Screening of copy number polymorphisms in human beta-defensin genes using modified real-time quantitative PCR.
Defensins are cationic antimicrobial peptides, which play an important role in host immune defense to some infectious diseases as well as immune disease and skin disease. Recent studies identified that the genes coding for human beta-defensin 2 (DEFB4), human beta-defensin 3 (DEFB103) and human beta-defensin 4 (DEFB104) showed variation in copy numbers. This variation may have an impact on gene expression levels. Here, we have demonstrated a real-time PCR-based method to measure beta-defensin gene copy number. Using this relative real-time quantitative PCR, we developed a new rapid and reliable approach, which involves amplification of the target locus (DEFB4 or DEFB103 or DEFB104) and the single-copy reference locus (human serum albumin, ALB) in a single PCR reaction. A calibrator was prepared by recombining one copy of the target gene and one copy of the reference gene into a plasmid. After correcting the PCR amplification efficiency, which differed between the defensin gene and ALB gene, and normalization by the calibrator, the ratio of the copy number of the target gene to that of the reference gene in an unknown sample was determined. This normalized ratio directly related to the gene copy number. The assay was validated using previously genotyped samples, which demonstrated high accuracy and reliability of the method. Furthermore, this method was used to screen the copy number variations of these three beta-defensin genes in healthy blood donors. This method proved to be a reliable and fast tool to genotype gene copy number variations in projects associating genomic variations with gene expression or with population phenotypes in epidemiologic studies.
Chen
QiXing
Q
Department of Anaesthesiology and Intensive Care Medicine, University of Bonn, 53105 Bonn, Germany.
Book
Malte
M
Fang
XiangMing
X
Hoeft
Andreas
A
Stuber
Frank
F
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D018628:Gene Dosage; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011110:Polymorphism, Genetic; D023083:beta-Defensins / Q000235:genetics*
C422950:DEFB103A protein, human; C106641:DEFB4A protein, human; D017931:DNA Primers; D015342:DNA Probes; D023083:beta-Defensins; D004247:DNA