Internal quality control of PCR-based genotyping methods in research studies and patient diagnostics.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

 
Udgivelsesdato: 2002-May
OriginalsprogEngelsk
TidsskriftThrombosis and Haemostasis
Vol/bind87
Udgave nummer5
Sider (fra-til)812-816
Antal sider4
ISSN0340-6245
StatusUdgivet - 1. maj 2002

Fingeraftryk

Polymerase Chain Reaction
DNA
Research
Reading
Edetic Acid
Restriction Fragment Length Polymorphisms
Alleles
Databases
Enzymes

Citer dette

@article{5ba8b4c0eb7b11dc86ef000ea68e967b,
title = "Internal quality control of PCR-based genotyping methods in research studies and patient diagnostics.",
abstract = "Genetic analyses are increasingly integrated in the clinical laboratory, and internal quality control programmes are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n = 500) by ammonium acetate precipitation and analysed for 18 polymorphisms by polymerase chain reaction (PCR), i. e. restriction fragment length polymorphisms, allele specific amplification, or amplification of insertion/deletion fragments. We evaluated the following aspects in the analytical procedures: sample handling and DNA-isolation (pre-analytical factors), DNA-amplification, digestion with restriction enzymes, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality (42 microg/ml blood, A260/A280 ratio >1.75, negative DNAsis tests), and the reagent blank was contaminated in <1{\%} of the results. Occasionally, results were re-analysed because of positive reagent blanks (< 1{\%}) or because of problems with the controls (<5{\%}). On confirmation, we observed 4 genotyping discrepancies. Control of data handling revealed 0.1{\%} reading mistakes and 0.5{\%} entry mistakes. Based on our experiences we propose an internal quality control programme for widely used PCR-based haemostasis polymorphism analyses.",
keywords = "Alleles, DNA, DNA Mutational Analysis, Diagnostic Errors, Genotype, Hemostasis, Humans, Polymerase Chain Reaction, Polymorphism, Genetic, Polymorphism, Restriction Fragment Length, Quality Control, Research, Research Design, Thrombophilia",
author = "Else-Marie Bladbjerg and J{\o}rgen Gram and J{\o}rgen Jespersen and Maat, {M de}",
year = "2002",
month = "5",
day = "1",
language = "English",
volume = "87",
pages = "812--816",
journal = "Thrombosis and Haemostasis",
issn = "0340-6245",
publisher = "Schattauer",
number = "5",

}

Internal quality control of PCR-based genotyping methods in research studies and patient diagnostics. / Bladbjerg, Else-Marie; Gram, Jørgen; Jespersen, Jørgen; Maat, M de.

I: Thrombosis and Haemostasis, Bind 87, Nr. 5, 01.05.2002, s. 812-816.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Internal quality control of PCR-based genotyping methods in research studies and patient diagnostics.

AU - Bladbjerg, Else-Marie

AU - Gram, Jørgen

AU - Jespersen, Jørgen

AU - Maat, M de

PY - 2002/5/1

Y1 - 2002/5/1

N2 - Genetic analyses are increasingly integrated in the clinical laboratory, and internal quality control programmes are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n = 500) by ammonium acetate precipitation and analysed for 18 polymorphisms by polymerase chain reaction (PCR), i. e. restriction fragment length polymorphisms, allele specific amplification, or amplification of insertion/deletion fragments. We evaluated the following aspects in the analytical procedures: sample handling and DNA-isolation (pre-analytical factors), DNA-amplification, digestion with restriction enzymes, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality (42 microg/ml blood, A260/A280 ratio >1.75, negative DNAsis tests), and the reagent blank was contaminated in <1% of the results. Occasionally, results were re-analysed because of positive reagent blanks (< 1%) or because of problems with the controls (<5%). On confirmation, we observed 4 genotyping discrepancies. Control of data handling revealed 0.1% reading mistakes and 0.5% entry mistakes. Based on our experiences we propose an internal quality control programme for widely used PCR-based haemostasis polymorphism analyses.

AB - Genetic analyses are increasingly integrated in the clinical laboratory, and internal quality control programmes are needed. We have focused on quality control aspects of selected polymorphism analyses used in thrombosis research. DNA was isolated from EDTA-blood (n = 500) by ammonium acetate precipitation and analysed for 18 polymorphisms by polymerase chain reaction (PCR), i. e. restriction fragment length polymorphisms, allele specific amplification, or amplification of insertion/deletion fragments. We evaluated the following aspects in the analytical procedures: sample handling and DNA-isolation (pre-analytical factors), DNA-amplification, digestion with restriction enzymes, electrophoresis (analytical factors), result reading and entry into a database (post-analytical factors). Furthermore, we evaluated a procedure for result confirmation. Isolated DNA was of good quality (42 microg/ml blood, A260/A280 ratio >1.75, negative DNAsis tests), and the reagent blank was contaminated in <1% of the results. Occasionally, results were re-analysed because of positive reagent blanks (< 1%) or because of problems with the controls (<5%). On confirmation, we observed 4 genotyping discrepancies. Control of data handling revealed 0.1% reading mistakes and 0.5% entry mistakes. Based on our experiences we propose an internal quality control programme for widely used PCR-based haemostasis polymorphism analyses.

KW - Alleles

KW - DNA

KW - DNA Mutational Analysis

KW - Diagnostic Errors

KW - Genotype

KW - Hemostasis

KW - Humans

KW - Polymerase Chain Reaction

KW - Polymorphism, Genetic

KW - Polymorphism, Restriction Fragment Length

KW - Quality Control

KW - Research

KW - Research Design

KW - Thrombophilia

M3 - Journal article

C2 - 12038782

VL - 87

SP - 812

EP - 816

JO - Thrombosis and Haemostasis

JF - Thrombosis and Haemostasis

SN - 0340-6245

IS - 5

ER -