IMPROVED PRIMER-BASED AMPLIFICATION METHODS
The present invention provides methods for amplifying a target nucleic acid with greater efficiency and accuracy by using one or more flap primers.
1 . A method for amplification of a target nucleic acid in a sample, the method comprising:
(a) contacting a sample suspected of containing the target nucleic acid with an amplification reaction mixture comprising:
at least one flap primer having the formula:
5′-X—Y-3′ (I)
wherein X represents the 5′ sequence portion of the flap primer that is non-complementary to the target nucleic acid, Y represents the 3′ sequence portion of the flap primer that is complementary to the target nucleic acid, wherein X is from 3-40 nucleotides in length;
(b) incubating the reaction mixture under amplification conditions, thereby generating an amplified target nucleic acid; and
(c) optionally detecting the amplified target nucleic acid.
2 . The method of claim 1 , wherein a signal from the amplified target nucleic acid is at least about 1.25 to about 3-fold greater in comparison to a signal from an amplified target nucleic acid amplified in an amplification reaction mixture that does not comprise at least one flap primer.
3 . The method of claim 1 , wherein the amount of the amplified target nucleic acid is at least about 1.25 to about 3-fold greater in comparison an amount of an amplified target nucleic acid amplified in an amplification reaction mixture that does not comprise at least one flap primer.
4 . The method of claim 1 , wherein the reaction mixture comprises a forward flap primer and a reverse flap primer.
5 . The method of claim 1 , wherein Y comprises a larger number of nucleotides than X.
6 . The method of claim 1 , wherein X and Y are about an equal number of nucleotides in length.
7 . The method of claim 1 , wherein Y is more than 10 nucleotides in length.
8 . The method of claim 1 , wherein X is 9-15 nucleotides in length.
9 . The method of claim 8 , wherein X is 10-14 nucleotides in length.
10 . The method of claim 9 , wherein X is 11-13 nucleotides in length.
11 . The method of claim 10 , wherein X is 12 nucleotides in length.
12 . The method of claim 1 , wherein X comprises at least 70% adenine or thymine nucleotide bases, or modified bases thereof.
13 . The method of claim 12 , wherein X comprises at least 80% adenine or thymine nucleotide bases, or modified bases thereof.
14 . The method of claim 13 , wherein X comprises at least 90% adenine or thymine nucleotide bases, or modified bases thereof.
15 . The method of claim 1 , wherein the amplification of the target nucleic acid sequence is continuously monitored.
16 . The method of claim 1 , wherein the amplified target nucleic acid is detected via fluorescence-generating probe.
17 . The method of claim 16 , wherein the amplified target nucleic acid is detected via a hybridization-based fluorescent probe.
18 . The method of claim 16 , wherein the amplified target nucleic acid is detected via a DNA binding fluorescent compound.
19 . The method of claim 1 , wherein the target nucleic acid is selected from the group consisting of DNA, mRNA, tRNA and rRNA.
20 . The method of claim 1 , wherein the target nucleic acid is less than 30 nucleotides in length.
21 . The method of claim 20 , wherein the target nucleic acid is miRNA or siRNA.
22 . The method of claim 1 , further comprising
(c) contacting the amplified target nucleic acid of step (b) with reaction mixture comprising:
(i) a primer comprising a sequence complementary to the target nucleic acid of step (b); and
(ii) a primer comprising a sequence complementary to the target nucleic acid of step (b) and a minor groove binder; and
(d) incubating the reaction mixture of step (c) under amplification conditions, thereby generating a second amplified target nucleic acid; and
(e) optionally detecting the amplified target nucleic acid of step (d).
23 . The method of claim 22 , wherein the primer of step (c)(i) is a second flap primer of formula I.
24 . The method of claim 23 , wherein the first primer of formula I and the second flap primer of formula I are the same.
25 . The method of claim 22 , wherein the primer of step (c)(i) is a MGB-primer.
26 . The method of claim 22 , wherein the primer of step (c)(ii) is a detection primer further comprising a fluorophore, wherein the fluorophore is quenched by the MGB and insertion of the MGB into a minor groove unquenches the fluorophore.
27 . A method for amplification of a target nucleic acid in a sample, the method comprising:
(a) contacting the sample suspected of containing the target nucleic acid with an amplification reaction mixture comprising:
at least one flap primer comprising an annealed helper oligonucleotide and having the formula:
5′-X—Y-3′
3′-X′-5′ (II)
wherein X represents the 5′ sequence portion of the flap primer that is non complementary to the target nucleic acid, X′ represents the helper oligonucleotide sequence that is complementary to X and comprises at least one modified nucleoside base, and Y represents the 3′ sequence portion of the flap primer that is complementary to the target nucleic acid, wherein X is from 3-40 nucleotides in length;
(b) incubating the reaction mixture under amplification conditions, thereby generating an amplified target nucleic acid; and
(c) optionally detecting the amplified target nucleic acid.
28 . The method of claim 27 , wherein X′ comprises a smaller number of nucleoside bases than X.
29 . The method of claim 27 , wherein X′ comprises a nucleoside base selected from the group consisting of: 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol (Super A); 6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d-]pyrimidin-4-one; 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione (Super T), and combinations thereof.
30 . The method of claim 27 , wherein the reaction mixture comprises a forward flap primer and a reverse flap primer.
31 . The method of claim 27 , wherein the forward flap primer and the reverse flap primer are independently selected from the group consisting of: Formula I and Formula II.
32 . The method of claim 27 , wherein Y comprises a larger number of nucleotides than X.
33 . The method of claim 27 , wherein X and Y are about an equal number of nucleotides in length.
34 . The method of claim 27 , wherein Y is more than 10 nucleotides in length.
35 . The method of claim 27 , wherein X is 9-15 nucleotides in length.
36 . The method of claim 35 , wherein X is 10-14 nucleotides in length.
37 . The method of claim 36 , wherein X is 11-13 nucleotides in length.
38 . The method of claim 37 , wherein X is 12 nucleotides in length.
39 . The method of claim 27 , wherein X comprises at least 70% adenine or thymine nucleotide bases, or modified bases thereof.
40 . The method of claim 39 , wherein X comprises at least 80% adenine or thymine nucleotide bases, or modified bases thereof.
41 . The method of claim 40 , wherein X comprises at least 90% adenine or thymine nucleotide bases, or modified bases thereof.
42 . The method of claim 27 , wherein the amplification of the target nucleic acid sequence is continuously monitored.
43 . The method of claim 27 , wherein the amplified target nucleic acid is detected via fluorescence-generating probe.
44 . The method of claim 43 , wherein the amplified target nucleic acid is detected via a hybridization-based fluorescent probe.
45 . The method of claim 43 , wherein the amplified target nucleic acid is detected via a DNA binding fluorescent compound.
46 . The method of claim 27 , wherein the target nucleic acid is selected from the group consisting of DNA, mRNA, tRNA and rRNA.
47 . The method of claim 27 , wherein the target nucleic acid is less than 30 nucleotides in length.
48 . The method of claim 47 , wherein the target nucleic acid is selected from the group consisting of siRNA and miRNA.
49 . The method of claim 27 , further comprising
(c) contacting the amplified target nucleic acid of step (b) with reaction mixture comprising:
(i) a primer comprising a sequence complementary to the target nucleic acid of step (b); and
(ii) a primer comprising a sequence complementary to the target nucleic acid of step (b)and a minor groove binder; and
(d) incubating the reaction mixture of step (c) under amplification conditions, thereby generating a second amplified target nucleic acid; and
(e) optionally detecting the amplified target nucleic acid of step (d).
50 . The method of claim 49 , wherein the primer of step (c)(i) is a second flap primer of formula I.
51 . The method of claim 50 , wherein the first flap primer of formula I and the second flap primer of formula I are the same.
52 . The method of claim 49 , wherein the primer of step (c)(i) is a MGB-primer.
53 . The method of claim 49 , wherein the primer of step (c)(ii) is a detection primer further comprising a fluorophore, wherein the fluorophore is quenched by the MGB and insertion of the MGB into a minor groove unquenches the fluorophore.
54 . A method for amplification of a target nucleic acid in a sample, the method comprising:
(a) contacting a sample suspected of containing the target nucleic acid with an amplification reaction mixture comprising:
a detection primer comprising a sequence complementary to the target nucleic acid, a minor groove binder, and fluorophore, wherein the fluorophore is quenched by the MGB and insertion of the MGB into a minor groove unquenches the fluorophore;
(b) incubating the reaction mixture under amplification conditions, thereby generating an amplified target nucleic acid; and
(c) optionally detecting the amplified target nucleic acid.
55 . The method of claim 54 , wherein the target nucleic acid is selected from the group consisting of DNA, mRNA, tRNA and rRNA.
56 . The method of claim 54 , wherein the target nucleic acid is less than 30 nucleotides in length.
57 . The method of claim 56 , wherein the target nucleic acid is selected from the group consisting of siRNA and miRNA.