Detection of the TLR4 1196C>T polymorphism by mismatched-polymerase chain reaction using plasmid DNA as internal control in restriction fragment length polymorphism assays.
BACKGROUND
Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays.


RESULTS
Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C > T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK- plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 microL PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 microL PCR product. In standardized conditions, TLR4 1196C>T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C>T.
Lima-Neto
Lidio G
LG
Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, Brazil. lidio@usp.br
Hirata
Rosario D
RD
Luchessi
Andre D
AD
Silbiger
Vivian N
VN
Pastorelli
Carla P
CP
Sampaio
Marcelo F
MF
Armaganijan
Dikran
D
Rezende
Adriana A
AA
Doi
Sonia Q
SQ
Hirata
Mario H
MH
D000328:Adult; D000483:Alleles; D016022:Case-Control Studies; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D005787:Gene Frequency; D006801:Humans; D008297:Male; D009203:Myocardial Infarction / Q000097:blood; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012015:Reference Standards; D051197:Toll-Like Receptor 4 / Q000235:genetics*
D051197:Toll-Like Receptor 4; D004247:DNA