Switch to
| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
2
|
| pubmed:dateCreated |
1994-6-15
|
| pubmed:abstractText |
Multiple fluorescence-based polymerase chain reaction single-strand conformation polymorphism (MF-PCR-SSCP) was developed. The target sequence was amplified by PCR using forward and reverse primers labeled with two different fluorescent dyes at their 5' ends. The amplified products were then heat-denatured, mixed with internal standard DNA markers labeled with a third fluorescent dye and applied to a temperature-controlled gel in an automated DNA sequencer, with a gel-temperature-controlling system. Mutations were detected as positional shifts of two-colored peaks in the electrophoretogram. The image data were analyzed by the computer program GENESCAN 672. The peak positions were standardized to internal DNA size markers. MF-PCR-SSCP analysis of 7 human tumor cell lines with 7 different single base mutations of the human K-ras oncogene detected all mutations even under the same electrophoresis conditions. Complete loss of heterozygosity was detected in two cell lines simultaneously. A gel temperature at 20 degrees C and polyacrylamide concentration of 10% gave the best separation. MF-PCR-SSCP is superior to the current PCR-SSCP in several ways: it does not involve radioactivity, migration patterns are standardized to internal standard DNA markers, there is a strict temperature-controlling system and the higher percentage of the gel enables better separation with resultant 100% detection of mutations most likely under one set of electrophoresis conditions.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/DNA,
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Neoplasm,
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Single-Stranded,
http://linkedlifedata.com/resource/pubmed/chemical/DNA Primers,
http://linkedlifedata.com/resource/pubmed/chemical/Glycerol
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Feb
|
| pubmed:issn |
0736-6205
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:volume |
16
|
| pubmed:geneSymbol |
K-ras
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
296-7, 300-5
|
| pubmed:dateRevised |
2006-11-15
|
| pubmed:meshHeading |
pubmed-meshheading:8179893-Base Sequence,
pubmed-meshheading:8179893-Biotechnology,
pubmed-meshheading:8179893-DNA,
pubmed-meshheading:8179893-DNA, Neoplasm,
pubmed-meshheading:8179893-DNA, Single-Stranded,
pubmed-meshheading:8179893-DNA Primers,
pubmed-meshheading:8179893-Electrophoresis, Polyacrylamide Gel,
pubmed-meshheading:8179893-Genes, ras,
pubmed-meshheading:8179893-Glycerol,
pubmed-meshheading:8179893-Humans,
pubmed-meshheading:8179893-Molecular Sequence Data,
pubmed-meshheading:8179893-Nucleic Acid Conformation,
pubmed-meshheading:8179893-Point Mutation,
pubmed-meshheading:8179893-Polymerase Chain Reaction,
pubmed-meshheading:8179893-Polymorphism, Genetic,
pubmed-meshheading:8179893-Reference Standards,
pubmed-meshheading:8179893-Sensitivity and Specificity,
pubmed-meshheading:8179893-Temperature,
pubmed-meshheading:8179893-Tumor Cells, Cultured
|
| pubmed:year |
1994
|
| pubmed:articleTitle |
Multiple fluorescence-based PCR-SSCP analysis.
|
| pubmed:affiliation |
Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
|
| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
|