Large-scale, cost-effective screening of PCR products in marker-assisted selection applications
Identifieur interne : 000195 ( Istex/Corpus ); précédent : 000194; suivant : 000196Large-scale, cost-effective screening of PCR products in marker-assisted selection applications
Auteurs : W. K. Gu ; N. F. Weeden ; J. Yu ; D. H. WallaceSource :
- Theoretical and Applied Genetics [ 0040-5752 ] ; 1995-08-01.
English descriptors
- KwdEn :
- Teeft :
- Allele, Allele specificity, Amplification, Annealing, Annealing temperature, Annealing temperatures, Appl, Appropriate allele, Arbitrary primers, Asap, Asap primers, Asaps, Bromide, Clone, Common bean, Cornell university, Correct size, Cycle parameters, Detection methods, Detection steps, Different plant, Direct measurement, Direct staining, Double helix, Enation mosaic virus, Error rate, Ethidium, Ethidium bromide, Extraction procedure, Fragment, Free nucleotides, Genet, Genetic markers, Genotype, Identical size, Large numbers, Leaf tissue, Marker, Marker detection, Marker sequence, Michelmore, Nucleic acids, Phaseolus vulgaris, Photoperiod, Photoperiod genes, Pisum sativum, Primer, Rapd, Rapd fragment, Rapd markers, Reaction mixture, Reaction product, Second amplification, Selection applications, Shorter primers, Simple procedures, Specific primers, Theor, Theor appl genet, Thermal cycler, Weak fluorescence, Weeden.
Abstract
Abstract: 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 of Wang et al. (Nucleic Acids Res 21:4153–4154, 1993). Template DNA is amplified using allelespecific 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 h, 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.).
Url:
DOI: 10.1007/BF00222974
Links to Exploration step
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<Para>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 of Wang et al. (Nucleic Acids Res 21:4153–4154, 1993). Template DNA is amplified using allelespecific 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 h, 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 (<Emphasis Type="Italic">Adh-1</Emphasis>
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<abstract lang="en">Abstract: 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 of Wang et al. (Nucleic Acids Res 21:4153–4154, 1993). Template DNA is amplified using allelespecific 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 h, 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.).</abstract>
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