Quantitative analysis of polymerase chain reaction products by dot blot.
Quantitative analysis of polymerase chain reaction (PCR) products is usually accomplished by gel electrophoresis and Southern blotting. We have developed an alternative technique that allows PCR products to be directly quantitated from unfractionated samples. The PCR was used to amplify genomic (endogenous) DNA sequences (actin) and exogenous DNA (herpes simplex virus-1 (HSV-1) ribonucleotide reductase) isolated from the trigeminal ganglia of rabbits to demonstrate the dot blot method of PCR product analysis. Two primer pairs (actin and ribonucleotide reductase) were coamplified, resulting in two different PCR products. Duplicate aliquots of the PCR products were applied to separate nylon membranes and hybridized with 32P-labeled oligonucleotide probes. Each radioactive probe was specific for target (HSV-1 DNA) or control (actin DNA) products. Quantitation using a laser scanning PhosphorImager and ImageQuant software demonstrated that the dot blot method can be used to rapidly analyze a large number of PCR samples.
Hill
J M
JM
Department of Microbiology, Louisiana State University Medical Center School of Medicine, New Orleans 70112, USA.
Halford
W P
WP
Wen
R
R
Engel
L S
LS
Green
L C
LC
Gebhardt
B M
BM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011817:Rabbits; D012264:Ribonucleotide Reductases / Q000235:genetics*
D017931:DNA Primers; D004279:DNA, Viral; D012264:Ribonucleotide Reductases