diplomsko delo univerzitetnega študijskega programa
Tjaša Savić (Author), Uroš Potočnik (Mentor)

Abstract

Analiza talilne krivulje visoke ločljivosti (HRM) je postala široko uporabljana metoda za genotipizacijo enonukleotidnih polimorfizmov (SNP), ki že zamenjuje verižno reakcijo s polimerazo v povezavi s polimorfizmom dolžin restrikcijskih fragmentov (PCR-RFLP). Namen tega diplomskega dela je bil optimizirati ter potrditi zanesljivost HRM za genotipizacijo treh izbranih SNPov, povezanih s kroničnima imunskima boleznima revmatoidnim artritisom in astmo. Študija opisuje teoretske osnove preiskovane problematike, uporabljene postopke laboratorijskega dela ter prikazuje analizo rezultatov. Genotipizacija je bila izvedena z referenčno metodo PCR-RFLP in HRM analizo, rezultati po obeh metodah so bili med sabo primerjani. Študija je skupno vključevala 463 DNA vzorcev slovenskih bolnikov oz. zdravih posameznikov (kontrole). Rezultati kažejo, da je bilo pri vseh treh SNPih doseženo visoko ujemanje med metodama (91%-98%). Izračunane frekvence dobljenih genotipov kažejo, da je bilo boljše ujemanje z referenčnimi frekvencami z NCBI pri dveh SNPih doseženo po HRM, kar potrjuje visoko zanesljivost te metode. Pri tem delu se je pojavilo nekaj težav pri optimizaciji PCR in pravilnem določanju genotipov po RFLP, kar še poudarja prednosti analize talilnih krivulj. HRM je zahtevala manj časa za optimizacijo, izvedbo in pridobivanje rezultatov, tako da je bila enostavnejša, posledično cenejša in v določenih primerih še bolj zanesljiva kot tradicionalna PCR-RFLP.

Keywords

chronic immune disease;single nucleotide polymorphisms;SNP;genotyping;high resolution melting curve analysis;HRM;polymerase chain reaction;PCR;restriction fragment lenght polymorphism;RFLP;

Data

Language: English
Year of publishing:
Source: Maribor
Typology: 2.11 - Undergraduate Thesis
Organization: UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher: [T. Savić]
UDC: 616.1(043.2)
COBISS: 16644118 Link will open in a new window
Views: 3100
Downloads: 356
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Other data

Secondary language: Slovenian
Secondary title: Optimization and accuracy of high resolution melting analysis for genotyping of selected DNA polymorphisms associated with chronic immune diseases
Secondary abstract: High Resolution Melting (HRM) analysis has become a widely used method for genotyping of single nucleotide polymorphisms (SNPs) replacing PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) method. The aim of this work was to optimize and confirm the accuracy of HRM for genotyping of selected SNPs associated with two chronic immune diaseases: rheumatoid arthritis and asthma. This study describes the theoretical background of the studied theme, work procedures of both methods used and analyses the results. Genotyping was performed by the reference PCR-RFLP method and HRM analysis and results were compared between the two methods. The study included alltogether 463 DNA samples of Slovene pacients and healthy individuals (controls) . The results show, that a high percentage in genotypes matching between both methods for all three SNPs (91%-98%) was achieved. The calculated genotype frequencies show that for two out of three SNPs a better matching with reference frequencies from NCBI was achieved by using HRM, which confirms the high accuracy of this method. During our experimental work some difficulties with PCR optimization and determination of the right genotype were encountered which accentuated the advantages of HRM analysis. HRM required less time for optimization, preparation and data acquirement, making it a much simpler and cheaper method and in some cases even more reliable and accurate than traditional PCR-RFLP.
Secondary keywords: kronične imunske bolezni;enonukleotidni polimorfizmi;genotipizacija;metoda analize talilne krivulje visoke ločljjivosti;verižna reakcija s polimerazo;polimorfizem dolžin restrikcijskih fragmentov;
URN: URN:SI:UM:
Type (COBISS): Bachelor thesis/paper
Thesis comment: Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages: IX, 47 str.
Keywords (UDC): applied sciences;medicine;technology;uporabne znanosti;medicina;tehnika;medical sciences;medicina;pathology;clinical medicine;patologija;klinična medicina;pathology of the circulatory system;blood vessels;cardiovascular complaints;bolezni krvnega obtoka in krvnih žil;kardiovaskularne bolezni;hematologija;
ID: 1002930