Linked Life Data

8681140

Source:http://linkedlifedata.com/resource/pubmed/id/8681140

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1996-8-20
pubmed:abstractText
Previous approaches to mutation detection in mRNA from the neurofibromatosis 1 (NF1) locus have required the PCR amplification of five or more overlapping cDNA segments to screen the entire 8.5-kb open reading frame (ORF). Systematically, these assays do not detect deletions that span the region of overlap (usually 1-3 exons) of any two consecutive target segments. In such cases, amplification from the mutant region of the disease-causing allele fails because binding sites for the PCR primers are missing, but amplification from the normal allele proceeds, yielding only the normal product. To alleviate this problem, we have developed a protocol to reverse transcribe and amplify the entire protein-coding sequence of NF1 as a single PCR product, starting with total RNA from lymphoblast cell lines or from whole blood. The 8.7-kb RT-PCR product was prepared from nine NF1 patients with known deletions or insertions, ranging in size from a 30-bp deletion within 1 exon to a 2.4-kb deletion that removes 12 exons. Agarose gel analysis of the initial products detected deletions as small as 341 bp. Restriction endonuclease digestion with Asel and Fspl, followed by agarose gel electrophoresis, revealed the predicted abnormal bands in all nine patients. All mutant bands were identified readily by observers with no knowledge of the patients' mutations. This simple assay should detect a great variety of insertion/deletion mutations in the NF1mRNA internal to the primer binding sites, including all possible single and multiple exon dropouts and approximately 30% of all previously reported NF1 mutations.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1088-9051
pubmed:author
pubmed:issnType
Print
pubmed:volume
6
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
58-66
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
Long RT-PCR of the entire 8.5-kb NF1 open reading frame and mutation detection on agarose gels.
pubmed:affiliation
Department of Human Genetics, University of Utah, Salt Lake City 84112, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.