Naslov
Detection of Loss of Heterozygosity in the Patched Gene by High-resolution Melting Approach

Autori
Čretnik, Maja ; Musani, Vesna ; Orešković, Slavko ; Leović, Dinko ; Levanat, Sonja

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
10th International Summer School on Biophysics : Supramolecular Structure and Function : Book of Abstracts / Pifat-Mrzljak, Greta ; Zahkradka, Ksenija - Zagreb : Institut Ruđer Bošković, 2009, 94-94

ISBN
978-953-6690-81-7

Skup
International Summer School on Biophysics : Supramolecular Structure and Function (10 ; 2009)

Mjesto i datum
Rovinj, Hrvatska, 19.09.2009. - 01.10.2009

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
high-resolution melting ; loss of heterozygosity ; Hh-Gli signaling

Sažetak
High-resolution melting (HRM) is based on melting curve analysis, which is generated by heating the PCR product with an intercalated fluorescent dye. It is a novel method for screening of mutations or polymorphisms. When heated, the DNA strands separate and the dye is released, which is registered as a decrease in fluorescence. Each DNA fragment has a characteristic melting curve shape, and a single nucleotide change can alter the appearance of the curve. The specificity and sensitivity of the method are very high, 95% and 99%, respectively (Reed and Wittwer, 2004), but it can differ slightly from system to system. Here, we used HRM for detection of mutations in the Ptch gene. Ptch is a receptor of the Hedgehog-Gli (Hh-Gli) signaling pathway, a developmental pathway that is also associated with stem cell maintenance. Deregulation of this pathway is associated with various developmental malformations (if it occurs during embryogenesis), and tumors (if it occurs in an adult organism). The signal transduction begins with binding of the ligand protein Hedgehog (Hh) to the transmembrane receptor Patched (Ptch). Ptch then releases its inhibition of Smoothened (Smo), and a cytoplasmatic cascade of phosphorylation events leads to activation of transcription factor Gli and transcription of target genes, which include Cyclin D, Cyclin E, members of Wnt and TGFβ signaling pathways, and Ptch itself. DNA was extracted from 22 tissue samples of baseocellular carcinoma (BCC) and 12 tissue samples of ovarian carcinoma (OC), and from matched blood samples from each patient. All DNA samples were analyzed using HRM. Ptch gene was split into 23 PCR reactions with fragments sized 187 – 425 bp. Most of the exons were uninformative, since sequence changes were not found. However, some of the exons showed common polymorphisms, and comparison between melting profiles of tissue vs. blood samples showed different patterns in some of the samples. These samples were sequenced, and loss of heterozygosity (LOH) analysis was performed to verify the results. Here we demonstrated that HRM can be used as a fast method for LOH detection, but only for heterozygous samples. Comparison of melting profiles in such cases can detect a loss of heterozygosity.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

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