Optimized PCR labeling in mutational and microsatellite analysis

Diana Liberata Esposito, Raffaele Palmirotta, Maria Concetta Verì, Sandra Mammarella, Franca D'Amico, Maria Cristina Curia, Gitana Aceto, Stefania Crognale, Beatrice Creati, Renato Mariani-Costantini, Pasquale Battista, Alessandro Cama

Research output: Contribution to journalArticlepeer-review

Abstract

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 μmol/L, with a peak at 1.8 μmol/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 [α35S]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 [α35S]dATP as the label.

Original languageEnglish
Pages (from-to)1381-1387
Number of pages7
JournalClinical Chemistry
Volume44
Issue number7
Publication statusPublished - 1998

ASJC Scopus subject areas

  • Clinical Biochemistry

Fingerprint Dive into the research topics of 'Optimized PCR labeling in mutational and microsatellite analysis'. Together they form a unique fingerprint.

Cite this