ABSTRACT
A new single nucleotide polymorphisms (SNP) genotyping method has been developed and validated using biological specimens directly as templates for TaqMan PCR without general DNA extraction and purification procedure from dried saliva samples attached on water-soluble papers. This new method can set up at ease and complete PCR analysis including data interpretation in under two hours with additional advantages of application for large-scale clinical research, diagnostics, and epidemiological studies at low cost. Specifically, SNP genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) were demonstrated by TaqMan PCR assay using dried saliva samples in the present investigation. In this protocol, by simplifying experimental operations and improving efficiency, omitting and simplifying the time and laborious DNA purification process, it is possible to shorten the experiment time and reduce the risk of human error such as contamination. Furthermore it became possible with great cost reduction. We succeeded in dramatically improving the judgment rate and accuracy of SNP genotyping by the master mix reagent for commercial available real-time TaqMan PCR. Moreover, it becomes possible to stably introduce template DNA into the reaction system, and it will be possible to apply it to copy number variation (CNV) by TaqMan probe method. The SNP analysis process using this optimized water-soluble paper will be applied to gene polymorphism analysis of drug metabolizing enzyme gene CYP, etc., to help efforts to realize personalized medicine.
Subject(s)
Alcohol Dehydrogenase/genetics , Alcohols/metabolism , Aldehyde Dehydrogenase, Mitochondrial/genetics , Genotype , Genotyping Techniques/methods , Polymorphism, Single Nucleotide/genetics , DNA Copy Number Variations , Humans , Real-Time Polymerase Chain Reaction , SalivaABSTRACT
DNA extraction and purification have been generally considered to be required for PCR assay. We demonstrated a new protocol using biological specimens directly as templates for real-time PCR with melting curve analysis. We confirmed the melting curve analysis was particularly suitable for the identification of the insertion/deletion (Ins/Del) polymorphism of the angiotensin-converting enzyme (ACE) gene. The new protocol we developed can be set up using simple and complete PCR analysis including data interpretation in under four hours with additional advantages of application for large-scale clinical research, diagnostics, and epidemiological studies at low cost.
Subject(s)
Genotyping Techniques/methods , Peptidyl-Dipeptidase A/genetics , Asian People , Female , Genotype , Humans , INDEL Mutation , Polymorphism, Genetic , Real-Time Polymerase Chain Reaction , Saliva/chemistryABSTRACT
A new efficient synthesis of 2-substituted tetrahydroquinolines has been achieved by the domino reaction of N-indanyl(methoxy)amines, which consists of three types of reactions: elimination of an alcohol, the rearrangement of an aryl group, and the addition of an organolithium or magnesium reagent. The synthetic utility of this approach is demonstrated by the stereoselective formal synthesis of (+/-)- martinellic acid.
