Linked Life Data

1490171

Source:http://linkedlifedata.com/resource/pubmed/id/1490171

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1993-2-26
pubmed:abstractText
We have found that under appropriate conditions, an allele-specific polymerase chain reaction (PCR) can achieve a sensitivity suitable for measuring specific, infrequent mutations in single cell systems or in animal tissues. Using the 12th codon GC-to-AT mutation in the rat c-Ha-ras gene as a model system, we have defined conditions that allow for measurement of mutations present at frequencies as low as one in 10(5) gene copies. Our approach involved the use of PCR primers that created a single mismatch with the mutated allele (GAA) but created a double mismatch with the wild-type allele (GGA). Five out of the six such double-mismatch primers we tested permitted amplification of the mutant allele (GAA) with a high degree of specificity. The specificity of the assay was further enhanced by using a two-step PCR cycle consisting of a denaturation step (1 min incubation at 94 degrees C) and an annealing/extension step (1 min incubation at 50 degrees C) in the presence of 10% (vol/vol) glycerol. Reconstruction experiments using genomic DNA demonstrate that this procedure cna measure the presence of 30 copies of the transforming ras allele present amongst 3 x 10(6) copies of the wild-type allele.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
1054-9803
pubmed:author
pubmed:issnType
Print
pubmed:volume
2
pubmed:geneSymbol
c-Ha-ras
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
14-20
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1992
pubmed:articleTitle
Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene.
pubmed:affiliation
Center for Environmental Health Sciences, Whitaker College of Health Science and Technology, Massachusetts Institute of Technology, Cambridge 02139.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.