Hot start PCR with heat-activatable primers: a novel approach for improved PCR performance.
The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3'-terminal and 3'-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)-PCR and in real-time PCR with SYBR Green I dye and Taqman(R) probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.
Lebedev
Alexandre V
AV
Department of Research and Development, TriLink BioTechnologies, Inc., 9955 Mesa Rim Road, San Diego, CA 92121, USA.
Paul
Natasha
N
Yee
Joyclyn
J
Timoshchuk
Victor A
VA
Shum
Jonathan
J
Miyagi
Kei
K
Kellum
Jack
J
Hogrefe
Richard I
RI
Zon
Gerald
G
D052160:Benzothiazoles; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry*; D004279:DNA, Viral / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003959:Diamines; D019281:Dimerization; D005456:Fluorescent Dyes; D015497:HIV-1 / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D009930:Organic Chemicals; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D009943:Organophosphorus Compounds; D011804:Quinolines; C434331:phosphoramidite; C098022:SYBR Green I; D004259:DNA-Directed DNA Polymerase
Nucleic Acids Res. 1976 Dec;3(12):3397-408
1005124
J Am Chem Soc. 1977 May 11;99(10):3526-8
853194
Nucleic Acids Res. 1991 Apr 11;19(7):1527-32
2027761
Nucleic Acids Res. 1993 Jun 25;21(12):2959-60
8332517
Nucleic Acids Res. 2005 Aug 08;33(14):e126
16087733
Nucleic Acids Res. 1986 Nov 25;14(22):9081-93
3786144
Chem Commun (Camb). 2008 Jan 28;(4):462-4
18188468
Biochemistry. 1982 May 11;21(10):2507-12
7046789
Org Lett. 2001 May 3;3(9):1287-90
11348216
J Org Chem. 2003 Dec 26;68(26):10123-9
14682709
Science. 1988 Jan 29;239(4839):487-91
2448875
Biotechniques. 1997 Jan;22(1):130-1, 134-8
8994660
Biochem Mol Biol Int. 1998 Feb;44(2):407-15
9530523
J Mol Biol. 1996 Nov 29;264(2):268-78
8951376
Nucleic Acids Res. 1989 Jun 26;17(12):4769-82
2748337
J Am Chem Soc. 1975 Dec 10;97(25):7332-7
172538
Appl Environ Microbiol. 2005 Dec;71(12):8966-9
16332901
Nucleic Acids Res. 1992 Apr 11;20(7):1717-23
1579465
Trends Biotechnol. 2006 Sep;24(9):395-402
16843558
Nucleic Acids Res. 2005 Nov 27;33(20):e181
16314311
Nucleic Acids Res. 1991 Jul 11;19(13):3749
1852616
Nucleic Acids Res. 2000 Nov 1;28(21):E94
11058144
Science. 2003 Sep 26;301(5641):1852-3
14512607
Bioorg Khim. 1985 Mar;11(3):358-69
3890861
Clin Chem. 2001 Dec;47(12):2089-97
11719471
Clin Microbiol Rev. 2000 Oct;13(4):559-70
11023957
J Biomol Tech. 2005 Sep;16(3):266-71
16461951
Anal Biochem. 2002 Aug 1;307(1):63-9
12137780
Bioorg Khim. 1987 Jan;13(1):45-57
3551963
Biotechniques. 1994 Jun;16(6):1134-7
8074881
Nucleic Acids Res. 1986 Sep 25;14(18):7405-20
3020514
J Org Chem. 2004 Apr 2;69(7):2509-15
15049652
Anal Biochem. 1997 Feb 15;245(2):154-60
9056205
J Am Chem Soc. 2001 Feb 7;123(5):783-93
11456611
PCR Methods Appl. 1993 Oct;3(2):133-4
8268790
Biotechniques. 2000 Nov;29(5):1018-20, 1022-4
11084864
Leg Med (Tokyo). 2003 Mar;5 Suppl 1:S191-3
12935586
Nucleic Acids Res. 1977 Aug;4(8):2757-65
909791
J Med Virol. 1994 May;43(1):72-6
8083652
Methods Mol Biol. 1997;67:3-15
9031125
Nucleic Acids Res. 2005 Jun 21;33(11):3550-60
15972797
Nature. 2004 Jul 29;430(6999):551-4
15282603
J Immunol Methods. 2004 Mar;286(1-2):203-17
15087233
Nucleic Acids Res. 2003 Nov 1;31(21):6139-47
14576300
Nucleic Acids Res. 1980 Mar 11;8(5):1097-105
7443541
Anal Biochem. 1999 Sep 10;273(2):307-10
10469503
Front Zool. 2008 Jul 20;5:12
18638418
Nucleic Acids Res. 1990 Sep 11;18(17):5197-205
2402444
J Org Chem. 2002 Sep 6;67(18):6430-8
12201764
Biochemistry. 1986 Sep 9;25(18):5083-91
3768334
Biotechniques. 1998 Oct;25(4):716-22
9793657
Pharmacogenomics J. 2002;2(2):103-10
12049172
Pharmacogenomics J. 2003;3(2):77-96
12746733
Genome Res. 1995 Oct;5(3):309-11
8593616
Nucleic Acids Res. 1986 Sep 25;14(18):7421-36
3763408
Biochemistry. 1977 Mar 22;16(6):1239-50
14676
J Biochem. 1999 Oct;126(4):762-8
10502686
Neurosci Lett. 2003 Mar 13;339(1):62-6
12618301
Nucleic Acids Res. 1981 Sep 25;9(18):4611-26
7301585
Mol Cell Probes. 2005 Jun;19(3):203-5
15797821
Genome Res. 2006 Feb;16(2):282-9
16369045