Journal article
Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes.
- Schneider M Division of Human Genetics, Department of Paediatrics Inselspital, University of Bern, Bern, Switzerland.
- von Känel T
- Sanz J
- Gallati S
- 2009-01-17
Published in:
- Clinica chimica acta; international journal of clinical chemistry. - 2009
Alleles
Cystic Fibrosis
Cystic Fibrosis Transmembrane Conductance Regulator
DNA, Complementary
Humans
Nucleic Acid Hybridization
Polymerase Chain Reaction
Reproducibility of Results
English
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.
CONCLUSION
This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.
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.
CONCLUSION
This assay represents an alternative to other fine quantitative methods such as ARMS PCR and Pyrosequencing.
- Language
-
- English
- Open access status
- closed
- Identifiers
-
- DOI 10.1016/j.cca.2008.12.017
- PMID 19146842
- Persistent URL
- https://sonar.ch/global/documents/1140
Statistics
Document views: 20
File downloads: