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(12) Patent Application Publication (10) Pub. No.: US 2017/0002401 A1 TRINH Et Al US 2017.0002401A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0002401 A1 TRINH et al. (43) Pub. Date: Jan.5, 2017 (54) COMPOSITIONS, KITS AND METHODS FOR (60) Provisional application No. 61/357,031, filed on Jun. SYNTHESIS AND/OR DETECTION OF 21, 2010. NUCLEC ACDS (71) Applicant: LIFE TECHNOLOGIES Publication Classification CORPORATION, Carlsbad, CA (US) (51) Int. Cl. (72) Inventors: Christopher TRINH, San Jose, CA CI2O I/68 (2006.01) (US); Tom XU, Castro Valley, CA (52) U.S. Cl. (US); Yating SHI, San Leandro, CA CPC ....... CI2O I/686 (2013.01); C12Y 207/07007 (US); Ferrier LE, San Jose, CA (US); Claire MARJORIBANKS, Campbell, (2013.01); C12O 1/6848 (2013.01) CA (US) (21) Appl. No.: 15/198,689 (57) ABSTRACT (22) Filed: Jun. 30, 2016 A composition comprising a thermostable DNA polymerase; Related U.S. Application Data and a PCR inhibitor blocking agent, wherein the PCR (62) Division of application No. 13/165,571, filed on Jun. inhibitor blocking agent is present in an amount effective to 21, 2011, now Pat. No. 9,410,194. enhance tolerance of an assembled PCR to a PCR inhibitor. Patent Application Publication Jan. 5, 2017. Sheet 1 of 13 US 2017/0002401 A1 37.5 Assay Pane O ACADV. A. APOE 35 v ASB7 C2 KXED3 32.5 WSF3 27.5 25 22.5 Taqian(8) Fast Advanced Taqiang Universal PCR ?iaster fix Master ?ix FG. 1 Patent Application Publication Jan. 5, 2017. Sheet 2 of 13 US 2017/0002401 A1 OOO ...i. --- 2 3 4 5 6 F 8 20 222 23 24 25 26 27 28 29 303 Cycle Number FG. 2 Patent Application Publication Jan. 5, 2017. Sheet 3 of 13 US 2017/0002401 A1 ------------------ XX XXX sia ea s s minimir fini-Hirii if ....A............... A ---------(1--4------1---|--|-- w 30 35 AO 5 20 25 Cycle FG, 3A s A. II.I.I.I.I.I.I...........i - / ....I.fif I.A. 5 20 25 Cycie 30 35 40 FG. 3B Patent Application Publication Jan. 5, 2017. Sheet 4 of 13 US 2017/0002401 A1 (...) re g Patent Application Publication Jan. 5, 2017. Sheet 5 of 13 US 2017/0002401 A1 H-E-HA!(sh F.G. 5A OOOO1 0. COO (i) OOC O. O Final Concentration (nM) FG, 5B Patent Application Publication Jan. 5, 2017. Sheet 6 of 13 US 2017/0002401 A1 OC () Cycle Number F.G. 6 Patent Application Publication Jan. 5, 2017. Sheet 7 of 13 US 2017/0002401 A1 8Kessw/ ZKesswy 9KessvyGKesswy?ygcÁessvy WnOL£ zKesswy Patent Application Publication Jan. 5, 2017. Sheet 8 of 13 US 2017/0002401 A1 O C s s Os ; C O e a if ; o N E d x e 2 ; f o w f N. o C s an (D a xxxxxxx wa s i. O ce o % S C 2 C E o E C N e C ce t C Patent Application Publication Jan. 5, 2017. Sheet 9 of 13 US 2017/0002401 A1 ZKesswy uue-,Xeidnci-uo??q?uulu??euueH 9KesswyGKessw/7KessvyC zKesswy |Kessw/ Patent Application Publication Jan.5, 2017. Sheet 10 of 13 US 2017/0002401 A1 N O e Exxxe ".... C N. Ce 8 D C O O e ; t O D C .......... 5s 6 ..... e O t s > C s Q CD O r i wa C > C O N (r T e E XXXXife XX. D II. --- D E. O (N var ce C Patent Application Publication Jan.5, 2017. Sheet 11 of 13 US 2017/0002401 A1 inhibitor Tolerance - Humic Acid ASSay 1 ASSay 2 Humic Acid Added w/o Additives No Humic Acid/ No Additives (Control) Humic Acid + Fish Gelatin/BSA FG. 8 Patent Application Publication Jan.5, 2017. Sheet 12 of 13 US 2017/0002401 A1 inhibitor Tolerance - Heparin Heparin W/o Additives No Heparin/No Additives (Control) Heparin Added w/ Fish Gelatin/BSA F.G. 9 Patent Application Publication Jan. 5, 2017. Sheet 13 of 13 US 2017/0002401 A1 (g9IN-INV-J)ELOV-XæIdndXE9ºsau?uunupeo>} (£9||W-INV-J)INZE-x0|dndXE9°SAJeuunupeo>| US 2017/0002401 A1 Jan. 5, 2017 COMPOSITIONS, KITS AND METHODS FOR and/or other secretory fluids. These sources may also contain SYNTHESIS AND/OR DETECTION OF compounds that inhibit PCR amplification. NUCLEC ACDS 0008 Accordingly, there is a need to identify agents that block or reduce the inhibition of PCR amplification by CROSS REFERENCE TO RELATED components found in Sources for nucleic acid samples. APPLICATIONS 0001. This application is a Divisional of U.S. patent SUMMARY application Ser. No. 13/165,571 filed Jun. 21, 2011, which 0009 Disclosed herein are compositions, kits, and meth claims benefit of priority under 35 U.S.C. 119(e) to US ods for the synthesis and/or detection of nucleic acids by Provisional Application No. 61/357,031 filed Jun. 21, 2010, polymerase chain reaction, comprising a DNA polymerase both which are incorporated hereby in their entirety. and a PCR inhibitor blocking agent. The PCR inhibitor blocking agent relieves inhibition of PCR caused by a BACKGROUND variety of compounds often found in Samples containing nucleic acids that are analyzed by PCR. The compositions, 0002 This disclosure relates to compositions, kits, and kits, and methods disclosed herein ensure sensitive and methods for the synthesis and/or detection of nucleic acids. reliable PCR results over a wide range of target nucleic acid 0003 For many medical, diagnostic, and forensic appli concentrations. The composition also allows for rapid cations, amplification of a particular DNA sequence is results in fast PCR thermal cyclers. The composition also essential to allow its detection in, or isolation from, a sample provides assembled PCR reactions that may be stable for up in which it is present in very low amounts. More recently, in to 72 hours or more at room temperature. vitro amplification of specific genes has provided powerful 0010 Disclosed herein are compositions comprising a and less costly means to facilitate the production of thera thermostable DNA polymerase; and a PCR inhibitor block peutic proteins by molecular biological techniques, and may ing agent, wherein the PCR inhibitor blocking agent is have applications in genetic therapy as well. present in an amount effective to enhance tolerance of an 0004. The polymerase chain reaction (PCR) technique is assembled PCR to a PCR inhibitor. disclosed in U.S. Pat. Nos. 4,683.202; 4,683, 195; 4,800,159; 0011. Also disclosed herein is a kit comprising a com and 4,965,188. The PCR method is also described in Saiki position comprising a thermostable DNA polymerase and a et al., 1985, Science 230:1350. In its simplest form, PCR is PCR inhibitor blocking agent, wherein the PCR inhibitor an in vitro method for the enzymatic synthesis of specific blocking agent is present in an amount effective to enhance DNA sequences using two oligonucleotide primers that tolerance of an assembled PCR to a PCR inhibitor. hybridize to opposite Strands and flank the region of interest 0012. Further disclosed herein is a kit comprising a in the target DNA. A repetitive series of reaction steps composition comprising a thermostable DNA polymerase involving template denaturation, primer annealing, and the and a PCR inhibitor blocking agent, wherein the PCR extension of the annealed primers by DNA polymerase inhibitor blocking agent is present in an amount effective to results in the exponential accumulation of a specific frag enhance tolerance of an assembled PCR to a PCR inhibitor, ment whose termini are defined by the 5' ends of the primers. a primer, and a labeled probe. PCR is capable of producing a selective enrichment of a 0013 Further disclosed herein is a method for nucleic specific DNA sequence by a factor of 10. acid synthesis comprising mixing a composition comprising 0005 Commercially available PCR master mixes a thermostable DNA polymerase and a PCR inhibitor block improve the efficiency and reduce the errors associated with ing agent, wherein the PCR inhibitor blocking agent is the assembly of large number of PCR reactions required for present in an amount effective to enhance tolerance of an high-throughput analysis. These master mixes contain a assembled PCR to a PCR inhibitor, with a nucleic acid combination of reagents that will be common to all PCR sample and a primer, and synthesizing a nucleic acid using reactions. For example, the master mix may contain a buffer, the nucleic acid sample as a template. In some embodiments, a salt Such as MgCl, deoxynucleoside triphosphates synthesis can occur up to 72 hours following the mixing of (dNTPs), and a thermostable DNA polymerase. Each well the composition with a nucleic acid sample and a primer or would contain the common master mix and a specific target primers. nucleic acid and primer pair. Typically, master mixes are 0014 Further disclosed herein is a method for the assem manufactured and distributed as concentrated Solutions or bly of a polymerase chain reaction (PCR) comprising adding lyophilized powders which are subsequently diluted or dis a composition comprising a thermostable DNA polymerase solved when final reactions are assembled. and a PCR inhibitor blocking agent, wherein the PCR 0006 For accurate analysis, PCR master mixes should inhibitor blocking agent is present in an amount effective to provide reliable, robust, and reproducible PCR results. Fur enhance tolerance of an assembled PCR to a PCR inhibitor, ther, PCR master mixes should allow for detection of low to a reaction vessel; and adding a nucleic acid sample and a copy number target nucleic acids. The PCR master mix primer to the reaction vessel. should also allow for fast PCR reaction cycles to allow rapid 0015.
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