Generation of high-sensitivity antisense cDNA probes by asymmetric PCR.
Monitoring changing levels of mRNA by hybridization analysis relies on the use of labeled probes. The development of antisense single-stranded cDNA probe methodologies (unidirectional polymerase chain reaction [PCR] amplification or asymmetric PCR amplification) allows the production of probes of high sensitivity and low background with excellent linearity of detection. Because the methods are PCR-based, templates do not need extensive modification: selection of oligonucleotide sequences determines the target amplified. Thus such methods are flexible, being successfully achieved on a variety of templates including recombinant plasmids or dsDNA from reverse-transcription PCR (RT-PCR). In addition these probes are relatively easily stripped from hybridization membranes, so allowing repeated probing in Northern analysis.
Bird
Ian M
IM
Department of Obstetrics and Gynecology, University of Wisconsin, Madison, USA.
D001345:Autoradiography; D015342:DNA Probes; D016373:DNA, Antisense / Q000737:chemistry*; D018076:DNA, Complementary; D004587:Electrophoresis, Agar Gel; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D016373:DNA, Antisense; D018076:DNA, Complementary