Target amplification of nucleic acid with mutant RNA polymerase

• US 20030124515A1
• Filed: 08/28/2002
• Published: 07/03/2003
• Est. Priority Date: 06/02/1999
• Status: Active Grant

• Current Assignee: PBI Technology, Inc.
• Original Assignee: Saigene Corporation
• Inventors: Haydock, Paul V.
• Granted Patent: US 7,078,170 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12N 9/1247 DNA-directed RNA polymerase (2.7.7.6)
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2537/155 Cyclic reactions

Abstract

This invention provides for a novel amplification procedure for nucleic acid. The method uses a mutant RNA polymerase designed to transcribe deoxynucleotides. Using primers that bind to target nucleic acid the primers form polymerase recognition sites that permit transcription of the target in an isothermal and logrithmic manner, with the added advantage of forming multiple single stranded copies, which are readily detected by hybridization assays.

Claims

1. An isothermal amplification method of copying a nucleic acid sequence comprising the steps of:

a. providing an aqueous solution comprising

i. a target nucleic acid for amplification said target comprising a double stranded DNA having a first 5′

end which bears a phage-encoded RNA polymerase recognition site and a second 5′

end which bears a phage-encoded RNA polymerase recognition sequence,

ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the 5′

end of the target sequence and the second primer is complementary to the antisense sequence of the 3′

end of the target sequence,

iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP and,

iv. an excess of dNTP;

b. repetitively allowing the polymerase to bind to its recognition site and to transcribe a first, short (−

) copy strand of the target nucleic acid yield a multiple copies of a primeness single (+) strand amplification product;

c. creating a first amplification duplex by allowing the second primer to bind to the primerless single (+) strand amplification products of step b and permitting the polymerase to (i) extend the primer to yield a polymerase primed (−

) amplification product and (ii) extend the primeness (+) strand to include a polymerase primer complement sequence creating a polymerase recognition site;

d. repetitively allowing the polymerase to bind to its recognition site on the first amplification duplex and to transcribe multiple copies of a primeness single stranded (−

) amplification product;

e. creating a second amplification duplex by allowing primer 1 to bind to the primeness single stranded (−

) amplification products of step h and permitting the polymerase (i) to extend primer 1 to yield a polymerase primed (+) amplification product and (ii) to extend the primeness (−

) strand to include a polymerase primer complement sequence creating a polymerase recognition site; and

,

f. repetitively allowing the polymerase to bind to its recognition site on the second amplification duplex and to transcribe multiple copies of a primeness single stranded (+) amplification product.

2. A method of claim 1 wherein the polymerase is a T7 RNA polymerase mutant.

3. A method of claim 2 wherein the polymerase is Y639F and S641A.

4. A method of claim 1 wherein the target nucleic acid is derived from a template nucleic acid having a subsequence as the target nucleic acid wherein the method further comprises the steps of placing the template nucleic acid in an aqueous solution comprising the first and second primers, the mutant phage polymerase and an excess of dNTP and permitting the polymerase and reactants to yield the target nucleic acid comprising a double stranded DNA having a first 5′

end which bears a phage-encoded RNA polymerase recognition site and a second 5′

end which bears a phage-encoded RNA polymerase recognition sequence.

5. A method of claim 4 wherein the target nucleic acid is single stranded DNA.

6. A method of claim 4 wherein the target nucleic acid is RNA and which method further comprises reverse transcription of the RNA by a reverse transcriptase and separation of the heteroduplex to a single stranded DNA intermediate.

7. A logarithmic, isothermal method of copying a nucleic acid sequence from a long strand of nucleic acid comprising the steps of:

a. providing an aqueous solution comprising;

i. template nucleic acid having a target sequence for amplification,

ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the 5′

end of the target sequence and the second primer is complementary to the antisense sequence of the 3′

end of the target sequence,

iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP and,

iv. an excess of dNTP;

b. allowing the first primers to bind to the template nucleic acid at the 3′

end of the target sequence;

c. creating target;

long strand duplex by allowing the polymerase to extend the 3′

end of the primer to create a first, long (+) strand complementary to the target subsequence;

d. displacing the template and the first, long (+) strand;

e. creating an intermediate duplex by allowing the second primer to bind to the long (+) strand at the 3′

end of the target sequence and using polymerase to extend the second primer in a 3′

direction to yield a first, short (−

) copy strand bound to the long (+) strand;

f. repetitively allowing the polymerase to bind to its recognition site and to transcribe the first, short (−

) copy strand of the intermediate duplex to yield a multiple copies of a primeness single (+) strand amplification product;

g. creating a first amplification duplex by allowing primer 2 to bind to the primerless single (+) strand amplification products of step f and permitting the polymerase to (i) extend the primer to yield a polymerase primed (−

) amplification product and (ii) extend the primeness (+) strand to include a polymerase primer complement sequence creating a polymerase recognition site;

h. repetitively allowing the polymerase to bind to its recognition site on the first amplification duplex and to transcribe multiple copies of a primeness single stranded (−

) amplification product;

i. creating a second amplification duplex by allowing primer 1 to bind to the primeness single stranded (−

) amplification products of step h and permitting the polymerase (i) to extend primer 1 to yield a polymerase primed (+) amplification product and (ii) to extend the primeness (−

) strand to include a polymerase primer complement sequence creating a polymerase recognition site; and

,

j. repetitively allowing the polymerase to bind to its recognition site on the second amplification duplex and to transcribe multiple copies of a primeness single stranded (+) amplification product.

8. A method of claim 7 wherein the polymerase is T7 RNA polymerase.

9. A method of claim 7 wherein the target nucleic acid is double stranded DNA and the method further comprises the step of separating the DNA prior to step b.

10. A method of claim 7 wherein the target nucleic acid is single stranded RNA and the method further comprises the steps of reverse transcribing the RNA into DNA using a reverse transcriptase and separating the DNA:

RNA duplex prior to step b.

11. A method of claim 7 wherein the reaction mixture further comprises a bumper oligonucleotide which is:

(i) able to hybridize to a DNA sequence about or adjacent to the 5′

end of the first long strand and (ii) able to serve as polymerase primer which displaces the first long strand when extended towards the 3′

end of the target nucleic acid.

12. A double stranded DNA having a first and second end comprising a phage RNA polymerase recognition sequences on both the first and second ends wherein at least one end has a complementary sequence that forms a phage polymerase recognition site.

13. A DNA of claim 12 wherein both ends have phage polymerase recognition sites.

14. A DNA of claim 12 wherein both ends have identical phage polymerase recognition sites.

15. A DNA of claim 12 wherein the DNA comprises a signature sequence for a specific genus or species of organism.

16. An aqueous reaction mixture comprising

i. a target nucleic acid for amplification;

ii. a first and second amplification primer each having a phage-encoded RNA polymerase recognition sequence wherein the first primer is complementary to the 5′

end of the target sequence and the second primer is complementary to the antisense sequence of the 3′

end of the target sequence;

iii. phage-encoded RNA polymerase mutated to recognize and polymerize dNTP; and

,

iv. an excess of dNTP.

17. A reaction mixture of claim 16 wherein the target nucleic acid comprises a double stranded DNA having a first 5′

end which bears a phage-encoded RNA polymerase recognition site and a second 5′

end which bears a phage-encoded RNA polymerase recognition sequence.

18. A kit for amplifying a target nucleic acid comprising a container containing a first primer having a sequence complementary to a 5′

end of the target nucleic acid and a phage polymerase recognition sequence and a container containing a second primer having a sequence which is the anti-complement to the 3′

end of the target nucleic acid and a phage polymerase recognition sequence.

19. A kit of claim 18 further comprising a mutant phage polymerase competent to incorporate dNTP into a template nucleic acid.

20. A kit of claim 18 which further comprises a bumper oligonucleotide which is able to hybridize to a template DNA sequence where that sequence is about or immediately adjacent to the 3′

base of the sequence to which one of the amplification primer binds.