Nucleic acid amplication utilizing intermediate duplexes
This invention provides for a novel amplification procedure for nucleic acid. The method uses a wild type or mutant RNA polymerase designed to transcribe both deoxyribonucleotides and ribonucleotides (see FIGS. 1 - 7 ).
1 . An oligonucleotide primer comprising in the following order from 5′ to 3′:
a phage-encoded RNA polymerase recognition sequence,
a spacer sequence comprising a sequence of from 12 to 21 nucleotides, and
a target complementary sequence which can bind a segment of a target nucleic acid.
2 . The primer of claim 1 , wherein said spacer sequence comprises a nucleotide sequence having the formula (XY) n ,
wherein n is from 6 to 10,
wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide,
wherein X and Y are not the same.
3 . The primer of claim 2 , wherein X is an adenine nucleotide and Y is a guanine nucleotide.
4 . The primer of claim 3 , wherein n is 9.
5 . The primer of claim 1 , wherein said spacer sequence comprises adenine nucleotides, guanine nucleotides, and either cytosine nucleotides, or thymidine nucleotides.
6 . The primer of claim 5 , wherein said spacer sequence comprises a nucleotide sequence having the formula (XYZ) n , wherein n is from 4 to 7,
and wherein X is an adenine nucleotide, Y is a guanine nucleotide, and Z is either a cytosine nucleotide or a thymidine nucleotide.
7 . The primer of claim 1 , wherein said spacer sequence comprises a nucleotide sequence having the formula (X) n ,
wherein n is from 12 to 20,
wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide.
8 . The primer of claim 7 , wherein n is 18.
9 . A method of amplifying a target nucleic acid in an aqueous solution with a first and a second primer, said method comprising:
i.) transcribing an intermediate duplex with a phage-encoded RNA polymerase to form a sense transcription product having a 5′ end and a 3′ end,
wherein said intermediate duplex comprises a double-stranded molecule, wherein said double-stranded DNA molecule comprises a first and a second strand,
wherein said first strand comprises in the following order from 5′ to 3:
a phage-encoded RNA polymerase recognition sequence,
a first spacer sequence comprising a sequence of from 12 to 20 nucleotides, and
a first target complementary sequence which can bind to a segment of said target nucleic acid,
wherein said second strand comprises in the following order from 5′ to 3′:
a second target complementary sequence which can bind to a segment of said target nucleic acid,
a second spacer sequence comprising a sequence of from 12 to 20 nucleotides, and
a phage-encoded RNA polymerase recognition sequence,
wherein said transcribing takes place in the presence of Mn ++ , of all four dNTPs, and of those rNTPs represented in said first spacer sequence;
ii.) hybridizing said second primer to said sense transcription product to form a second primer-sense transcription product complex,
wherein said second primer comprises in the following order from 5′ to 3′:
a phage-encoded RNA polymerase recognition sequence, said second spacer sequence, and
said second target complementary sequence which can bind to a 3′ segment of said target nucleic acid;
iii.) extending said second primer-sense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a first amplification duplex;
iv.) transcribing said first amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn ++ , of all four dNTPs, and of those rNTPs represented in said second spacer sequence, to form an antisense transcription product;
v.) hybridizing said first primer to said antisense transcription product to form a first primer-antisense transcription product complex,
wherein said first primer comprises in the following order from 5′ to 3′:
a phage-encoded RNA polymerase recognition sequence, said first spacer sequence, and
said first target complementary sequence which can bind to a 5′ segment of said target nucleic acid;
vi.) extending said second primer-antisense transcription product complex with a Reverse Transcriptase that lacks RNAseH activity to form a second amplification duplex; and
vii.) transcribing said second amplification duplex with a phage-encoded RNA polymerase, in the presence of Mn ++ , of all four dNTPs, and of those rNTPs represented in said first spacer sequence to form said sense transcription product.
10 . The method of claim 9 , wherein the method further comprises repetitively carrying out steps i to vii.
11 . The method of claim 9 , wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (XY) n ,
wherein n is from 6 to 10,
wherein X and Y are independently selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thyrmidine nucleotide,
wherein X and Y are not the same.
12 . The method of claim 11 , wherein X is an adenine nucleotide and Y is a guanine nucleotide.
13 . The method of claim 12 , wherein n is 9.
14 . The method of claim 9 , wherein said spacer sequence comprises adenine nucleotides, guanine nucleotides, and either cytosine nucleotides, or thymidine nucleotides.
15 . The method of claim 14 , wherein said spacer sequence comprises a nucleotide sequence having the formula (XYZ) n , wherein n is from 4 to 7,
and wherein X is an adenine nucleotide, Y is a guanine nucleotide, and Z is either a cytosine nucleotide or a thymidine nucleotide.
16 . The method of claim 9 , wherein said first or said second spacer sequence comprises a nucleotide sequence having the formula (X) n ,
wherein n is from 12 to 20,
wherein X is selected from the group consisting of an adenine nucleotide, a guanine nucleotide, a cytosine nucleotide, and a thymidine nucleotide.
17 . The method of claim 16 , wherein n is 18.
18 . The method of claim 9 , wherein said sense and antisense transcription products comprise a nucleic acid strand comprising both ribonucleotides and deoxyribonucleotides.
19 . The method of claim 9 , wherein said first and said second amplification duplexes consist of deoxyribonucleotides and ribonucleotides.
20 . The method of claim 9 , wherein said method is carried out at a single temperature.
21 . The method of claim 9 , wherein said method is carried out at a single temperature of between 25° C. and 55° C.
22 . The method of claim 9 , wherein the method is carried out at a single temperature of greater than 50° C.
23 . The method of claim 9 , wherein said intermediate duplex comprises a double-stranded DNA comprising one complete primer sequence followed by the entire sequence that is to amplified.
24 . The method of claim 9 , wherein said intermediate duplex is formed from double-stranded DNA, single-stranded DNA, or RNA.
25 . The method of claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of:
denaturing a double-stranded DNA target to form an upper strand and a lower strand;
hybridizing said first primer to said lower strand to form a first primer-lower strand complex;
extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex;
denaturing said first long sense strand product-lower strand complex into a first long sense strand product and said lower strand;
hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and
extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.
26 . The method of claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of:
denaturing a double-stranded DNA target to form an upper strand and a lower strand;
hybridizing said first primer to said lower strand to form a first primer-lower strand complex;
extending said first primer-lower strand complex with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to form a first long sense strand product-lower strand complex, wherein said first long sense strand product has a 5′ and a 3′ end;
displacing said first sense strand product from said lower strand by:
hybridizing a bumper oligonucleotide to a subsequence on said lower strand adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex;
extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first sense strand product; hybridizing said second primer to said first long sense strand product to form a second primer-first long sense strand product; and
extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.
27 . The method of claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of:
hybridizing said second primer to a target RNA molecule to form a second primer-RNA template complex;
extending said second primer-target RNA molecule complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex, wherein said first long antisense strand product has a 5′ and a 3′ end;
displacing said first long antisense strand product from said target RNA molecule by:
hybridizing a bumper oligonucleotide to a subsequence on said target RNA molecule adjacent to said 5′ end of said first sense strand product on the first sense strand product-lower strand complex;
extending said bumper oligonucleotide with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase, thereby displacing said first long antisense strand product;
hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and
extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.
28 . The method of claim 9 , wherein said intermediate duplex is formed by the process comprising the following steps of:
hybridizing said second primer to a single-stranded target RNA molecule to form a second primer-RNA template complex;
extending said second primer-RNA template complex with a Reverse Transcriptase that lacks RNAseH activity or a DNA Polymerase to form a first long antisense strand product-template complex;
denaturing said first long antisense strand product-RNA template complex into a first long antisense strand product and said single-stranded RNA molecule;
hybridizing said first primer to said first long antisense strand product to form a first primer-first long antisense strand product complex; and
extending said first primer-first long antisense strand product with a Reverse Transcriptase that lacks RNAseH activity or with a DNA Polymerase to yield said intermediate duplex.
29 . The method of claim 9 , wherein said phage-encoded RNA polymerase is polymerase selected from the group consisting of: a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase.
30 . The method of claim 29 , wherein said phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase that is competent to incorporate dNTPs into a template nucleic acid.
31 . The method of claim 30 , wherein said phage-encoded RNA polymerase is a T7 RNA polymerase.
32 . The method of claim 31 , wherein said T7 RNA polymerase contains a Y639F mutation.
33 . The method of claim 31 , wherein said T7 RNA polymerase contains a S641A mutation.
34 . The method of claim 31 , wherein said T7 RNA polymerase contains at least two mutations.
35 . The method of claim 9 , wherein said Mn ++ is present in a concentration of between 10 μM to 20 mM.
36 . The method of claim 35 , wherein said concentration is 10 mM.
37 . The method of claim 9 , wherein said target nucleic acid is single-stranded DNA.
38 . The method of claim 9 , wherein the target nucleic acid is comprised of RNA.
39 . The method of claim 9 , further detecting said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex,
wherein said detecting comprises hybridizing a detection oligonucleotide comprising a detectable moiety, wherein said detection oligonucleotide is complementary to a subsequence of said sense transcription product, said antisense transcription product, said first amplification duplex, or said second amplification duplex.
40 . A kit for copying a target nucleic acid comprising:
a container containing:
a first nucleotide primer comprising in the following order from 5′ to 3′:
a phage-encoded RNA polymerase recognition sequence,
a first spacer sequence comprising a sequence of from 12 to 20 nucleotides,
a first target complementary sequence which can bind to a segment of said target nucleic acid; and
a second primer comprising in the following order from 5′ to 3′:
a phage-encoded RNA polymerase recognition sequence,
a second spacer sequence comprising a sequence of from 12 to 20 nucleotides, and
a second target complementary sequence which can bind to a segment of said target nucleic acid.
41 . The kit of claim 40 , wherein said phage-encoded RNA polymerase is polymerase selected from the group consisting of: a T7 RNA polymerase, a T4 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase and a K11 RNA polymerase.
42 . The kit of claim 41 , wherein said phage-encoded RNA polymerase is a mutant phage-encoded RNA polymerase that is competent to incorporate dNTPs into a template nucleic acid.
43 . The kit of claim 41 , wherein said phage-encoded RNA polymerase is a T7 RNA polymerase.
44 . The kit of claim 43 , wherein said T7 RNA polymerase contains a Y639F mutation.
45 . The kit of claim 43 , wherein said T7 RNA polymerase contains a S641 A mutation.
46 . The kit of claim 43 , wherein said T7 RNA polymerase contains at least two mutations.
47 . The kit of claim 40 , further comprising a member selected from the group consisting of:
a DNA polymerase;
a Reverse Transcriptase that lacks RNAseH activity;
a phage-encoded RNA polymerase;
all four dNTPs;
those rNTPs represented in said first and second spacer sequences;
reaction buffer containing manganese in a concentration from 10 μM to 20 mM and magnesium in a concentration from 5 mM to 20 mM;
a positive control target nucleic acid; and
instructions for carrying out a method of copying a nucleic acid using said first primer and said second primer.