Real-time PCR-based genotyping assay for CXCR2 polymorphisms

Manish Gupta, Pengfei Song, Charles Yates, Bernd Meibohm

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Background: The human chemokine receptor CXCR2 (IL8RB) is a high affinity receptor for interleukin-8 as well as other CXC chemokines, and is involved in the chemotaxis of immune cells. Genetic variants of CXCR2 have potential relevance in various inflammatory human disorders. We developed a real-time polymerase chain reaction (PCR)-based allelic discrimination assay for the detection of the CXCR2 single nucleotide polymorphisms (SNPs) C785T, T1208C and G1440A. Methods: Polymorphisms were delineated using PCR amplification of specific alleles (PASA). Allele-specific primers were developed for both wild-type and mutant alleles. An additional nucleotide mismatch at the third position from the 3′ end of each primer was used to improve amplification specificity and to prevent generation of nonspecific products. Genotypes were assigned based on PCR growth curves and melt curve analysis performed on a SmartCycler® using SYBR Green I chemistry. Results: Genotyping assignments were successfully performed in a set of 20 human DNA samples, and were validated by comparison with results from direct DNA sequencing and agarose gel electrophoresis of PCR products. Conclusions: Due to its rapid and relatively inexpensive performance and accuracy, the presented allelic discrimination assay for CXCR2 polymorphisms has wide applicability, especially for high-throughput sample analysis in large population genotyping studies.

Original languageEnglish (US)
Pages (from-to)93-100
Number of pages8
JournalClinica Chimica Acta
Volume341
Issue number1-2
DOIs
StatePublished - Jan 1 2004

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Polymerase chain reaction
Polymorphism
Real-Time Polymerase Chain Reaction
Assays
Alleles
Polymerase Chain Reaction
Interleukin-8 Receptors
Amplification
CXC Chemokines
Nucleotides
Agar Gel Electrophoresis
Chemokine Receptors
Chemotaxis
DNA Sequence Analysis
Single Nucleotide Polymorphism
DNA
Electrophoresis
Reaction products
Genotype
Sepharose

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Real-time PCR-based genotyping assay for CXCR2 polymorphisms. / Gupta, Manish; Song, Pengfei; Yates, Charles; Meibohm, Bernd.

In: Clinica Chimica Acta, Vol. 341, No. 1-2, 01.01.2004, p. 93-100.

Research output: Contribution to journalArticle

Gupta, Manish ; Song, Pengfei ; Yates, Charles ; Meibohm, Bernd. / Real-time PCR-based genotyping assay for CXCR2 polymorphisms. In: Clinica Chimica Acta. 2004 ; Vol. 341, No. 1-2. pp. 93-100.
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