Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

• Published in: Biochemistry (Easton) 2008-12, Vol.47 (51), p.13666-13673
• Main Author: Kutyavin, Igor V
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - chemistry; Base Sequence; DNA - chemistry; DNA Primers - chemistry; Humans; Macroglobulins - metabolism; Models, Chemical; Molecular Sequence Data; Nucleic Acid Hybridization; Nucleotides - chemistry; Oligonucleotide Probes - chemistry; Polymerase Chain Reaction - methods; Pyrimidines - chemistry; Reverse Transcriptase Polymerase Chain Reaction; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi8017784

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.