Optimized PCR labeling in mutational and microsatellite analysis.
To optimize the labeling and visualization of PCR products we tested different variables, including deoxynucleotide concentration and ratio, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols. Labeling was achieved using a fixed amount of labeled dATP, whose relative specific activity was varied by adding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 micromol/L, with a peak at 1.8 micromol/L. This concentration corresponded to an optimal ratio between the increase in specific activity and the decrease in DNA yield. Nucleotide imbalances >1:2 were not advantageous. Mutational analysis by single-strand conformational polymorphism (SSCP) was used to validate PCR-labeling protocols. The limiting nucleotide concentrations did not affect SSCP. Clear SSCP patterns were obtained using DNA templates of different sizes derived from several genes. SSCP patterns obtained using one-step or nested PCR-labeling protocols were equivalent and were visualized after overnight exposure, using [alpha35S]dATP as the label. Dilutions of labeled products ranging between 1:10 and 1:2.5 influenced SSCP patterns, and the lowest dilution tested produced better-defined and more-intense signals. Optimized SSCP conditions allowed the detection of novel and previously characterized nucleotide variants. Clear microsatellite typing was also obtained using optimized protocols and [alpha35S]dATP as the label.
Esposito
D L
DL
Department of Oncology and Neurosciences, Faculty of Medicine, University Gabriele D' Annunzio, Chieti, Italy.
Palmirotta
R
R
Verì
M C
MC
Mammarella
S
S
D'Amico
F
F
Curia
M C
MC
Aceto
G
G
Crognale
S
S
Creati
B
B
Mariani-Costantini
R
R
Battista
P
P
Cama
A
A
D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D006801:Humans; D018895:Microsatellite Repeats; D009711:Nucleotides / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D013462:Sulfur Radioisotopes
D004273:DNA, Neoplasm; D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D013462:Sulfur Radioisotopes