DUAL ENZYMATIC AMPLIFICATION
Provided are methods for validating the presence and character of genomic mutations, particularly single nucleotide polymorphisms (SNPs), by parallel amplification of a portion or the whole genome with at least two different DNA polymerases.
1 . A method for verifying the presence of a genomic mutation in cells of a rare cell population comprising:
a) amplifying a portion or the whole genome of the cells of the rare cell population with a first DNA polymerase;
b) amplifying a portion or the whole genome of the cells of the rare cell population with a second DNA polymerase, wherein the second DNA polymerase is different from the first DNA polymerase;
c) comparing the amplified genomic sequences obtained in steps a) and b) with an unamplified genomic sequence obtained from a control population of cells comprising normal somatic genomic DNA, wherein identification of a nucleotide polymorphism that is identical in the genomic sequences obtained in steps a) and b), but different from a nucleotide polymorphism at the same nucleotide position in the genomic sequence obtained the unamplified genomic sequence verify the presence of a genomic mutation in cells of the rare cell population.
2 . The method of claim 1 , wherein the amplified and unamplified genomic sequences are compared by one or more procedures comprising sequencing, amplification and/or hybridization.
3 . The method of any one of claims 1 to 2 , wherein the presence or absence of the genomic mutation is detected by PCR.
4 . The method of any one of claims 1 to 2 , wherein the presence or absence of the genomic mutation is detected by microarray.
5 . The method of any one of claims 1 to 2 , wherein the presence or absence of the genomic mutation is detected by sequencing.
6 . A method for verifying the presence of a genomic mutation in cells of a rare cell population comprising:
a) amplifying and sequencing a portion or the whole genome of the cells of the rare cell population with a first DNA polymerase;
b) amplifying and sequencing a portion or the whole genome of the cells of the rare cell population with a second DNA polymerase, wherein the second DNA polymerase is different from the first DNA polymerase;
c) sequencing without amplifying a portion or the whole genome of a control cell population comprising normal somatic genomic DNA;
d) comparing the genomic sequences obtained in steps a), b) and c), wherein identification of a nucleotide polymorphism that is identical in the genomic sequences obtained in steps a) and b), but different from a nucleotide polymorphism at the same nucleotide position in the genomic sequence obtained in step c) verify the presence of a genomic mutation in cells of the rare cell population.
7 . The method of any one of claims 1 to 6 , wherein the first DNA polymerase and the second DNA polymerase have different error correction rates.
8 . The method of any one of claims 1 to 7 , wherein the first DNA polymerase and the second DNA polymerase have different nucleic acid copying fidelities.
9 . The method of any one of claims 1 to 8 , wherein the first DNA polymerase and/or the second DNA polymerase have 5′→3′ exonuclease activity.
10 . The method of any one of claims 1 to 9 , wherein the first DNA polymerase and/or the second DNA polymerase do not have 3′→5′ exonuclease activity.
11 . The method of any one of claims 1 to 10 , wherein the first DNA polymerase and/or the second DNA polymerase have helicase and/or strand displacement activity.
12 . The method of any one of claims 1 to 11 , wherein the first DNA polymerase and the second DNA polymerase are selected from the group consisting of a Φ29 DNA polymerase, a Thermus aquaticus (Taq) DNA polymerase, a Thermus flavus (Tfl) DNA polymerase, a Thermus thermophilus (rTth) DNA polymerase, a Thermus litoris (Tli) DNA polymerase, a Thermotoga maritima (Tma) DNA polymerase, a Pyrococcus furiosus (Pfu) DNA polymerase, a Bacillus stearothermophilus (Bst) DNA polymerase, PHUSION® High-Fidelity DNA polymerase, Vent R ® DNA polymerase, Deep Vent R ™ DNA polymerase, a Q5™ High-Fidelity DNA polymerase, and REPLI-g DNA polymerase.
13 . The method of any one of claims 1 to 12 , wherein the first DNA polymerase and the second DNA polymerase are selected from the group consisting of a Φ29 DNA polymerase, a Thermus aquaticus (Taq) DNA polymerase, a Thermus thermophilus (rTth) DNA polymerase, a Pyrococcus furiosus (Pfu) DNA polymerase, a Bacillus stearothermophilus (Bst) DNA polymerase, and a PHUSION® High-Fidelity DNA polymerase.
14 . The method of any one of claims 1 to 13 , wherein the first polymerase is a Φ29 DNA polymerase and the second DNA polymerase is a Thermus aquaticus (Taq) DNA polymerase.
15 . The method of any one of claims 1 to 13 , wherein the first polymerase is a Φ29 DNA polymerase and the second DNA polymerase is a PHUSION® High-Fidelity DNA polymerase.
16 . The method of any one of claims 1 to 15 , further comprising the step of isolating the genomic DNA from the cells of a rare cell population.
17 . The method of any one of claims 1 to 16 , further comprising the step of isolating the cells of the rare cell population.
18 . The method of any one of claims 1 to 17 , further comprising the step of obtaining the cells of the rare cell population from a subject.
19 . The method of any one of claims 1 to 18 , wherein the rare cell population is circulating tumor cells (CTC).
20 . The method of claim 19 , wherein the CTC are obtained from a blood sample of a subject.
21 . The method of any one of claims 19 to 20 , wherein the CTC are isolated based on their surface expression of Epithelial cell adhesion molecule (Ep-CAM).
22 . The method of any one of claims 1 to 21 , wherein the genomic mutation is a single nucleotide polymorphism (SNP).
23 . The method of any one of claims 1 to 22 , wherein the somatic genomic DNA is from white blood cells (WBC).
24 . The method of any one of claims 1 to 22 , wherein the somatic genomic DNA is from buccal swab.
25 . The method of any one of claims 1 to 22 , wherein the somatic genomic DNA is from hair bulb or hair follicle.
26 . The method of any one of claims 1 to 25 , wherein the whole genome of the cells in steps a) and b) is amplified and sequenced.
27 . The method of any one of claims 1 to 26 , wherein a portion of the whole genome of the cells in steps a) and b) is amplified and sequenced.
28 . The method of any one of claims 1 to 27 , wherein the portion or the whole genome of the cells is sequenced by performing Next Generation Sequencing.
29 . A method for verifying the presence of a genomic mutation in cells of a rare cell population comprising:
a) amplifying a portion or the whole genome of the cells of the rare cell population two or more iterations with a first DNA polymerase;
b) comparing the genomic sequences obtained in step a) with an unamplified genomic sequence obtained from a control population of cells comprising normal somatic genomic DNA, wherein identification of a nucleotide polymorphism that is identical in the genomic sequences obtained in step a), but different from a nucleotide polymorphism at the same nucleotide position in the genomic sequence obtained the unamplified genomic sequence verify the presence of a genomic mutation in cells of the rare cell population.
30 . The method of claim 29 , wherein the amplified and unamplified genomic sequences are compared by one or more procedures comprising sequencing, amplification and/or hybridization.
31 . The method of any one of claims 29 to 30 , wherein the presence or absence of the genomic mutation is detected by PCR.
32 . The method of any one of claims 29 to 30 , wherein the presence or absence of the genomic mutation is detected by microarray.
33 . The method of any one of claims 29 to 30 , wherein the presence or absence of the genomic mutation is detected by sequencing.
34 . A method for verifying the presence of a genomic mutation in cells of a rare cell population comprising:
a) amplifying and sequencing a portion or the whole genome of the cells of the rare cell population two or more iterations with a first DNA polymerase;
b) sequencing without amplifying a portion or the whole genome of a control cell population comprising normal somatic genomic DNA;
c) comparing the genomic sequences obtained in steps a) and b) with an unamplified genomic sequence obtained in step c), wherein identification of a nucleotide polymorphism that is identical in the genomic sequences obtained in step a), but different from a nucleotide polymorphism at the same nucleotide position in the genomic sequence obtained in step b) verify the presence of a genomic mutation in cells of the rare cell population.
35 . The method of any one of claims 29 to 34 , wherein the first DNA polymerase has 5′→3′ exonuclease activity.
36 . The method of any one of claims 29 to 35 , wherein the first DNA polymerase does not have 3′→5′ exonuclease activity.
37 . The method of any one of claims 29 to 36 , wherein the first DNA polymerase has helicase and/or strand displacement activity.
38 . The method of any one of claims 29 to 37 , wherein the first DNA polymerase is selected from the group consisting of a Φ29 DNA polymerase, a Thermus aquaticus (Taq) DNA polymerase, a Thermus flavus (Tfl) DNA polymerase, a Thermus thermophilus (rTth) DNA polymerase, a Thermus litoris (Tli) DNA polymerase, a Thermotoga maritima (Tma) DNA polymerase, a Pyrococcus furiosus (Pfu) DNA polymerase, a Bacillus stearothermophilus (Bst) DNA polymerase, PHUSION® High-Fidelity DNA polymerase, Vent R ® DNA polymerase, Deep Vent R ™ DNA polymerase, a Q5™ High-Fidelity DNA polymerase, and REPLI-g DNA polymerase.
39 . The method of any one of claims 29 to 38 , wherein the first DNA polymerase is selected from the group consisting of a Φ29 DNA polymerase, a Thermus aquaticus (Taq) DNA polymerase, a Thermus thermophilus (rTth) DNA polymerase, a Pyrococcus furiosus (Pfu) DNA polymerase, a Bacillus stearothermophilus (Bst) DNA polymerase, and a PHUSION® High-Fidelity DNA polymerase.
40 . The method of any one of claims 29 to 39 , further comprising the step of isolating the genomic DNA from the cells of a rare cell population.
41 . The method of any one of claims 29 to 40 , further comprising the step of isolating the cells of the rare cell population.
42 . The method of any one of claims 29 to 41 , further comprising the step of obtaining the cells of the rare cell population from a subject.
43 . The method of any one of claims 29 to 42 , wherein the rare cell population is circulating tumor cells (CTC).
44 . The method of claim 43 , wherein the CTC are obtained from a blood sample of a subject.
45 . The method of any one of claims 29 to 44 , wherein the CTC are isolated based on their surface expression of Epithelial cell adhesion molecule (Ep-CAM).
46 . The method of any one of claims 29 to 45 , wherein the genomic mutation is a single nucleotide polymorphism (SNP).
47 . The method of any one of claims 29 to 46 , wherein the somatic genomic DNA is from white blood cells (WBC).
48 . The method of any one of claims 29 to 46 , wherein the somatic genomic DNA is from a buccal swab.
49 . The method of any one of claims 29 to 46 , wherein the somatic genomic DNA is from a hair bulb or hair follicle.
50 . The method of any one of claims 29 to 49 , wherein the whole genome of the cells in steps a) and b) is amplified and sequenced.
51 . The method of any one of claims 29 to 50 , wherein a portion of the whole genome of the cells in steps a) and b) is amplified and sequenced.
52 . The method of any one of claims 29 to 51 , wherein the portion or the whole genome of the cells is sequenced by performing Next Generation Sequencing.