Large-scale, cost-effective screening of PCR products in marker-assisted selection applications

• Published in: Theoretical and applied genetics Vol. 91; no. 3; pp. 465 - 470
• Main Authors: Gu, W.K, Weeden, N.F, Yu, J, Wallace, D.H. (Cornell Univ., Geneva, NY (USA). Dept. of Horticultural Sciences)
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01-08-1995; Berlin
• Subjects: Biological and medical sciences; Classical genetics, quantitative genetics, hybrids; ELECTROFORESIS; ELECTROPHORESE; Fundamental and applied biological sciences. Psychology; GENE; GENES; Genetics of eukaryotes. Biological and molecular evolution; MARCADORES GENETICOS; MARQUEUR GENETIQUE; PHASEOLUS VULGARIS; PISUM SATIVUM; Pteridophyta, spermatophyta; SELECCION; SELECTION; Vegetals; Genetic marker; Artificial selection; Genotype; Method; Screening; Leguminosae; Vegetable crop; Random amplified polymorphic DNA; Phaseolus vulgaris; Dicotyledones; Pisum sativum; Angiospermae; Spermatophyta; Polymorphism
• ISSN: 0040-5752; 1432-2242
• DOI: 10.1007/bf00222974

A simple, PCR-based method has been developed for the rapid genotyping of large numbers of samples. The method involves a alkaline extraction of DNA from plant tissue using a slight modification of the procedure. Template DNA is amplified using allele specific associated primers (ASAPs) which, at stringent annealing temperatures, generate only a single DNA fragment and only in those individuals possessing the appropriate allele. This approach eliminates the need to separate amplified DNA fragments by electrophoresis. Instead, samples processing the appropriate allele are identified by direct staining of DNA with ethidium bromide. Total technician time required for extraction, amplification and detection of 96 samples is about 4 hours, and this time requirement can be reduced by automation. Excluding labor, cost per sample is less than $0.40. The method is tested using the codominant isozyme marker, alcohol dehydrogenase (Adh-1) gene in pea (Pisum sativum), and applied to the screening of photoperiod genes in common bean (Phaseolus vulgaris L.).