Claims for Patent: 8,530,194

Summary for Patent: 8,530,194

Title:	Oligonucleotides as temperature-sensitive inhibitors for DNA polymerases
Abstract:	Aspects of the invention relate to the use of novel oligonucleotides as temperature-sensitive inhibitors for thermostable DNA polymerases. Some inhibitors exhibit temperature-dependent and, in some cases, reversible inhibitory property by changing the conformation of at least a portion of the oligonucleotides from double-stranded to single stranded or in some cases vice versa in a temperature-dependent manner. Aspects also relate to the use of an the inhibitors in a hot-start PCR compositions, wherein the inhibitor may act to suppress the activity of the thermostable DNA polymerase below a desired activation temperature, Tact, and wherein the inhibitor is thermally inactivated above Tact, thus liberating the polymerase activity and initiating the DNA amplification process. Aspects further relate to a procedure for formulating the composition of a hot-start PCR reaction mixture. The hot-start PCR methods disclosed herein are generally faster, more flexible and lower in cost than existing methods.
Inventor(s):	Mao; Fei (Fremont, CA), Xin; Xing (Foster City, CA)
Assignee:	AlleLogic Biosciences Corporation (Hayward, CA)
Application Number:	12/088,213
Patent Claims:	1. A DNA polymerase inhibitor suitable for binding to a Pol domain and a 5'-exo domain of a DNA polymerase, said inhibitor comprising: at least one oligonucleotide, said oligonucleotide including: a first section of DNA, and a second section of DNA, wherein: said first section comprises a primer-like oligonucleotide of about 8 to about 50 nucleotides, and further comprises an oligonucleotide complementary to said primer-like oligonucleotide, and wherein said first section of DNA forms a double stranded DNA structure capable of binding to the Pol domain of a DNA polymerase, said second section comprises a 5'-exo oligonucleotide of about 3 to about 50 nucleotides and an oligonucleotide complementary to said 5'-exo oligonucleotide, and wherein said second section of DNA forms a double stranded structure capable of binding to the 5'-exo domain of said polymerase, and said second section includes at least two nucleotides joined by a backbone linkage wherein at least one of said backbone linkages is resistant to 5'-exonuclease activity. 2. The DNA polymerase inhibitor according to claim 1, wherein the cumulative nucleotide count of the contiguous portion of DNA spanning the oligonucleotide complementary to said primer-like oligonucleotide and the oligonucleotide complementary to said 5'-exo oligonucleotide is greater than the sum of the lengths of the primer-like oligonucleotide and the 5'-exo oligonucleotide. 3. The DNA polymerase inhibitor according to claim 1, further including a Flap region of DNA, wherein said Flap region is at least one nucleotide long and said Flap region is located downstream of said 5'-exo oligonucleotide. 4. The DNA polymerase inhibitor according to claim 1, further including an exo-loop, wherein said exo-loop comprises at least one unpaired nucleotide located between said 5'-exo oligonucleotide and said oligonucleotide complementary to said 5'exo oligonucleotide. 5. The inhibitor according to claim 2, wherein the 5'-exo oligonucleotide backbone linkages between at least some neighboring nucleotide pairs are selected from the group of said neighboring nucleotide pairs consisting of nucleotides -1, +1, +2 and +3. 6. The inhibitor according to claim 1, wherein the 5'-exonuclease resistant backbone linkages include at least one linkage selected from the group consisting of thiophosphate bonds, peptide bonds and alkylated phosphates. 7. The inhibitor according to claim 3, wherein said Flap region ranges from a single nucleotide to an oligonucleotide of about 50 bases, and the Flap and the 5'-exo oligonucleotide are joined by at least one exonuclease resistant linkage. 8. The inhibitor according to claim 2, wherein the 3'-terminus nucleotide of said primer-like oligo is selected from the group of nucleotides consisting of natural 2'-deoxynucleotides and 2',3'-dideoxynucleotides. 9. A method of formulating a thermally activatable PolA DNA polymerase complex, comprising the steps of: adding a DNA polymerase inhibitor according to claim 1 to a concentrated PolA DNA polymerase solution; and incubating the resulting solution before initiating a reaction catalyzed by DNA polymerase. 10. A kit for carrying out PCR, comprising: an inhibitor according to claim 1; a thermostable DNA polymerase; and at least one additional reagent selected from the group consisting of sterile water, 2'-deoxynucleoside triphosphates, MgCl.sub.2, and buffer.

Details for Patent 8,530,194

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	ZOSTAVAX	zoster vaccine live	For Injection	125123	05/25/2006		2025-09-26