Using the web based program NebCutter we selected the restriction enzyme Ase1 for the production of restriction fragments.
The forward primer was 5′-GGCCAGAGGCAACTAGAGTAT-3′ and 5′-ATACTCTAGTTGCCTCTGGCC -3′ served as the reverse primer. We describe, therefore, a quick cost effective RFLP based method to detect the SLCO1B1 c.1929A>C variant.īoth forward and reverse PCR primers were designed using the PrimerQuest Tool (Integrated DNA Technology, Iowa, USA) and tested for specificity using the NCBI Nucleotide Blast tool. Although methods such as mass arrays and sequencing offer more robust, automated and at times pre-validated options, restriction fragment length polymorphism (RFLP) remains an attractive method of choice for resource limited settings. Currently, genotyping of the SLCO1B1 c.1929A> C is done using the relatively more expensive methods such as Sanger sequencing, 5′-nuclease assays, microarrays. To do this we required a quick and cheap method to detect SLCO1B1 c.1929A> C in our study population. The c.1929C allele has been associated with increased hepatic uptake of the antifolate methotrexate (P = 0.028) in cancer patients and increased uptake of atorvastatin. The SLCO1B1 ( c.1929A> C, p.Leu643Phe) SNP has been associated with altered OATP1B1 transporter activity. The RFLP method is quick and cost effective.Īs part of a larger pharmacogenetics study, we opted to explore the possible impact of single nucleotide polymorphism c.1929A>C within the gene Solute carrier organic anion transporter family member 1B1 ( SLCO1B1) on rosuvastatin pharmacokinetics. The SLCO1B1 c.1929C allele was associated with a 75% reduction (P C may therefore play a significant role in rosuvastatin response. The frequency of the SLCO1B1 c.1929C allele amongst Zimbabweans was 6%.
A student’s t test with Welch correction was used to establish association between the SLCO1B1 c.1929A> C variant and rosuvastatin exposure.
We describe a restriction fragment length polymorphism method to genotype SLCO1B1 c.1929A> C polymorphism using the restriction enzyme Ase1. This study was designed to investigate the effect of the polymorphism SLCO1B1 c.1929A> C on interindividual variability in rosuvastatin pharmacokinetics in healthy volunteers of African descent. The aim of this study therefore was to design and validate a cost effective RFLP for genotyping the SLCO1B1 c.1929A> C polymorphism. Currently SLCO1B1 c.1929A> C is genotyped using direct sequencing techniques and 5′ nuclease based assays which can be cost prohibiting in resource limited settings. The polymorphism SLCO1B1 c.1929A> C has been associated with increased activity resulting in increased hepatic uptake of drugs. This study describes a restriction fragment polymorphism protocol for rapidly screening the polymorphism SLCO1B1 c.1929A> C in genomic DNA samples.
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