Use of base-modified duplex-stabilizing deoxynucleoside 5'-triphosphates to enhance the hybridization properties of primers and probes in detection polymerase chain reaction.
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.
Kutyavin
Igor V
IV
perpetualgenomics@comcast.net
D000255:Adenosine Triphosphate / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry*; D006801:Humans; D008259:Macroglobulins / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009711:Nucleotides / Q000737:chemistry; D015345:Oligonucleotide Probes / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011743:Pyrimidines / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D017931:DNA Primers; D008259:Macroglobulins; D009711:Nucleotides; D015345:Oligonucleotide Probes; D011743:Pyrimidines; D000255:Adenosine Triphosphate; D004247:DNA