Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes.
BACKGROUND
As for Cystic Fibrosis (CF) and many other hereditary diseases there is still a lack in understanding the relationship between genetic (e.g. allelic) and phenotypic diversity. Therefore methods which allow fine quantification of allelic proportions of mRNA transcripts are of high importance.


METHODS
We used either genomic DNA (gDNA) or total RNA extracted from nasal cells as starting nucleic acid template for our assay. The subjects included in this study were 9 CF patients compound heterozygous for the F508del mutation and each one F508del homozygous and one wild type homozygous respectively. We established a novel ligation based quantification method which allows fine quantification of the allelic proportions of ss and ds CFTR cDNA. To verify reliability and accuracy of this novel assay we compared it with semiquantitative fluorescent PCR (SQF-PCR).


RESULTS
We established a novel assay for allele specific quantification of gene expression which combines the benefits of the specificity of the ligation reaction and the accuracy of quantitative real-time PCR. The comparison with SQF-PCR clearly demonstrates that LASQ allows fine quantification of allelic proportions.


CONCLUSIONS
This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.
Schneider
Mircea
M
Division of Human Genetics, Department of Paediatrics Inselspital, University of Bern, Bern, Switzerland.
von Känel
Thomas
T
Sanz
Javier
J
Gallati
Sabina
S
D000483:Alleles; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation; D015203:Reproducibility of Results
D018076:DNA, Complementary; D019005:Cystic Fibrosis Transmembrane Conductance Regulator