ABSTRACT
We developed a rapid and low-cost panel of three assays for visual genotyping of the three most common genetic risk factors in thrombophilia, namely, the single-point mutations in the FV (Leiden factor), FII and MTHFR genes. A triplex PCR was developed for simultaneous amplification of three fragments spanning the interrogated loci. Allele discrimination was accomplished by a 5-min primer extension reaction on each locus. Detection of the extension products was performed by means of a dual-allele dipstick-type DNA biosensor. The biosensor is disposable and allows visual detection and confirmation of the genotyping products within 15 min by hybridization without the need for specialized instruments or expensive reagents. The proposed method was evaluated by genotyping 40 samples with a variety of genotypes for all three mutations. The genotyping results were in full concordance with those obtained by restriction fragment length polymorphism analysis and sequencing.
Subject(s)
Biosensing Techniques/instrumentation , DNA Mutational Analysis/methods , Oligonucleotide Array Sequence Analysis/instrumentation , Reagent Kits, Diagnostic , Refractometry/instrumentation , Thrombophilia/diagnosis , Thrombophilia/genetics , Equipment Design , Equipment Failure Analysis , Humans , Visual PerceptionABSTRACT
In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)(30) segment at the 5' end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.
