, pH 7.5, and

control DNA template,

wherein the composition for stabilization comprises 800 mM Tris-HCl, pH 4,

wherein the reaction mix comprises 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4 mM deoxynucleotide triphosphates, and 0.2 mM random hexamer primers,

wherein the DNA polymerase is φ

29 DNA polymerase.