US008715987B2 (12) United States Patent (10) Patent No.: US 8,715,987 B2 Johnson et al. (45) Date of Patent: May 6, 2014 (54) SOLUBILIZED PHOSPHOLIPIDS FOR WO WOO 132887 A1 5, 2001 STABILIZING NUCLECACID WO WO2O080 13885 A2 1, 2008 POLYMERASES WO WO2O08077O17 A2 6, 2008 OTHER PUBLICATIONS (75) Inventors: Donald Johnson, Brookline, MA (US); Thomas C. Evans, Jr., Topsfield, MA Manual Translation CN 101570792A, Nov. 2009.* Barnes, WM. “The fidelity of Taq polymerase catalyzing PCR is (US) improved by an N-terminal deletion”. Gene, 112(1):29-35, 1992. Cann, et al., “A heterodimeric DNA polymerase: evidence that mem (73) Assignee: New England Biolabs, Inc., Ipswich, bers of Euryarchaeota possess a distinct DNA polymerase.”. Proc. MA (US) Natl. Acad. Sci. USA,95: 14250-5, 1998. Capitani.etal, “Effect of phosphatidylcholine vesicles on the activity (*) Notice: Subject to any disclaimer, the term of this of DNA polymerase-O.", Mol and Cell Biochem 27:137-138, 1979. patent is extended or adjusted under 35 Chien, et al., “Deoxyribonucleic acid polymerase from the extreme U.S.C. 154(b) by 0 days. thermophile Thermus aquaticus”,Journal of Bacteriology, 127:1550, 1976. Diaz, et al., "Accuracy of replication in the polymerase chain reaction. (21) Appl. No.: 13/450,549 Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase.”. Brazilian Journal of Medical (22) Filed: Apr. 19, 2012 and Biological Research, 31:1239, 1998. Eckert , et al. “The Fidelity of DNA Polymerases used in the (65) Prior Publication Data polymerase chain reactions'. Oxford University Press, New York, US 2012/O282669 A1 Nov. 8, 2012 NY. pp. 225-244, 1991. Flaman, et al. "A Rapid PCR Fidelity Assay”. Nucleic Acids Research, 22(15):3259-3260, 1994. Related U.S. Application Data Gill, et al. "Nucleic acid isothermal amplification technologies.”. (60) Provisional application No. 61/481,437, filed on May Nucleosides, Nucleotides and Nucleic Acids 27(3): 224-43, 2008. 2, 2011, provisional application No. 61/494.458, filed Hinnisdaels, et al., “Direct Cloning of PCR Products Amplified with Pwo DNA Polymerase”, Biotechniques 20:186-8, 1996. on Jun. 8, 2011. Kim, et al., “Isothermal DNA amplification in bioanalysis: Strategies and applications.”. Bioanalysis3(2): 227-39, 2011. (51) Int. Cl. Kornberg, et al., “DNA Replication.”. 2nd edition, W. H. Freeman, CI2N 9/96 (2006.01) New York, N.Y., pp. 101-115, 150-152, 157, 165, 169-173, 192-193, (52) U.S. Cl. 223-225, 1992. USPC .......................................................... 435/188 Lawyer, et al. “High-level expression, purification, and enzymatic (58) Field of Classification Search characterization of full-length. Thermus aquaticus DNA polymerase None and a truncated form deficient in 5' to 3' exonuclease activity”. PCR See application file for complete search history. Meth. Appl. 2:275-287, 1993. Lecomte, et al., “Selective inactivation of the 3' to 5' exonuclease (56) References Cited activity of Escherichia coli DNA polymerase I by heat”. Nucleic Acids Res., 11:7505, 1983. U.S. PATENT DOCUMENTS Lundberg, et al. “High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.”. Gene 4,889,818 A 12/1989 Gelfand et al. 108(1):1-6, 1991. 5,047,342 A 9/1991 Chatterjee MacDonald, et al. “Small-volume extrusion apparatus for prepara 5,079,352 A 1/1992 Gelfand et al. tion of large, unilamellar vesicles...Biochim. Biophys. Acta 5,270,179 A 12/1993 Chatterjee 1061:297-303, 1991. 5,374,553 A 12/1994 Gelfand et al. Myers, et al., “Reverse transcription and DNA amplification by a 5,436,149 A 7, 1995 Barnes Thermus thermophilus DNA polymerase.”. Biochemistry, 30:7661 5,512.462 A 4/1996 Cheng 7666, 1991. 5,614,365 A 3, 1997 Tabor et al. Nordstrom, et al., "Characterization of bacteriophage T7 DNA 5,792,612 A 8, 1998 Kinnunen et al. polymerase purified to homogeneity by antithioredoxin 6,127,155 A 10, 2000 Gelfand et al. 6,242.235 B1 6, 2001 Shultz et al. immunoadsorbent chromatography'. Journal of Biological Chemis 6,881,559 B2 4/2005 Sobek et al. try, 256:3112, 1981. 7,429,468 B2 9, 2008 Sobek et al. 7,541,170 B2 6/2009 Wang et al. (Continued) 7,666,645 B2 2/2010 Wang 7,846,703 B2 12/2010 Kobayashi et al. Primary Examiner — Suzanne M Noakes 2004/009 1873 A1 5/2004 Sorge et al. (74) Attorney, Agent, or Firm — New England Biolabs, Inc: 2005, 004.8530 A1 3/2005 Borns 2007/O141591 A1 6, 2007 Borns Harriet M. Strimpel 2008, OO64071 A1 3/2008 Hogrefeet al. 2010.0099150 A1 4/2010 Fang et al. (57) ABSTRACT 2010. O159528 A1 6, 2010 Liu et al. Compositions and methods are provided that relate to solu bilized phospholipids and their use in stabilizing nucleic acid FOREIGN PATENT DOCUMENTS polymerases. For example, a phospholipid with a tail contain CN 1O1570792 A * 11, 2009 ing at least 8 carbons can be solubilized in the presence of an WO WO9206188 A2 4f1992 amphipathic molecule. WO WO9206200 A1 4f1992 WO WO961064.0 A1 4f1996 10 Claims, 14 Drawing Sheets US 8,715,987 B2 Page 2 (56) References Cited licheniformis and Bacillus stearothermophilus.”. Biochimica et Biophysica Acta, 475:32, 1977. Takagi, et al. “Characterization of DNA polymerase from Pyrococ OTHER PUBLICATIONS cus sp. strain KOD1 and its application to PCR”. Applied and Envi Stenesh, et al., “DNA polymerase from mesophilic and thermophilic ronmental Microbiology, 63:4504, 1997. bacteria. III. Lack of fidelity in the replication of synthetic polydeoxyribonucleotides by DNA polymerase from Bacillus * cited by examiner U.S. Patent May 6, 2014 Sheet 1 of 14 US 8,715,987 B2 s w--- six-- U.S. Patent May 6, 2014 Sheet 2 of 14 US 8,715,987 B2 ***** U.S. Patent May 6, 2014 Sheet 5 of 14 US 8,715,987 B2 -------------------------------? s: 8. & U.S. Patent May 6, 2014 Sheet 6 of 14 US 8,715,987 B2 U.S. Patent May 6, 2014 Sheet 7 of 14 US 8,715,987 B2 }}}}} U.S. Patent May 6, 2014 Sheet 8 of 14 US 8,715,987 B2 xxx U.S. Patent May 6, 2014 Sheet 9 of 14 US 8,715,987 B2 S::::::::::: s ;: f ; : : : : :::::::: U.S. Patent May 6, 2014 Sheet 10 of 14 US 8,715,987 B2 * S$8.8 x: . BR, ANDR, COULD BE ANY OF TE FOLLOWING: * 88: www. 2n-1 & --- ww. WWW 3 U.S. Patent May 6, 2014 Sheet 11 of 14 US 8,715,987 B2 *::: 8:8; 8.8 x, S.883 : , , co.) BEA OF THE FOLLOENG. U.S. Patent May 6, 2014 Sheet 12 of 14 US 8,715,987 B2 F. *::: Si:8:3: x8, S8,888 i. S:::::::::::8 : F, NE f ::::::::: :: * : *:::::::::: * ...an 3.. 3 it, co, BE NY OF E FOLLOWING: Pisis . straitsa,3'ss :- .. 8 U.S. Patent May 6, 2014 Sheet 13 of 14 US 8,715,987 B2 . , 388x8 : R, ANR, COLD BE ANY OF THE FOLLONG: in 8. : 8 COLLO BE ANY OF THE FOLLENG: -ti -: - in t; U.S. Patent May 6, 2014 Sheet 14 of 14 US 8,715,987 B2 US 8,715,987 B2 1. 2 SOLUBLIZED PHOSPHOLIPIDS FOR the Solubilized long chain phospholipid is a phosphatidyl STABILIZING NUCLECACID choline POLYMERASES the composition includes a solubilizer Such as an amphip athic molecule Such as a lipid having a tail with a carbon CROSS REFERENCE backbone of no more than 8 carbons; the polymerase is a thermostable polymerase; This application claims priority from U.S. Provisional the activity of the polymerase is greater in the presence of Application No. 61/481,437 filed May 2, 2011, herein incor the solubilized phospholipid than in the absence of the porated by reference, and U.S. Provisional Application No. Solubilized phospholipid as can be measured in a repli 10 cation assay by incorporation of deoxynucleoside triph 61/494.458 filed Jun. 8, 2011. osphate (dNTP). The enhancement in polymerase activity in the presence of BACKGROUND the solubilized phospholipid is at least 3-fold, for example at least 5-fold. Polymerases may have suboptimal activity associated with In a further aspect, a method includes stabilizing a poly their stability under reaction conditions. See for example, 15 merase in a buffer by adding a solubilized long-chain phos Eckert and Kunkel, “The Fidelity of DNA Polymerase and the pholipid in which the solubilized phospholipid has a tail with Polymerases used in the PCR in Polymerase Chain Reaction at least 8 carbons. I: A Practical Approach, McPherson, et al., eds., Oxford Various embodiments may include: University Press, New York, N.Y., (1991), pp. 235-244. adding a solubilizer Such as a lipid having one or more Early work reported by Capitani, et al., (Molecular and hydrophobic tails where the tail or tails have no more than 8 Cellular Biochemistry, 27:137-138 (1979)) described the use carbons forming the backbone of the tail; or obtaining an of phosphatidylcholine in the form of vesicles to stimulate the enhancement of the activity of the polymerase. activity of DNA polymerase-C. U.S. Pat. No. 5,792,612 In a further aspect, a kit includes a polymerase in a buffer reported the use of phospholipids in a suspension or matrix in containing a solubilized phospholipid having at least one or the form of unilamellar liposomes which are combined with 25 more tails with a backbone of at least 8 carbons forming the polymerases and DNA or RNA to increase the efficiency of tail and a stabilizer. polymerase-dependent amplification reactions. The unila In a further aspect, a composition, is provided that includes mellar liposomes were prepared by extrusion through 0.1 uM a solubilized long-chain phospholipid in which the phospho polycarbonate membranes with a LiposoFast matrix homog lipid has at least one hydrophobic tail containing at least 8 enizer as reported by MacDonald, et al., (Biochimica et Bio 30 carbons physica Acta-Biomembranes, 1061:297-303 (1991) and were added prior to initiation of the reaction.