Methods for normalized amplification of nucleic acids

• US 20060216724A1
• Filed: 07/29/2005
• Published: 09/28/2006
• Est. Priority Date: 07/30/2004
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Christians, Frederick C., Mei, Rui, Walsh, Sean
• Application Number:
  US11/192,854
• Publication Number:
  US 20060216724A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Methods of preparing normalized mixtures from a plurality of nucleic acid samples are disclosed. Nucleic acids are amplified so that similar amounts of a target nucleic acid are generated in a plurality of different reactions. Separate amplification reactions are performed to amplify the same or different targets in a plurality of different reactions. The amounts of amplified product are approximately normalized during the amplification without the need to empirically measure the amount of amplified target.

Claims

1. A method of amplifying a target sequence from a complex nucleic acid sample comprising:

obtaining a first amplification product that is enriched for the target sequence by incubating the complex nucleic acid sample in a first amplification reaction, wherein said first amplification reaction comprises polymerase chain reaction with a pair of primers that are specific for said target; and

,

obtaining a second amplification product by amplifying an aliquot of the first amplification product with a strand displacing DNA polymerase, in the presence of dNTPs and at least one primer in a second amplification reaction.

2. The method of claim 1 wherein the strand displacing DNA polymerase is a phi29 polymerase.

3. The method of claim 1 wherein the yield of the second amplification reaction is limited by the amount of dNTPs added to said second amplification reaction.

4. The method of claim 1 wherein the at least one primer is a collection of random primers.

5. The method of claim 1 wherein the yield of amplified target sequence in the second amplification reaction is about 1 to 2 μ

g of amplified target sequence per μ

l reactionn volume.

6. The method of claim 1 wherein the yield of amplified target sequence in the second amplification reaction is about 0.5 to 1 μ

g of amplified target sequence per μ

l reaction volume.

7. The method of claim 1 wherein the strand displacing polymerase is Bst DNA polymerase.

8. A method of obtaining a pooled sample comprising approximately equal molar amounts of a plurality of amplified target sequences comprising:

(a) amplifying each target sequence according to the method of claim 1 wherein the amount of dNTPs present in the second amplification of each target sequence is approximately the same;

(b) obtaining an estimate of the molecular weight of each target;

(c) determining a volume of the second amplification reaction to add to a pooled sample for each of the targets, so that each will be present at approximately the same molar amount in the pooled sample, using the estimated molecular weight of each target and assuming that the amount of DNA in each of the second amplification reactions is the same; and

(c) obtaining the pooled sample by mixing the volumes of the second amplification reaction calculated in (c) to a new tube.

9. The method of claim 8 wherein the yield of amplified target sequence in the second amplification reaction for each target sequence in the plurality of amplified target sequences is about 0.5 to 1 μ

g of amplified target sequence per μ

l reaction volume.

10. The method of claim 8 wherein the yield of amplified target sequence in the second amplification reaction for each target sequence in the plurality of amplified target sequences is about 1 to 2 μ

g of amplified target sequence per μ

l reaction volume.

11. The method of claim 8 wherein the volume of the second amplification reaction added to the pooled sample is proportionate to the molecular weight of the target in said second amplification reaction.

12. The method of claim 8 wherein each target is between 1 and 30 kilobases in length.

13. The method of claim 8 further comprising analyzing the pooled sample by fragmenting the targets in the pooled sample to generate fragments, labeling the fragments to generate labeled fragments and hybridizing the labeled fragments to a resequencing array.

14. The method of claim 8 wherein an automated liquid handling device is used for mixing the volumes of the second reaction.

15. A method for obtaining a pooled sample comprising approximately equimolar amounts of a first amplified target sequence and a second amplified target sequence comprising

Amplifying said first target sequence in a first amplification reaction to generate a first amplification product wherein the first target is amplified by PCR with a pair of primers that are specific for said first target sequence;

amplifying said second target sequence in a second reaction to generate a second amplification product wherein the second target sequence is amplified by PCR with a pair of primers that are specific for said second target sequence;

amplifying an aliquot of said first amplification product in a third amplification reaction, to generate a third amplification product, wherein the third amplification reaction comprises a mixture of random primers, a strand displacing DNA polymerase, and a first amount of dNTPs;

amplifying an aliquot of said second amplification product in a fourth amplification reaction, to generate a fourth amplification product, wherein the fourth amplification reaction comprises a mixture of random primers, a strand displacing DNA polymerase, and a second amount of dNTPs; and

mixing a volume of the third amplification product with a volume of the fourth amplification product to generate a mixture of amplified first and second target sequences wherein the first and second target amplicons are present in approximately equal molar amounts in the mixture.

16. The method of claim 15 wherein said strand displacing DNA polymerase is selected from the group consisting of a phi29 polymerase and a Bst polymerase.

17. The method of claim 15 wherein said first amount of dNTPs and said second amount of dNTPs are approximately equal.

18. The method of claim 15 wherein said first amount of dNTPs is proportional to the molecular weight of the first target sequence and said second amount of dNTPs is proportionate to the molecular weight of the second target sequence.

19. The method of claim 18 wherein the volume of the third amplification product and the volume of the fourth amplification product that are added to the mixture are approximately equal.

20. The method of claim 15 wherein the first target sequence and the second target sequence are between 1 and 5 kilobases in length.

21. The method of claim 15 wherein the first target sequence and the second target sequence are between 5 and 15 kilobases in length.

22. The method of claim 15 wherein the yield of the third amplification product and the yield of the fourth amplification product are approximately equal.