{"title":"Development of a rapid DNA screening procedure for the Factor V Leiden mutation.","authors":"G A Scobie, S T Ho, G Dolan, N A Kalsheker","doi":"10.1136/mp.49.6.m361","DOIUrl":null,"url":null,"abstract":"<p><p>Aim-To develop a rapid, simple and highly specific DNA screening procedure based on the amplification refractory mutation system (ARMS) to detect the Leiden mutation in whole blood.Methods-ARMS PCR amplification primers with additional mismatches at either -2 or -3, which greatly improves specificity, were constructed to detect the normal Factor V gene and the Leiden mutation in whole blood samples from patients with abnormal clotting results.Results-Construction of ARMS primers with either an additional mismatch at -2 or -3 at the 3' end of the primer could be used to detect the Leiden mutation in 0.5 mu1 whole blood in under three hours. Primers destabilised at position -3 could be used at a lower annealing temperature, which gave greater sensitivity and are now routinely used. A control set of primers was included in the same reaction to act as a positive control.Conclusions-This rapid and specific assay for the factor V Leiden mutation is a useful addition to the investigation of patients with or at risk from thrombovascular disease.</p>","PeriodicalId":87395,"journal":{"name":"Clinical molecular pathology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1996-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1136/mp.49.6.m361","citationCount":"10","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinical molecular pathology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1136/mp.49.6.m361","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 10
Abstract
Aim-To develop a rapid, simple and highly specific DNA screening procedure based on the amplification refractory mutation system (ARMS) to detect the Leiden mutation in whole blood.Methods-ARMS PCR amplification primers with additional mismatches at either -2 or -3, which greatly improves specificity, were constructed to detect the normal Factor V gene and the Leiden mutation in whole blood samples from patients with abnormal clotting results.Results-Construction of ARMS primers with either an additional mismatch at -2 or -3 at the 3' end of the primer could be used to detect the Leiden mutation in 0.5 mu1 whole blood in under three hours. Primers destabilised at position -3 could be used at a lower annealing temperature, which gave greater sensitivity and are now routinely used. A control set of primers was included in the same reaction to act as a positive control.Conclusions-This rapid and specific assay for the factor V Leiden mutation is a useful addition to the investigation of patients with or at risk from thrombovascular disease.