Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes

Schneider, Mircea; von Känel, Thomas; Sanz, Javier; Gallati, Sabina (2009). Ligation dependent allele specific quantification (LASQ) of CFTR cDNA on the LightCycler using MLPA hybridization probes. Clinica chimica acta, 402(1-2), pp. 47-53. Amsterdam: Elsevier 10.1016/j.cca.2008.12.017

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Gynaecology, Paediatrics and Endocrinology (DFKE) > Clinic of Paediatric Medicine

UniBE Contributor:

von Känel, Thomas and Gallati, Sabina

ISSN:

0009-8981

ISBN:

19146842

Publisher:

Elsevier

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 15:03

Last Modified:

06 Dec 2013 13:52

Publisher DOI:

10.1016/j.cca.2008.12.017

PubMed ID:

19146842

Web of Science ID:

000264618700009

URI:

https://boris.unibe.ch/id/eprint/27202 (FactScience: 104950)

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