Institut for Biomedicin

Lars Bolund

Detection of point mutations in amplified single copy genes by biotin-labelled oligonucleotides: Diagnosis of variants of alpha-1-antitrypsin

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

  • Niels Gregersen
  • Vibeke Winter, Aarhus Universitet
  • ,
  • Kirsten Bruun Petersen, Aarhus Universitet
  • ,
  • Jørn Koch
  • ,
  • Steen Kølvraa
  • ,
  • Niels Rüdiger, Aarhus Universitet
  • ,
  • Else Marie Heinsvig
  • ,
  • Lars Bolund

Specific analysis for point mutations in genomic DNA has until recently been a difficult and time-consuming task, using large amounts of unstable, hazardous and expensive 32P. By enzymatically amplifying the mutation-bearing sequence of the DNA the sensitivity of the analysis is increased several 100-fold, making the detection possible with stable, non-radioactive and inexpensive biotinylated oligonucleotides. We have applied this method (polymerase chain reaction (PCR)) to the detection of the Z-mutation in the alpha-1-antitrypsin gene. After amplification, dot-blots of amplified DNA were subjected to hybridization with allele specific biotinylated oligonucleotide probes and washed at temperatures giving allele specificity. The bound biotin was visualized with avidin conjugated alkaline phosphatase using 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium as colour reagents. The detection can be performed on < 1 μg genomic DNA, and is therefore applicable on small amounts of blood, fibroblasts and chorionic villus biopsies.

OriginalsprogEngelsk
TidsskriftClinica Chimica Acta
Vol/bind182
Nummer2
Sider (fra-til)151-164
Antal sider14
ISSN0009-8981
DOI
StatusUdgivet - 30 jun. 1989

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