Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1994-3-28
pubmed:abstractText
A 985A-->G transition is the single prevalent mutation representing 89% of all variant medium-chain acyl-CoA dehydrogenase (MCAD) alleles that causes MCAD deficiency. We and others previously devised a molecular method for the detection of the 985G allele that involves PCR coupled with NcoI digestion. The method has been widely used. However, when used for the analysis of dried blood samples, it sometimes produced ambiguous results with weak target bands in the presence of numerous artifact bands. An improved version of the method has been developed here, involving two stages of amplification using two different sets of primers. In the first stage, the entire exon-11 was amplified. A small aliquot (5 microliters) of the first PCR products were directly used as a template for the second PCR amplification. The second PCR is similar to the original method, but utilizes a pair of primers encompassing a smaller section within exon-11. The upstream primer incorporates a substitution at 981, so that a NcoI site involving 985G is introduced in the copies of the variant allele, as in the original PCR/NcoI method. The improved 2-stage method produces an intense target band with a very high sensitivity, yet devoid of artifacts, providing clean, unequivocal results.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0009-8981
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
220
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
165-74
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1993
pubmed:articleTitle
Improved PCR/NcoI method for the molecular diagnosis of medium chain acyl-CoA dehydrogenase deficiency using dried blood samples: two-stage amplification using two different sets of primers improves accuracy and sensitivity.
pubmed:affiliation
Yale University School of Medicine, Department of Genetics, New Haven, CT 06510.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't