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professional thesis
Validacija metode za probir dinamičkih mutacija u genu FMR1
Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry, 2017. urn:nbn:hr:163:822131
Škrlec, Ivana
University of Zagreb
Faculty of Pharmacy and Biochemistry

Institutional repository: Repository of Faculty of Pharmacy and Biochemistry University of Zagreb

Cite this document

Škrlec, I. (2017). Validacija metode za probir dinamičkih mutacija u genu FMR1 (Professional thesis). Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry. Retrieved from https://urn.nsk.hr/urn:nbn:hr:163:822131

Škrlec, Ivana. "Validacija metode za probir dinamičkih mutacija u genu FMR1." Professional thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2017. https://urn.nsk.hr/urn:nbn:hr:163:822131

Škrlec, Ivana. "Validacija metode za probir dinamičkih mutacija u genu FMR1." Professional thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2017. https://urn.nsk.hr/urn:nbn:hr:163:822131

Škrlec, I. (2017). 'Validacija metode za probir dinamičkih mutacija u genu FMR1', Professional thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, accessed 25 November 2024, https://urn.nsk.hr/urn:nbn:hr:163:822131

Škrlec I. Validacija metode za probir dinamičkih mutacija u genu FMR1 [Professional thesis]. Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry; 2017 [cited 2024 November 25] Available at: https://urn.nsk.hr/urn:nbn:hr:163:822131

I. Škrlec, "Validacija metode za probir dinamičkih mutacija u genu FMR1", Professional thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, 2017. Available at: https://urn.nsk.hr/urn:nbn:hr:163:822131

Metadata
TitleValidacija metode za probir dinamičkih mutacija u genu FMR1
Title (english)Validation of screening method for dynamic mutation in the FMR1 gene
AuthorIvana Škrlec
MentorKarmela Barišić (mentor)
MentorJasenka Wagner (mentor)
MBZ: 268083
Committee memberSandra Šupraha Goreta (predsjednik povjerenstva)
Committee memberIngeborg Barišić (član povjerenstva)
Committee memberJadranka Sertić (član povjerenstva)
MBZ: 124856
GranterUniversity of Zagreb
Faculty of Pharmacy and Biochemistry
Zagreb
Defense date and country2017, Croatia
Scientific / art field,
discipline and subdiscipline		BIOMEDICINE AND HEALTHCARE
Pharmacy
Medical Biochemistry
Abstract
Ciljevi istraživanja su sljedeći: a) validacija metode izravnoga PCR-a s tri početnice (engl. direct triplet-primed PCR, dTP-PCR) za određivanje dinamičkih mutacija u genu FMR1, te b) usporedba rezultata dobivenih metodom dTP-PCR s rezultatima dobivenim Southern blot metodom. Uzorak ispitanika sastojao se od 40 pacijenata upućenih u Klinički bolnički centar (KBC) Osijek s dijagnozom intelektualnog zaostajanja. Ispitanici su nasumično izabrani i validacija je provedena slijepim istraživanjem. Izolacija genomske DNA napravljena je iz pune krvi. Broj tripleta CGG gena FMR1 utvrđen je metodom izravne lančane reakcije polimeraze u stvarnom vremenu s tri početnice (dTP-PCR) uz analizu krivulje mekšanja DNA. Analizom krivulje mekšanja utvrđena je temperatura mekšanja za svaki umnoženi uzorak DNA ispitanika. Temeljem razlike u temperaturi mekšanja ispitivanoga uzorka i kontrolnih uzoraka s poznatim brojem ponavljanja tripleta CGG (30, 41 i 53 ponavljanja) svi su ispitanici svrstani u četiri kategorije (normalan broj CGG tripleta ≤ 44 CGG, siva zona od 45 do 54 CGG, premutacija od 55 do 200 CGG i puna mutacija > 200 CGG). Prisutnost proširenih alela (> 200 tripleta CGG) kod osoba oba spola imala je drugačiju krivulju mekšanja DNA od normalnih alela (< 30 tripleta CGG). Klinička značajnost testa na 40 prethodno analiziranih ispitanika pokazala je 100 %-tnu osjetljivost i 90,48 %-tnu specifičnost kod utvrđivanja povećanja broja ponavljanja tripleta CGG (> 30) u genu FMR1. Metoda dTP-PCR-a primjerena je za brzi probir promjene broja ponavljanja tripleta CGG, kao i za otkrivanje nositelja premutacija i mutacija u genu FMR1 u populaciji ispitanika koja obuhvaća osobe s intelektualnim poteškoćama nepoznatoga uzroka.
Abstract (english)
Objectives of this study are validation of the direct triplet-primed PCR method (dTP-PCR) for determination of dynamic mutation in the FMR1 gene, and comparison of the results of dTP-PCR method and Southern blot analysis. In this study 40 patients from Osijek University Hospital with diagnosis of the intellectual disability were included. The patients were chosen randomly and validation of the method was conducted as a blind study. DNA was extracted from the whole blood. The number of the CGG repeats in the FMR1 gene is determined by direct triplet-primed PCR method and melting curve analysis. Cut-off temperature between normal and permutation of the CGG repeats is determined by control samples with known number of CGG repeats. All patients are classified in four categories based on DNA melting curve (normal number of CGG repeats ≤44, grey zone 45 to 54 CGG repeats, premutation 55 to 200 CGG repeats, and full mutation >200 CGG repeats). Presence of expanded alleles (>200 CGG repeats) in both sex had different DNA melting curve than the melting curve of normal allele (<30 CGG repeat). The clinical performance of the assay was established with 40 previously analyzed samples, yielding results of 100% sensitivity and 90,48% specificity in detection expansions of CGG (>30) repeats in FMR1 gene. This method is appropriate for quick determination of allelic changes in the FMR1 gene, screening population and defining mutations or permutation carriers in the population with intellectual disabilities with unknown cause.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:163:822131
Study programmeTitle: Molecular diagnostics
Study programme type: university
Study level: postgraduate specialist
Academic / professional title: sveučilišni magistar/sveučilišna magistra molekularne dijagnostike (sveučilišni magistar/sveučilišna magistra molekularne dijagnostike)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2017-09-18 14:23:19