Individually synthesized primers to be used in whole genome amplification

• US 9,593,366 B2
• Filed: 10/26/2009
• Issued: 03/14/2017
• Est. Priority Date: 10/30/2008
• Status: Active Grant

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Nazarenko, Irina, O'Neil, Dominic, Thai, Ha, Lorincz, Attila
• Primary Examiner(s): Priest, Aaron
• Application Number:
  US13/126,165
• Publication Number:
  US 20110256591A1
• Time in Patent Office: 2,696 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences

Abstract

Disclosed are compositions and methods for random amplification of nucleic acid sequences of interest using random-G-deficient primers. Also disclosed are methods of randomly amplifying a target nucleic acid sequence using random G-deficient primers alone or in combination with random, partially random, or specific primers.

Claims

1. An in vitro method of randomly amplifying a target nucleic acid sequence, the method comprising, bringing into contact a DNA polymerase, a target sample, and a set of random G-deficient primers, and incubating the target sample under conditions that promote replication of the target sequence, wherein replication of the target sequence results in replicated strands, wherein all of the primers of the primer set:

a. are exonuclease-resistant,

b. have at least one guanine residue,

c. do not contain two or more consecutive guanine residues at the 3′

end, and

d. do not comprise three or more consecutive guanine residues.

2. The method of claim 1, wherein at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.

3. The method of claim 1, wherein the target sample is not subjected to denaturing conditions.

4. The method of claim 1, wherein the primers each contain at least one modified nucleotide.

5. The method of claim 4, wherein the at least one modified nucleotide renders the primers resistant to 3′

-5′

exonuclease.

6. The method of claim 1, wherein the target sample is not subjected to heat denaturing conditions.

8. The method of claim 1, wherein the conditions that promote replication of the target sequence involve thermal cycling.

19. The method of claim 1, wherein the set of primers comprises three or more primers.

20. The method of claim 19, wherein each of the three or more primers has a different sequence.

7. The method of claim wherein the conditions that promote replication of the target sequence are substantially isothermic.

9. The method of claim further comprising:

diluting the replicated strands, contacting the diluted replicated strands with a second set of primers and DNA polymerase, and incubating the replicated strands under conditions that promote replication of the target sequence,

wherein replication of the target sequence results in additional replicated strands, wherein during replication at least one of the additional replicated strands is displaced from the target sequence by strand displacement replication of another additional replicated strand.

10. The method of claim 9, wherein the primers of the second set of primers are random G-deficient primers.

11. A method of randomly amplifying a target nucleic acid sequence, the method comprising, bringing into contact a DNA polymerase, a target sample, and a set of random G-deficient primers, and incubating the target sample under conditions that promote replication of the target sequence, wherein nucleic acids in the target sample are not separated from other material in the target sample, wherein all of the primers of the primer set:

a. are exonuclease-resistant,

b. have at least one guanine residue,

c. do not contain two or more consecutive guanine residues at the 3′

end, and

d. do not comprise three or more consecutive guanine residues.

12. The method of claim 11, wherein the target sample is a crude cell lysate.

13. The method of claim 11, wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand.

14. A method of randomly amplifying messenger RNA, the method comprising,

reverse transcribing messenger RNA to produce a first strand cDNA,

bringing into contact a set of random G-deficient primers, DNA polymerase, and the first strand cDNA, and incubating under conditions that promote replication of the first strand cDNA,

wherein replication of the first strand cDNA results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the first strand cDNA by strand displacement replication of another replicated strand, and wherein all of the primers of the primer set:

a. are exonuclease-resistant,

b. have at least one guanine residue,

c. do not contain two or more consecutive guanine residues at the 3′

end, and

d. do not comprise three or more consecutive guanine residues.

15. A method of randomly amplifying a target nucleic acid sequence, the method comprising,

(a) mixing a set of random G-deficient primers with a target sample, to produce a primer-target sample mixture, and incubating the primer-target sample mixture under conditions that promote hybridization between the random G-deficient primers and the target sequence in the primer-target sample mixture, wherein all of the primers of the primer set are exonuclease-resistant, have at least one guanine residue, do not contain two or more consecutive guanine residues at the 3′

end, and do not comprise three or more consecutive guanine residues; and

(b) mixing DNA polymerase with the primer-target sample mixture, to produce a polymerase-target sample mixture, and incubating the polymerase-target sample mixture under conditions that promote replication of the target sequence,

wherein replication of the target sequence results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the target sequence by strand displacement replication of another replicated strand,

wherein the target sequence is a nucleic acid sample of substantial complexity.

16. The method of claim 15, further comprising the step of:

(c) thermal cycling to allow hybridization between the random G-deficient primers and the replicated strands, and incubating under conditions that promote replication of the target sequence and replicated strands.

17. The method of claim 15, wherein the conditions that promote replication of the target sequence are substantially isothermic.

18. The method of claim 15, wherein the conditions that promote replication of the target sequence involve thermal cycling.

21. A kit comprising a lysis solution, a stabilization solution, a DNA polymerase, and a set of random G-deficient primers, wherein all of the primers of the primer set:

a. are exonuclease-resistant,

b. have at least one guanine residue,

c. do not contain two or more consecutive guanine residues at the 3′

end, and

d. do not comprise three or more consecutive guanine residues.

22. The kit of claim 21, further comprising deoxynucleotide triphosphates.

23. The kit of claim 21, further comprising one or more detection probes.