# US 10,767,217 B2 · Compositions, methods and kits for real time polymerase chain reaction (PCR) > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,767,217 B2 - Document ID: US10767217B2 - Status: Granted - Kind: B2 - Application number: 15/927,572 - Filed: 2018-03-21 - Published: 2018-10-04 - Granted: 2020-09-08 - Earliest claimed: 2012-06-14 - Assignees: Life Technologies Corporation - Applicants: Life Technologies Corporation - Inventors: Carole Bornarth; Michael Lau; Junko Stevens - Web page: https://patentgrep.com/patent/US10767217B2 ## CPC - C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction - C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies - C12N 9/00: Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00) - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present disclosure is directed to compositions, methods and kits for amplifying target nucleic acids while reducing non-specific amplification and undesired amplification products using a dual hot start reaction mixture that comprise at least two different hot start mechanisms. ## Claims ### Claim 1 A method for amplifying a target nucleic acid comprising: a) contacting the target nucleic acid with a first nucleic acid polymerase, a dual hot start reaction mixture, at least one primer and at least one dNTP at a first temperature, thereby forming a reaction composition, wherein the dual hot start reaction mixture comprises at least two hot start mechanisms that, together, both inhibit activity of the first nucleic acid polymerase; b) heating the reaction composition to a second temperature; and c) amplifying the target nucleic acid in the reaction composition. ### Claim 2 (depends on 1) The method of claim 1, wherein the first nucleic acid polymerase is thermostable. ### Claim 3 (depends on 1) The method of claim 1, wherein the first nucleic acid polymerase is a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. ### Claim 4 (depends on 1) The method of claim 1, wherein the first nucleic acid polymerase is selected from the group consisting of Taq DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, and Pfu DNA polymerase. ### Claim 5 (depends on 1) The method of claim 1, wherein the at least two hot start mechanisms are different. ### Claim 6 (depends on 5) The method of claim 5, wherein the at least two different hot start mechanisms are selected from the group consisting of antibodies or combinations of antibodies that block DNA polymerase activity at lower temperatures, oligonucleotides that block DNA polymerase activity at lower temperatures, reversible chemical modifications of the DNA polymerase that dissociate at elevated temperatures, amino acid modifications of the DNA polymerase that provide reduced activity at lower temperatures, fusion proteins that include hyperstable DNA binding domains and topoisomerase, temperature dependent ligands that inhibit the DNA polymerase, single stranded binding proteins that sequester primers at lower temperatures, modified primers, or modified dNTPs. ### Claim 7 (depends on 1) The method of claim 1, wherein the reaction composition further comprises a nucleic acid binding dye. ### Claim 8 (depends on 1) The method of claim 1, wherein the reaction composition further comprises a reporter probe. ### Claim 9 (depends on 4) The method of claim 4, wherein the reaction composition further comprises a reverse transcriptase. ### Claim 10 (depends on 1) The method of claim 1, wherein the at least two different hot start mechanisms inhibit activity of the first nucleic acid polymerase by binding to the first nucleic acid polymerase. ### Claim 11 (depends on 9) The method of claim 9, wherein binding to the first nucleic acid polymerase is reversible. ### Claim 12 (depends on 1) The method of claim 1, wherein binding to the first nucleic acid polymerase is temperature-dependent. ### Claim 13 (depends on 11) The method of claim 11, wherein the at least two different hot start mechanisms bind to the first nucleic acid polymerase at a temperature less than about 40° C. and do not bind to the first nucleic acid polymerase at a temperature greater than about 40° C. ### Claim 14 (depends on 1) The method of claim 1, wherein the method further comprises a step for detecting the amplified target nucleic acid.