Rapid and inexpensive real-time PCR for genotyping functional polymorphisms within the Toll-like receptor -2, -4, and -9 genes.
The discovery of the human Toll-like receptors (TLRs) and the recognition of their pivotal role in sensing microbial pathogens during the last 5 years have resulted in a renewed appreciation of innate immunity. Due to their central role in both, triggering innate immunity as well as linking innate and adaptive immunity, genetic variations within the TLR genes, known to be associated with a variety of infectious diseases, are currently of great interest. Several single nucleotide polymorphisms (SNPs) within TLR genes have been described and seem to be associated with susceptibility to inflammatory diseases. However, methods for genotyping SNPs within the TLR genes, e.g. direct sequencing or polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis, are time-consuming. In this work, we report novel real-time PCR methods for genotyping five TLR SNPs within TLR-2, TLR-4 and TLR-9 that have been associated with various diseases using fluorescence labeled hybridization probes and the LightCycler instrument. In addition, we provide protocols employing a standard Taq polymerase in order to reduce substantially the costs for real-time PCR.
Hamann
Lutz
L
Institute for Microbiology and Hygiene, Charite Medical Center, Humboldt University, Dorotheenstrasse, Berlin 96 10117, Germany. lutz.hamann@charite.de
Hamprecht
Axel
A
Gomma
Abuzeid
A
Schumann
Ralf R
RR
D017931:DNA Primers; D005838:Genotype; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors
D017931:DNA Primers; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C495371:TLR9 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D019914:Taq Polymerase