# Patent

## Title
Pyrophosphorolysis-activated polymerization (PAP) using ribonucleic acid (RNA) template

## Abstract

A new method of RNA-PAP was developed that can directly amplify RNA template without additional treatment. RNA-PAP brings in a new mechanism for amplification of RNA template in which RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization are serially coupled using 3′ blocked primers. Due to this serial coupling, RNA-PAP has high selectivity against mismatches on the RNA template, providing highly specific amplification of RNA template. In addition, mutant polymerases were genetically engineered for higher efficiency of RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional patent application Ser. No. 61/724,292, filed on Nov. 8, 2012.


SEQUENCE LISTING
This application is being filed along with a Sequence Listing and its electronic format entitled SequenceListing.txt.
BACKGROUND
The present invention relates to the field of molecular biology and particularly to methods for amplification of ribonucleic acid (RNA).
PAP Technology for Amplification of DNA Template
Pyrophosphorolysis activated polymerization is a method for nucleic acid amplification1 2 where pyrophosphorolysis and polymerization are serially coupled by DNA polymerase using 3′ blocked primers. A primer is blocked at the 3′ end with a non-extendable nucleotide (3′ blocker), such as a dideoxynucleotide, and cannot be directly extended by DNA polymerase. When the 3′ blocked primer anneals to its complementary DNA template, DNA polymerase can remove the 3′ blocker from the 3′ blocked primer in the presence of pyrophosphate, which reaction is called pyrophosphorolysis. The DNA polymerase can then extend the 3′ unblocked primer on the DNA template. In addition to references cited herein, PAP has been described in U.S. Pat. Nos. 6,534,269, 7,033,763, 7,105,298, 7,238,480, 7,504,221, 7,914,995, and 7,919,253.
The serial coupling of pyrophosphorolysis and extension using the 3′ blocked primer in PAP results in an extremely high selectivity1 3 because a significant nonspecific amplification (Type II error) requires mismatch pyrophosphorolysis followed by mis-incorporation by the DNA polymerase, an event with a frequency estimated to be 3.3×10−11.
The bi-directional form of PAP (Bi-PAP) is especially suitable for allele-specific amplification that uses two opposing 3′ blocked primers with a single-nucleotide overlap at their 3′ ends3 4. Bi-PAP can detect one copy of a mutant allele in the presence of 109 copies of the wildtype DNA without false positive amplifications.
PAP was initially tested with Tfl and Taq polymerases using DNA template of the human dopamine D1 gene1, proving the principle that DNA-dependent DNA pyrophosphorolysis and DNA-dependent DNA polymerization can be serially coupled. The efficiency of PAP was greatly improved using TaqFS, a genetically engineered polymerase containing contain a F667Y mutation5, which were demonstrated using other DNA templates2 4 6.
However, no evidence has showed that PAP can work using RNA template, a long-felt but unsolved need. For PAP to work using RNA template, it is also required RNA-dependent DNA polymerization and RNA-dependent DNA pyrophosphorolysis which latter feasibility has not been demonstrated.
RNA-Dependent DNA Polymerization or Reverse Transcription Used in RT-PCR
The first step of RT-PCR is DNA polymerization or primer extension on RNA template that is catalyzed by RNA-dependent DNA polymerase or reverse transcriptase. Avian myeloblastosis virus (AMV) and moloney murine leukemia virus (MMLV) reverse transcriptases, two mesophilic retroviral transcriptases, are commonly used in this first step to convert the RNA template into its complimentary DNA (cDNA) product7 8. Native Taq, a thermophilic DNA polymerase, has measurable reverse transcriptase activity particularly in the presence of Mn2+ divalent ion9. rTth, another thermophilic DNA polymerase, shows over 100-fold greater reverse transcriptase activity than Taq even though they have significant amino acid sequence similarity10. Furthermore, Taq and rTth polymerases were also genetically engineered in order for higher reverse transcriptase activity11 12 13 14.
RNA-Dependent DNA Pyrophosphorolysis
Taq, Tfl, TaqFS, Pfu, and Vent polymerases can catalyze DNA-dependent DNA pyrophosphorolysis1 2 3 15. HIV and HCV reverse transcriptases were also reported to catalyze DNA-dependent DNA pyrophosphorolysis that removes 3′ dideoxynucleotide from DNA primer on synthetic DNA (rather than RNA) template16 17.
However, there was no report of RNA-dependent DNA pyrophosphorolysis of polymerase that removes 3′ deoxyribonucleotide or 3′ dideoxynucleotide or 3′ acyclonucleotide from a primer using RNA as template.
Advantages of the Invention
It is advantageous that RNA-PAP can direct amplify RNA template without additional treatment. In addition, RNA-PAP has high selectivity against mismatches on the RNA template, providing highly specific amplification of RNA template. Furthermore, we genetically engineered mutant polymerases for higher RNA-PAP efficiency.
SUMMARY OF THE INVENTION
A new method of RNA-PAP was developed that can directly amplify RNA template. RNA-PAP brings in a new mechanism for amplification of RNA template in which RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization are serially coupled using 3′ blocked primers.
The RNA-PAP method for synthesizing nucleic acid using RNA template comprises:
(a) a 3′ blocked primer that has a non-extendable nucleotide at the 3′ end (3′ blocker) anneals to its complementary RNA template, (b) RNA-dependent DNA pyrophosphorolysis removes the 3′ blocker from the 3′ blocked primer to produce a 3′ unblocked primer, and (c) RNA-dependent DNA polymerization extends the 3′ unblocked primer.
Of the RNA-PAP method, the steps (a), (b), and (c) can be repeated until a desired level of the extended nucleic acid is achieved.
In an embodiment, the RNA-PAP method can further include a second primer in the reaction: (d) the second primer anneals to the nucleic acid product of the step (c), and (e) DNA-dependent DNA polymerization extends the second primer, thereby an exponential amplification can be achieved.
In another embodiment, the RNA-PAP method can further include a second 3′ blocked primer: (f) the second 3′ blocked primer anneals to the nucleic acid product of the step (c), (g) DNA-dependent DNA pyrophosphorolysis removes the 3′ blocker from the blocked primer to produce a 3′ unblocked primer, and (h) DNA-dependent DNA polymerization extends the 3′ unblocked primer.
Of the RNA-PAP method, steps (b) and (c) are catalyzed by a polymerase.
In a preferred embodiment, steps (b), (c), (d), (e), (g), and (h) are catalyzed by a polymerase in one reaction tube.
The polymerase may be a genetically engineered DNA polymerase selected from the group consisting of TaqFS, TaqFS681G, TaqFS681K, TaqFS681V, TaqFS681Y, TaqFS608V681 G, TaqFS599V602A605A608V681G, TaqFS742S747I corresponding to Taq polymerase according to SEQ ID NO: 10.
In a preferred embodiment, the genetically engineered polymerase is selected from the group of mutants consisting of 599V, 602A, 605A, 608V, 667Y, 681G, 681K, 681V, 681R, 742S, and 747I mutations corresponding to Taq polymerase according to SEQ ID NO: 10.
In another preferred embodiment, the genetically engineered polymerase is selected from group of mutants consisting of amino acids located at 599, 602, 605, 608, 667, 681, 742, and 747 corresponding to Taq polymerase according to SEQ ID NO: 10.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the principle of RNA-PAP. The DNA primer is blocked at the 3′ end with, e.g., a dideoxynucleotide, preventing it from extended by polymerase. When the 3′ blocked primer anneals to its complementary RNA template, the 3′ dideoxynucleotide can be removed by RNA-dependent DNA pyrophosphorolysis. Then the unblocked DNA primer can be extended on the RNA template by RNA-dependent DNA polymerization.
FIG. 2 illustrates optimal dNTP concentration for RNA-dependent DNA polymerization. RT-PCR Assays II (panels A to D) and III (panels E to H) amplified from 0.25 ng of total RNA in 25 μl of reaction. One Unit of each polymerases of Taq (panels A and E), rTth (panels B and F), TaqFS (panels C and G), and TaqFS742S747I (panels D and H) was used. The amplification plot is showed with cycle number and SybrGreen fluorescence unit for the given cycle.
FIG. 3 illustrates RNA-PAP feasibility with various polymerases. RNA-PAP Assays IV (panels A to D), V (panels E to H) and VI (panels I to L) amplified from 0.25 ng of total RNA. For each assay, four polymerases of TaqFS (panels A, E, and I), TaqFS681G (panels B, F, and J), TaqFS681K (panels C, G, and K), and TaqFS742S747I (panels D, H, and L) were tested. The amount of each enzyme was 2-fold serially diluted from 1 U to 1/16 U in 25 μl of reaction.



DETAILED DESCRIPTION OF THE INVENTION
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
PCR refers to polymerase chain reaction.
RT-PCR refers to reverse transcription polymerase chain reaction.
Pyrophosphorolysis is the reverse reaction of deoxyribonucleic acid polymerization. In the presence of pyrophosphate, the 3′ nucleotide is removed by a polymerase from duplex DNA to generate a triphosphate nucleotide and a 3′ unblocked duplex DNA: [dNMP]n+PPi→[dNMP]n-1+dNTP18.
DNA-dependent DNA pyrophosphorolysis refers to pyrophosphorolysis of DNA using DNA template. It is the reverse reaction of DNA-dependent DNA polymerization.
RNA-dependent DNA pyrophosphorolysis refers to pyrophosphorolysis of DNA using RNA template. It is the reverse reaction of RNA-dependent DNA polymerization.
Polymerase refers to a polymerase characterized as polymerization or extension of deoxyribonucleic acids.
DNA-dependent DNA polymerase refers to a polymerase characterized as DNA polymerization using DNA template.
RNA-dependent DNA polymerase or reverse transcriptase refers to a polymerase characterized as DNA polymerization using RNA template.
3′ blocked primer refers to an oligonucleotide with a 3′ non-extendable nucleotide (3′ blocker), such as a dideoxynucleotide. The 3′ nucleotide could not be directly extended, but it can be removed by pyrophosphorolysis and then the unblocked primer can be extended by polymerase.
PAP refers to pyrophosphorolysis activated polymerization.
RNA-PAP refers to PAP using RNA template.
cDNA refers to a complementary DNA molecule synthesized using RNA template.
Thermostable refers to an enzyme that is heat stable or heat resistant.
Protein mutation refers to a change in amino acid residue at a location of a protein, like Taq polymerase. The change in amino acid residue is defined with respect to a naturally occurring protein. A protein having a mutation is referred to as a “mutant” protein.
Unless indicated otherwise, an amino acid position should be interpreted as the analogous position in all DNA polymerases, even though the single letter amino acid code specifically relates to the amino acid residue at the indicated position in Taq polymerase.
Principle of RNA-PAP
The invention is a RNA amplification method of synthesizing desired nucleic acid on RNA template (FIG. 1).
The method comprises the following steps: a) hybridization: annealing to the RNA template a complementary 3′ blocked primer that has a 3′ non-extendable nucleotide (3′ blocker), b) RNA-dependent DNA pyrophosphorolysis: removing the 3′ blocker from the 3′ blocked primer, and c) RNA-dependent DNA polymerization: extending the 3′ unblocked primer to synthesize the desired nucleic acid (FIG. 1).
The method yields a single-stranded complementary DNA (cDNA), or an RNA/cDNA duplex. Subsequent to step c, the cDNA product can be further amplified, for example, by PAP or PCR.
The method may employ a polymerase that catalyzes both RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization.
3′ blockers include but not limited to 3′ dideoxynucleotides. Other 3′ blockers, such as acyclonucleotides that substitute a 2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar3, may also be used.
A biological sample may contain RNA molecules. The RNA may be heterogeneous in which the target RNA is a small portion.
EXAMPLES
The practice of the present invention employs, unless otherwise indicated, common techniques of chemistry, molecular biology, and recombinant DNA, e.g., those by Sambrook et al., Molecular Cloning, 2nd Ed.19; Sambrook and Russell, Molecular Cloning, 3rd Ed.20; Ausubel et al., Current Protocols in Molecular Biology21.
Example 1
Materials and Methods
Preparation of Primers
Each 3′ blocked primer, 30 nucleotides long and blocked with ddCMP at the 3′ end, was chemically synthesized and HPLC purified by Integrated DNA Technologies. The regular DNA primers were also synthesized by the same vendor (Table 1).







TABLE 1







List of primers and assays
















Product





Sequence (5′ to 3′)

size
Starting


Assay
Name b
(SEQ ID NO:)
Location
(bp)
template





I.
P53(13280)20D
AGGGTCCCCAGGCCTCTG
Intron
208
Genomic


PCR

AT (1)
5

DNA



P53(13488)22U
GCCACTGACAACCACCCT
Intron






TAAC (2)
6




 


II.
ACTB(426)25D
CAACCGCGAGAAGATGA
Exons
 63
Total


RT-

CCCAGATC (3)
3 and 4

RNA


PCR a
ACTB(488)25U
GCAACGTACATGGCTGG
Exon 4






GGTGTTGA (4)





 


III.
ACTB(426)25D
CAACCGCGAGAAGATGA
Exons
280
Total


RT-

CCCAGATC (3)
3 and 4

RNA


PCR
ACTB(705)25U
TTCCCGCTCGGCCGTGGT
Exon 4






GGTGAAG (5)





 


IV.
ACTB(426)25D
CAACCGCGAGAAGATGA
Exons
 63
Total


RNA-

CCCAGATC (3)
3 and 4

RNA


PAP a
ACTB(488)30U
GCAACGTACATGGCTGG
Exon 4






GGTGTTGAAGGTddC (6) c





 


V.
ACTB(426)25D
CAACCGCGAGAAGATGA
Exons
147
Total


RNA-

CCCAGATC (3)
3 and 4

RNA


PAP a
ACTB(572)30U
ACAGTGTGGGTGACCCC
Exon 4






GTCACCGGAGTCddC (7)





 


VI.
GAPDH(50)-
ACCATGGGGAAGGTGAA
Exon 2
 61
Total


RNA-
30D
GGTCGGAGTCAAddC (8)


RNA


PAP a
GAPDH(110)-
TGGTGACCAGGCGCCCA
Exon 3





30U
ATACGACCAAATddC (9)





a In order to avoid non-specific amplification from potentially contaminated genomic DNA, RT-PCR and RNA-PAP primers were designed to anneal to sequences of the transcript that span intron regions.


b For example, P53 means the human P53 gene; (13280), 5′ end of the primer begins at nucleotide 13280 according to GenBank accession: x54156; D, downstream (i.e., in the direction of transcription). The ACTB mRNA is from GenBank accession: NM_001101.3. The GAPDH mRNA is from GenBank accession: AB062273.1. The precise sizes and locations of the PCR fragment can be obtained from the informative names.


c Due to the availability of chemical synthesis, ddC was exampled.






Total RNA Extraction

The total RNA was extracted from blood white cells using QIAamp RNA kit according to Qiagen' protocol (QIAamp RNA Blood Mini Handbook). Within the process, RNase-Free DNase was used to remove contaminated genomic DNA. The concentration of the total RNA was measured by a spectrophotometer at 260 nm. The quality was controlled with A260/A280 ratio between 1.8 and 2.1. It was stored at −20° C. until used.
Construction of Recombinant Plasmids
The amino acid sequence of the wildtype form of Taq polymerase is as described in GenBank accession AAA27507.1 (SEQ ID NO: 10). The numbering starts at the amino terminus residue. The letter is the single letter amino acid code for the amino acid residue at the indicated position.
Recombinant plasmid that encodes the wildtype form of Taq polymerase was constructed by ligating the Taq polymerase coding sequence, a 2.4 kb DNA segment from 121 nt to 2619 nt of GenBank accession J04639, into pET14b vector at the Nde I and BamH I restriction sites according to Navagen pET system user manual. Then the recombinant plasmid transformed DH5α E. coli cells and its plasmid DNA was extracted. The ligated Taq polymerase coding sequence was confirmed by ABI Sanger sequencing analysis.
Eight recombinant plasmids that encode mutant forms of Taq polymerase were constructed using mutagenic primers designed by QuikChange primer design program and QuikChange lightning site-directed mutagenesis kit according to Stratagene's user manual. Then each mutated plasmid transformed DH5α E. coli cells and its plasmid DNA was extracted. The mutant DNA sequence was confirmed by ABI Sanger sequencing analysis.
TaqFS contained G46D and F667Y mutations, TaqFS681G contained G46D, F667Y and E681G mutations, TaqFS681K contained G46D, F667Y and E681K mutations, TaqFS681V contained G46D, F667Y and E681V mutations, TaqFS681Y contained G46D, F667Y and E681Y mutations, TaqFS608V681G contained G46D, 608V, F667Y and E681G mutations, TaqFS599V602A605A608V681G contained G46D, I599V, E602A, L605A, A608V, F667Y and E681G mutations, and TaqFS742S747I contained G46D, F667Y, E742S, and M747I mutations. Individual substitution mutations are indicated by the form of a letter/number/letter combination. The letters are the single letter code for amino acid residues. The numbers indicate the amino acid residue position of the mutation site according to SEQ ID NO: 10.
Expression and Extraction of Mutant Polymerases
The mutant Taq polymerases were expressed in transformed T7 Express lysY/Iq E. coli cells according to BioLabs' manual. Before the start codon of Taq polymerase, 6× His-Tag residues were located and co-expressed. Because the expressed polymerase contained His-Tag at the N-terminus, it was purified using Qiagen Ni-NTA His-Tag technology. A typical yield of the purified polymerase was 4 mg from 500 ml of induced E. coli culture. SDS-PAGE analysis of the purified protein showed one major band of about 95,000 Daltons after Coomassie Blue staining, indicating ≧90% purity.
The enzyme was stored in the storage buffer containing 20 mM Tris-HCl (pH 8.0 at 25° C.), 100 mM KCl, 0.1 mM EDTA, 50% Glycerol, 0.5% Tween-20, and 0.5% NP-40 at −20° C. until use.
PCR to Calibrate DNA-Dependent DNA Polymerase Activity
The primers of Assay I were designed to amplify a 209-bp region of exon 6 of the human P53 gene. Each PCR mixture contained a total volume of 25 μl: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 200 μM each dNTPs (dATP, dTTP, dGTP and dCTP), 0.1 μM each primer, 0.1× SybrGreen I dye, 0.02% Twee-20, 0.02% NP-40, 20 ng of genomic DNA, and various units of polymerase.
A Bio-Rad CFX96 real time PCR detection system was used for quantification of the amplified product. Analysis mode: SybrGreen fluorophore, Baseline setting: baseline subtracted curve fit, Threshold cycle (Ct) determination: single threshold, Baseline method: SYBR auto calculated, Threshold setting: auto calculated.
The cycling entailed denaturation at 94° C. for 15 seconds, annealing at 55° C. for 30 seconds, and elongation at 72° C. for 1 minute for 30 cycles. Before thermocycling, a step of 94° C. for 2 minutes was applied to completely denature the genomic DNA.
For the activity calibration, Taq polymerase (Roche) was taken as standard and its amount was 2-fold serially diluted from 1 U to 1/16 U in 25 μl of reaction. For comparison, other enzymes were diluted in the same way. The minimum unit of a polymerase with a measurable Ct within 30 cycles reflected its compatible level of DNA-dependent DNA polymerase activity.
To confirm the amplified product, melting curving analysis was followed from 68° C. to 95° C. with increment 0.5° C. and holding 5 seconds.
RT-PCR to Measure RNA-Dependent DNA Polymerization
Unless stated otherwise, the RT-PCR reaction mixture of 25 μl contained 880 mM Tris-HCl (pH 8.0 at 25° C.), 10 mM (NH4)2SO4, 1.2 mM MgCl2, 25 μM each dNTPs (dATP, dTTP, dGTP and dCTP), 0.1 μM each primer of Assay II or III, 90 μM Na4PPi, 0.1× SybrGreen I dye, 0.02% Twee-20, 0.02% NP-40, 1 U of polymerase, and 0.25 ng of total RNA template.
A Bio-Rad CFX96 real time PCR detection system was used for quantification of the amplified product. Analysis mode: SybrGreen fluorophore, Baseline setting: baseline subtracted curve fit, Threshold cycle (Ct) determination: single threshold, Baseline method: SYBR auto calculated, Threshold setting: auto calculated.
The cycling entailed 96° C. for 12 seconds, 60° C. for 30 seconds, 64° C. for 30 seconds, and 68° C. for 30 seconds for a total of 30-35 cycles. A denaturing step of 96° C. for 2 minutes was added before the first cycle.
To confirm the amplified product, melting curving analysis was followed from 68° C. to 95° C. with increment 0.5° C. and holding 5 seconds.
For further confirmation, the product was electrophoresed through a standard 3% agarose gel. The gel was stained with ethidium bromide for UV photography by a charge-coupled device camera.
RNA-PAP
Unless stated otherwise, the PAP reaction mixture of 25 μl contained 880 mM Tris-HCl (pH 8.0 at 25° C.), 10 mM (NH4)2SO4, 1.2 mM MgCl2, 25 μM each dNTPs (dATP, dTTP, dGTP and dCTP), 0.1 μM each primer of Assay IV, V, or VI, 90 μM Na4PPi, 0.1× SybrGreen I dye, 0.02% Twee-20, 0.02% NP-40, various units of polymerase, and 0.25 ng of total RNA template.
A Bio-Rad CFX96 real time PCR detection system was used for quantification of the amplified product. Analysis mode: SybrGreen fluorophore, Baseline setting: baseline subtracted curve fit, Threshold cycle (Ct) determination: single threshold, Baseline method: SYBR auto calculated, Threshold setting: auto calculated.
The cycling entailed 96° C. for 12 seconds, 60° C. for 30 seconds, 64° C. for 30 seconds, and 68° C. for 30 seconds for a total of 30-35 cycles. A denaturing step of 96° C. for 2 min was added before the first cycle.
To confirm the amplified product, melting curving analysis was followed from 68° C. to 95° C. with increment 0.5° C. and holding 5 seconds to confirm the amplified product.
For further confirmation, the product was electrophoresed through a standard 3% agarose gel. The gel was stained with ethidium bromide for UV photography by a charge-coupled device camera.
Example 2
Optimal Conditions for RNA-Dependent DNA Polymerization
RT-PCR includes two steps of 1) reverse transcription of RNA template into cDNA product and 2) regular PCR amplification of the cDNA product. Reverse transcription is considered as the efficiency limiting reaction of the whole process.
In reverse transcription, AMV7 and MMLV8 reverse transcriptases as well as Taq9, Tth10 polymerases and their genetically engineered forms11 12 13 14 are commonly used with each dNTP concentration to be 200 μm. In RT-PCR, this concentration of dNTPs is suitable to the reverse transcription step and the subsequent PCR step. However, it severely inhibited pyrophosphorolysis1. Thus, an optimal dNTP concentration was needed to explore.
To search for optimal reaction conditions, we designed two RT-PCR Assays II and III (Table 1). RT-PCR Assay II amplified a 63-bp ACTB mRNA region with primers ACTB(426)25D and ACTB(488)25U, and Assay III amplified a 280-bp ACTB mRNA region with primers ACTB(426)25D and ACTB(705)25U. Each assay had a forward primer and a reverse primer with the reverse primer matching the RNA template.
First, we examined the effect of various dNTP concentrations in the presence of Mg2+ divalent ion. In FIG. 2, three dNTP conditions of 25 μM each dNTP with 90 μM PPi, 25 μM each dNTP without PPi, as well as 200 μM dNTP without PPi were tested. Of four polymerases of Taq, rTth, TaqFS, and Taq681G, one unit was added to 25 μl of reaction. The polymerase activity for DNA-dependent DNA polymerization was calibrated according to Taq's activity (Roche) by amplifying a 209-bp region of exon 6 of the P53 gene (PCR Assay I) from human genomic DNA under standard PCR conditions. In addition, one unit of Taq DNA polymerase is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75° C. (Roche). With the two RT-PCR Assays II and III, we found that the RNA-dependent DNA polymerase activity was higher with 25 μM each dNTP than with 200 μM. Thus, 25 μM rather than 200 μM each dNTP was used for the following testing.
Second, we examined the effect of various polymerase amounts. Eight polymerases of TaqFS, TaqFS681G, TaqFS681K, TaqFS681V, TaqFS681Y, TaqFS608V681G, TaqFS599V602A605A608V681G were tested. The amount of each enzyme was 2-fold serially diluted from 1 U to 1/16 U in 25 μl of reaction (Table 2). The minimum unit of a polymerase with a measurable Ct within 30 cycles was considered to reflect the compatible level of RNA-dependent DNA polymerization because it is the efficiency limiting reaction of the whole process. We found that the eight enzymes showed different levels of RNA-dependent DNA polymerase activity.
In addition, in melting curving analysis, only one melting peak showed the Tm value from the amplified product (Tm=80.5-82° C. for Assays II and Tm=89.5-90° C. for Assay III). No-template-negative control did not show any measurable Ct.
Example 3
RNA-PAP
The process of RNA-PAP can be divided into two steps. In the first step with RNA as starting template, RNA-dependent DNA pyrophosphorolysis removes the 3′ blocker from the 3′ blocked primer, and then RNA-dependent DNA polymerization extends the 3′ unblocked primer to generate a DNA product. In the second step with the DNA product as template, DNA-dependent DNA pyrophosphorolysis removes the 3′ blocker from the 3′ blocked primer and then DNA-dependent DNA polymerization extends the 3′ unblocked primer. Thus, four distinct reactions are involved that can be catalyzed by a polymerase.
For proof of principle, we designed three RNA-PAP Assays IV, V, and VI (Table 1) that amplified from RNA template. Each assay had a forward primer and a reverse primer for exponential amplification. The reverse primer, blocked by a dideoxynucleotide at the 3′ end, matched the RNA template.
For each assay, eight polymerases of TaqFS, TaqFS681G, TaqFS681K, TaqFS681V, TaqFS681Y, TaqFS608V681G, TaqFS599V602A605A608V681G, and TaqFS742S747I were examined. The amount of each enzyme was 2-fold serially diluted from 1 U to 1/16 U in 25 μl of reaction.
RNA-PAP Assay VI amplified a 63-bp ACTB mRNA region with primers ACTB(426)25D and ACTB(488)30U (Table 2, FIG. 3A). In the first step, RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide from the 3′ blocked primer ACTB(488)30U and then RNA-dependent DNA polymerization extended the unlocked primers by 34 bases.
RNA-PAP Assay V amplified a 147-bp ACTB mRNA region with primers ACTB(426)25D and ACTB(572)30U (Table 2, FIG. 3B). In the first step, RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide from the 3′ blocked primer ACTB(572)30U and then RNA-dependent DNA polymerization extended the unlocked primers by 118 bases.
RNA-PAP Assay VI amplified a 61-bp GAPDH mRNA region with primers GAPDH(50)-30D and GAPDH(110)-30U (Table 2, FIG. 3C). In the first step, RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide from the 3′ blocked primer GAPDH(110)-30U and then RNA-dependent DNA polymerization extended the unlocked primers by 32 bases.
With Assays IV, V, and VI, we proved the principle of RNA-PAP and particularly demonstrated the feasibility of 1) RNA-dependent DNA pyrophosphorolysis and 2) the serial coupling of RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization. We also found that five enzymes of TaqFS681K, TaqFS681V, TaqFS681Y, TaqFS608V681G, and TaqFS742S747I showed higher activities particularly in RNA-dependent DNA pyrophosphorolysis.
In addition, in melting curving analysis, only one melting peak showed the Tm value from the amplified product (Tm=80.5-81.5° C. for Assay IV, Tm=87.5-88° C. for Assay V, and Tm=82-83° C. for Assay VI). No-template-negative control did not show any measurable Ct.







TABLE 2







The minimum units of polymerases required for RT-PCR and


RNA-PAPa














RT-






RT-
PCR



PCR
Assay
RNA-PAP
RNA-PAP
RNA-PAP


Polymerase
Assay II
III
Assay IV
Assay V
Assay VI





TaqFS
0.125Ub
1U
0.5U
1U
0.5U


TaqFS681G
0.25U
NA
1U
NA
NA


TaqFS681K
0.125U
0.25U
0.0625U
0.125U
0.125U


TaqFS681V
0.125U
0.5U
0.25U
0.5U
0.25U


TaqFS681Y
0.125U
0.5U
0.125U
0.25U
0.25U


TaqFS608V681G
0.125U
0.5U
0.125U
0.125U
0.0625U


TaqFS599V602A
NAc
NA
1U
NA
NA


605A608V


681G


TaqFS742S747I
0.25U
0.5U
0.25U
0.5U
0.25U





aThe unit is defined as DNA-dependent DNA polymerase activity calibrated according to Taq's activity.


bwith 0.25 ng of total RNA, the amount of enzyme was 2-fold serially diluted from 1U to 1/16U in 25 μl of reaction. The minimum required unit of a polymerase with a measurable Ct was counted within 30 cycles.


cno Ct was called within 30 cycles.






RNA-Dependent DNA Pyrophosphorolysis vs. RNA-Dependent DNA Polymerization

The process of RNA-PAP can be divided into two steps. In the first step with RNA as starting template are two distinct reactions of RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization catalyzed serially by a polymerase.
To show their relative importance, the minimum units of polymerases were compared for RT-PCR and RNA-PAP (Table 3). In RT-PCR Assay II, RNA-dependent DNA polymerization extended 38 bases from the 3′ end of the primer ACTB(488)25U. On the other hand in RNA-PAP Assay IV, RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide from the 3′ blocked primer ACTB(488)30U and then RNA-dependent DNA polymerization extended the unlocked primers by 34 bases. In RNA-PAP Assay VI, RNA-dependent DNA pyrophosphorolysis removed the 3′ ddC nucleotide from the 3′ blocked primer GAPDH(110)-30U and then RNA-dependent DNA polymerization extended the unlocked primers by 32 bases.







TABLE 3







Ratio of minimum units required for RT-PCR and RNA-PAP
















Ratio of
Ratio of






Assay
Assay



RT-PCR
RNA-PAP
RNA-PAP
II to
II to


Polymerase
Assay II
Assay IV
Assay VI
IV
VI





TaqFS
0.125U
0.5U
0.5U
1⁄4
1⁄4


TaqFS681G
0.25U
1U
NA
1⁄4


TaqFS681K
0.125U
0.0625U
0.125U
2
1


TaqFS681V
0.125U
0.25U
0.25U
1⁄2
1⁄2


TaqFS681Y
0.125U
0.125U
0.25U
1
1⁄2


TaqFS608V681G
0.0625U
0.125U
0.0625U
1⁄2
1⁄2


TaqFS599V602A
NA
1U
NA


605A608V


681G


TaqFS742S747I
0.25U
0.25U
0.25U
1
1









Because of the small amplicon sizes, the ratios of minimum required units between RT-PCR Assay II and RNA-PAP Assay IV or VI indicate the relative importance between RNA-dependent DNA pyrophosphorolysis and RNA-dependent DNA polymerization (Table 3). Polymerases with the ratios of minimum units to be ≧1 are preferred, such as TaqFS681K, for RNA-PAP.
REFERENCES


    1. Liu, Q., and Sommer, S. S. (2000). Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification. BioTechniques 29, 1072-1080.
    2. Liu, Q., and Sommer, S. S. (2002). Pyrophosphorolysis-activatable oligonucleotides may facilitate detection of rare alleles, mutation scanning and analysis of chromatin structures. Nucleic acids research 30, 598-604.
    3. Liu, Q., and Sommer, S. S. (2004). Pyrophosphorolysis by Type II DNA polymerases: implications for pyrophosphorolysis-activated polymerization. Analytical biochemistry 324, 22-28.
    4. Liu, Q., and Sommer, S. S. (2004). Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues. BioTechniques 36, 156-166.
    5. Tabor, S., and Richardson, C. C. (1995). A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proceedings of the National Academy of Sciences of the United States of America 92, 6339-6343.
    6. Shi, J., Liu, Q., and Sommer, S. S. (2007). Detection of ultrarare somatic mutation in the human TP53 gene by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification. Human mutation 28, 131-136.
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    9. Jones, M. D., and Foulkes, N. S. (1989). Reverse transcription of mRNA by Thermus aquaticus DNA polymerase. Nucleic acids research 17, 8387-8388.
    10. Myers, T. W., and Gelfand, D. H. (1991). Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30, 7661-7666.
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    12. Vichier-Guerre, S., Ferris, S., Auberger, N., Mahiddine, K., and Jestin, J. L. (2006). A population of thermostable reverse transcriptases evolved from Thermus aquaticus DNA polymerase I by phage display. Angewandte Chemie 45, 6133-6137.
    13. Smith, E. S. E., C. M. Gelfand, D. H. Higuchi, R. G. Myers, T. W. Schonbrunner, N. J. Wang, A. M. (2007). High temperature reverse transcription using mutant DNA polymerases. U.S. Pat. No. 7,179,590.
    14. Sauter, K. B., and Marx, A. (2006). Evolving thermostable reverse transcriptase activity in a DNA polymerase scaffold. Angewandte Chemie 45, 7633-7635.
    15. Gardner, A. F., Joyce, C. M., and Jack, W. E. (2004). Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase. The Journal of biological chemistry 279, 11834-11842.
    16. Anion, D., Kaushik, N., McCormick, S., Borkow, G., and Parniak, M. A. (1998). Phenotypic mechanism of HIV-1 resistance to 3′-azido-3′-deoxythymidine (AZT): increased polymerization processivity and enhanced sensitivity to pyrophosphate of the mutant viral reverse transcriptase. Biochemistry 37, 15908-15917.
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## Claims

1. A method for synthesizing nucleic acid on a RNA template, comprising:
(a) annealing to the RNA template a complementary 3′ blocked primer wherein the 3′ blocked primer has a dideoxynucleotide at the 3′ end, which, if not removed, would prevent the 3′ blocked primer from being extended;
(b) removing the dideoxynucleotide from the 3′ end of the 3′ blocked primer by pyrophosphorolysis to produce a 3′ unblocked primer; and
(c) extending the 3′ unblocked primer by polymerization to produce a nucleic acid product, wherein steps (b) and (c) are catalyzed by a mutant Taq DNA polymerase having RNA-dependent DNA polymerase activity, and wherein the mutant DNA polymerase has a 667Y mutation and another mutation selected from the group consisting of 599V, 602A, 605A, 608V, 681G, 681K, 681V, 681R, 742S and 747I mutations corresponding to Taq DNA polymerase according to SEQ ID NO: 10.

2. The method of claim 1, wherein the steps (a), (b), and (c) are repeated one or more times.

3. The method of claim 1, further comprising:
(d) annealing a second primer to the nucleic acid product of the step (c), and
(e) extending the second primer, wherein steps (a) to (e) occur in a single reaction tube.

4. The method of claim 1, further comprising:
(f) annealing a second 3′ blocked primer having a dideoxynucleotide at the 3′ end to the nucleic acid product of the step (c),
(g) removing the dideoxynucleotide from the 3′ end of the second 3′ blocked primer by pyrophosphorolysis to produce a second 3′ unblocked primer, and
(h) extending the second 3′ unblocked primer, wherein steps (a) to (c) and (f) to (h) occur in a single reaction tube.

5. The method of claim 1, wherein steps (b) and (c) are catalyzed by a DNA polymerase in the presence of Mg++ ion rather than Mn++ ion,
whereby the dideoxynucleotide from the 3′ end of the blocked primer is removed by pyrophosphorolysis and then the 3′ unblocked primer is extended by polymerization.

6. The method of claim 1, wherein dATP, dTTP, dGTP, and dCTP, or their analogs are used as substrates for the extending.

7. The method of claim 1, wherein pyrophosphate or its analogs are used as substrates for the pyrophosphorolysis.

8. The method of claim 1, wherein 25 μM dATP, dTTP, dGTP, and dCTP, or their analogs are used as substrates for the extending.

9. The method of claim 1, wherein 90 μM pyrophosphate or its analogs are used as substrates for the pyrophosphorolysis.