Reference Measurement Systems in Genetic Testing: Development of DNA-based Reference Materials

Dr. Márki-Zay János
Reference Measurement Systems in Genetic Testing: Development of DNA-based Reference Materials.
Doktori értekezés, Szegedi Tudományegyetem.
(2011)

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Absztrakt (kivonat) idegen nyelven

Recent progress in molecular pathology and biotechnology has made DNA-based assays important tools in patient care. DNA tests allow the detection of genetic alterations responsible for inheritable disease conditions, higher risk of developing diseases or altered drug effects. These tests have a high impact on clinical decision-making and thousands of different tests are already in routine use. Thus, quality issues and standardisation of DNA tests are particularly important. Reference materials (RMs) and certified reference materials (CRMs) are recognised as an excellent tool for checking analytical accuracy and are valuable in creating the necessary reference points in the development of comprehensive measurement systems. (C)RMs are used for method development and validation, calibration, statistical quality control within a laboratory and to assess the performance of laboratories in proficiency testing schemes. At present, the lack of (C)RMs for molecular genetic tests means that there are limitations in benchmarking against which a laboratory can judge the performance of its assays. Thus, the need for appropriate (C)RMs became increasingly urgent. In the frame of these projects, prototypes for different types of genetic RMs were processed for inherited diseases and genetic risk factors, such as hereditary hemochromatosis, fragile X syndrome, cystic fibrosis, the factor V Leiden (G1691A) mutation and Factor II (prothrombin) G20210A variant. These prototype RMs were evaluated for their suitability and different possibilities for the preservation and packaging of purified DNA were investigated in order to improve the stability and recovery rate of DNA-based RMs. Utilizing these results and experiences, a set of plasmid-type RMs for the analysis of the human prothrombin gene G20210A mutation were developed and produced. These candidate CRMs were characterised using quantitative real-time PCR techniques for homogeneity, stability, sequence identity and fitness for purpose. The material is homogeneous and stable at -20 °C. The sequence of the insert was confirmed by DNA sequence analysis. Each vial contains an indicative volume of 50 μL corresponding to approximately 1 ng of plasmid DNA. This series of plasmid CRMs was certified according to the ISO Guides 30−35 and is now available from the IRMM to support the validation and the harmonisation of polymerase chain reaction (PCR)-based methods used for detection of the G20210A mutation in the human prothrombin gene. The CRMs IRMM/IFCC-490 (G20210/G20210 wild-type), IRMM/IFCC-491 (20210A/20210A homozygous mutant) and IRMM/IFCC-492 (G20210/20210A heterozygous mutant) have been the first clinical genetic CRMs introduced worldwide. In addition, these reference plasmids were used also for the production of quality control materials for rare sequence variants to carry out a European proficiency study for the assessment of the competence of clinical laboratories to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our experiences and knowledge gained from these projects can be used in the development and production of further DNA-based CRMs for any molecular genetic test.

Mű típusa: Disszertáció (Doktori értekezés)
Doktori iskola: Multidiszciplináris Orvostudományok Doktori Iskola
Tudományterület / tudományág: orvostudományok > elméleti orvostudományok
orvostudományok > klinikai orvostudományok
Magyar cím: Referencia mérési rendszerek a genetikai diagnosztikában: dns-alapú referencia anyagok fejlesztése
Idegen nyelvű cím: Reference Measurement Systems in Genetic Testing: Development of DNA-based Reference Materials
Témavezető(k):
Témavezető neveBeosztás, tudományos fokozat, intézményEmail
Dr. Dux Lászlótanszékvezető egyetemi tanár, Phd. DSc., SZTE ÁOK Biokémia Intézet, Szeged, Magyarországdux.laszlo@med.u-szeged.hu
Dr. Klein Christoph L.Scientific Officer, PhD, MBA, European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, BelgiumChristoph.KLEIN@ec.europa.eu
EPrint azonosító (ID): 1118
Publikációban használt név : Dr. Márki-Zay János
A mû MTMT azonosítója: 1930875
doi: 10.14232/phd.1118
A feltöltés ideje: 2011. nov. 05. 08:08
Utolsó módosítás: 2015. jún. 05. 10:47
Egyebek (raktári szám): B 4971
URI: http://doktori.bibl.u-szeged.hu/id/eprint/1118
Védés állapota: védett

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