Allele specific PCR with microfluorometry: application to the detection of del F508 mutation in cystic fibrosis.
BACKGROUND
A simple and practical screening method, allowing the mass detection of targeted DNA mutations, was developed by combined use of allele specific PCR (ASP) and subsequent fluorogenic intercalation to the amplicon.


METHODS
Crude DNAs were extracted from dried blood spots (DBS) by a simple boil method. Highly specific polymerase chain reaction (PCR) amplification was achieved by adopting Taq DNA polymerase (Amersham Pharmacia) modified with TaqStart Antibody (Clontech). A fluorogenic DNA intercalator, SYBR Green I (Molecular Probes), was directly added to the PCR products and the resultant fluorescence was measured by a conventional fluorometric microplate reader, microfluorometry (MFL).


RESULTS
The most common mutation in cystic fibrosis (CF), del F508, was successfully detected with clear differentiation as homozygotes (n=4) and heterozygotes (n=9) from control subjects (n=18). Fluorescence intensities, with mean+/-S.D. in arbitrary unit, were 895+/-249, 900+/-184 and 257+/-53, respectively. Those from control newborns (n=352) were 250+/-27 with the range of 188-475.


CONCLUSIONS
The proposed ASP/MFL provides a simple, objective and economical detection of known mutations or single nucleotide polymorphisms (SNPs). The usefulness of this method was clearly shown in the detection of del F508 in CF.
Yamaguchi
Akihiro
A
Sapporo Immunodiagnostic Laboratory, Shinkawa 2-2-12-20, Kita, Sapporo 001-0922, Japan.
Nepote
Juan Alberto
JA
Kadivar
Maliheh
M
Tagami
Yasuko
Y
Fukushi
Masaru
M
Kikuchi
Yuko
Y
Sato
Noriyuki
N
Kikuchia
Kokichi
K
D000293:Adolescent; D000328:Adult; D000483:Alleles; D052160:Benzothiazoles; D001800:Blood Specimen Collection; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D003365:Costs and Cost Analysis; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics*; D003592:Cytophotometry; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods / Q000592:standards; D003959:Diamines; D005456:Fluorescent Dyes; D005820:Genetic Testing / Q000191:economics / Q000379:methods / Q000592:standards; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D009154:Mutation; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D017384:Sequence Deletion / Q000235:genetics
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I