Open AccessUse of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction
Author(s) -
Igor V. Kutyavin
Publication year - 2008
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/bi8017784
Subject(s) - taq polymerase , amplicon , primer dimer , chemistry , oligonucleotide , taqman , polymerase chain reaction optimization , klenow fragment , dna , primer (cosmetics) , polymerase , polymerase chain reaction , microbiology and biotechnology , nucleotide , molecular beacon , base pair , dna polymerase , combinatorial chemistry , biochemistry , biology , thermus aquaticus , multiplex polymerase chain reaction , gene , organic chemistry , exonuclease
Several base-modified duplex-stabilizing deoxyribonucleoside 5'-triphosphates (dNTPs) have been evaluated as agents for enhancing the hybridization properties of primers and probes in real-time polymerase chain reaction (PCR). It was shown that pyrimidines substituted at the 5-position with bromine or iodine atoms and methyl or propynyl groups are incorporated into PCR amplicons by Taq DNA polymerase as efficiently as natural dNTPs. The dNTP of 2-aminoadenosine was incorporated somewhat less efficiently than dATP but still supported PCR. Incorporation of these modified nucleotides into the amplified DNA represents a simple and inexpensive way to stabilize duplexes of primers and probes and is particularly effective in improving the amplification and detection of A/T-rich sequences. This technology permits the use of higher PCR annealing temperatures or alternatively a reduction in the length of the oligonucleotide components. Examples of successful application in TaqMan and Scorpion real-time detection assays are provided. Limits of the approach are identified and discussed. For example, application of the 5-bromo and 5-iodo derivatives may be limited to relatively G/C-rich DNA targets and, in particular, to those lacking long runs of adenylate and/or thymidylate. Simultaneous use of base-modified analogues of dATP and dTTP should be avoided in PCR due to "overstabilization" of the amplicon.