Aptamers for the reversible inhibition of DNA polymerases
Described herein are methods and compositions for improved polymerase chain reaction (PCR). In one aspect the methods and compositions include improved aptamers to reversibly inhibit polymerase and exonuclease activity of the polymerase enzyme.
1 . A composition comprising:
a thermostable DNA polymerase and an aptamer;
wherein the aptamer is selected from a nucleotide sequence comprising at least 90% identity to SEQ ID NO:8 or SEQ ID NO:9.
2 . The composition of claim 1 , wherein the aptamer is selected from a nucleotide sequence comprising SEQ ID NO:8 or SEQ ID NO:9.
3 . The composition of claim 1 , wherein the thermostable DNA polymerase is a Phusion DNA polymerase.
4 . The composition of claim 2 wherein the Phusion DNA polymerase is Pfu-sso7d.
5 . An aptamer selected from a nucleotide sequence comprising at least 90% identity to SEQ ID NO:8 or SEQ ID NO:9.
6 . The aptamer of claim 5 , wherein the aptamer is selected from a nucleotide sequence comprising SEQ ID NO:8 or SEQ ID NO:9.
7 . A method for amplifying a target DNA sequence, said method comprising the steps of:
(a) providing a reaction mixture comprising:
(i) a thermostable DNA polymerase,
(ii) a thermostable DNA polymerase aptamer selected from a nucleotide sequence comprising at least 90% identity to SEQ ID NO:8 or SEQ ID NO: 9, wherein the thermostable DNA polymerase aptamer binds to the thermostable DNA polymerase to form a blocked thermostable DNA polymerase;
(iii) at least one oligonucleotide primer,
(iv) one or more sample nucleic acids that may or may not comprise a target sequence complementary to the at least one oligonucleotide primer;
(b) hybridizing the at least one oligonucleotide primer to the sample nucleic acids that comprise a target DNA sequence complementary to the at least one oligonucleotide primer;
(c) elevating the temperature to release the thermostable DNA polymerase aptamer from the blocked thermostable DNA polymerase; and
(d) initiating DNA polymerase activity and extending the primer with the DNA polymerase.
8 . The method of claim 7 , wherein the thermostable DNA polymerase aptamer is selected from a nucleotide sequence comprising SEQ ID NO:8 or SEQ ID NO:9.
9 . The method of claim 7 , wherein the thermostable DNA polymerase is a Phusion DNA polymerase.
10 . The method of claim 9 , wherein the Phusion DNA polymerase is Pfu-sso7d.
11 . The method of claim 7 , wherein the elevated temperature to release the thermostable DNA polymerase aptamer from the blocked thermostable DNA polymerase is greater than 45° C.
12 . The method of claim 7 , wherein the elevated temperature to release the thermostable DNA polymerase aptamer from the blocked thermostable DNA polymerase is from 50-55° C.
13 . The method of claim 7 , wherein the initiating DNA polymerase activity and extending the primer with the DNA polymerase is performed at a temperature of greater than 65° C.
14 . The method of claim 7 , wherein the reaction mixture further comprises, a buffer mixture and dNTPs.
15 . The method of claim 14 , wherein:
the buffer mixture comprises: 10 mM Tris-HCl (pH 8.4 at 25° C.), 1.5 mM MgCl 2 , 110 mM KCl, 0.08% (w/v) Brij-58, 0.2% (w/v) PEG-8000, and 0.1% (v/v) propylene glycol); and
the dNTPs comprise 0.8 mM dNTPs.