期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 308, 期 1-2, 页码 231-240出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2005.11.001
关键词
copy number polymorphisms; human beta-defensin genes; DEFB4; DEFB103; DEFB104; 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 P-defensin 3 (DEFB 103) and human P-defensin 4 (DEFB 104) 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 DEFB 103 or DEFB 104) 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. (c) 2005 Elsevier B.V. All rights reserved.
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