Quantification of polymerase chain reaction products by affinity-based hybrid collection.
We have used oligonucleotides modified with biotin in the 5'-end as primers in the polymerase chain reaction (PCR)-amplification. This results in the synthesis of 5'-biotinylated DNA molecules, which are detected by hybridization to a labelled probe in solution. The formed hybrids are collected on an avidin-matrix by mediation of the biotin residue of the target molecules. The affinity-based hybrid collection method is quantitative and makes it possible to measure the amount of DNA produced in the PCR-amplification. At low concentrations of template the efficiency of the process is close to 100%, making it possible to detect the presence of a few molecules of target DNA in 25 cycles. With high template concentrations the efficiency of the process is low.
Syvänen
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AC
Orion Genetic Engineering Laboratory, Orion Corporation Ltd, Helsinki, Finland.
Bengtström
M
M
Tenhunen
J
J
Söderlund
H
H
D000345:Affinity Labels; D001483:Base Sequence; D001710:Biotin; D003587:Cytomegalovirus / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D019914:Taq Polymerase
D000345:Affinity Labels; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D001710:Biotin; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase