Isothermal Single Nucleotide Polymorphism Genotyping and Direct PCR from Whole Blood Using a Novel Whole-Blood Lysis Buf
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Isothermal Single Nucleotide Polymorphism Genotyping and Direct PCR from Whole Blood Using a Novel Whole-Blood Lysis Buffer Sylvia T. Victor,1,2 Alexander Lezhava,1 Takefumi Ishidao,1,3 Ryuta Endo,1 Yasumasa Mitani,1,3 Yoshihiro Kawaoka2 and Yoshihide Hayashizaki1 1 Technology Development Unit, Omics Science Center, RIKEN Yokohama Institute, Yokohama, Japan 2 Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 3 K.K. Dnaform, Yokohama, Japan
Abstract
Cell lysis and subsequent release of genomic DNA is an ongoing dilemma for molecular biological techniques. In most cases, technologies such as PCR and other amplification techniques require DNA extraction and purification steps. The Smart Amplification Process Version 2 (SmartAmp2) is an isothermal and integrated amplification technology that eliminates the need for time-consuming sample preparation for the rapid detection of nucleic acids, including single nucleotide polymorphisms (SNPs), mutations, and other targets. In addition, DNA amplification directly from whole blood is beneficial and lessens the risk of cross-contamination. Traditional SmartAmp2 assays entail two steps and require an alkali pretreatment step at 981C prior to the 601C run. To make SmartAmp2 truly isothermal and to simplify DNA amplification, we hereby introduce the SmartAmp Isothermal Lysis Buffer (SIL-B), a newly developed chaotropic lysis buffer that enables the simultaneous recovery and denaturation of genomic material directly from whole blood at a uniform 601C. The improved method for isolating nucleic acids from whole blood is a critical milestone in making SmartAmp2 truly isothermal from start to finish at one temperature, increasing its potential to be routinely used in field point-of-care testing. Furthermore, pretreatment with SIL-B enables the PCR amplification of genomic material directly from whole blood.
Innovative diagnostic methods that integrate isothermal amplification and detection in a single closed-tube assay are likely to play a major role in routine point-of-care testing (POCT) in the future. Currently, POCT using whole blood requires complete turn-key technology development for the simultaneous cell lysis and denaturation of template genomic material. Components of blood can interfere with cell lysis and inactivate thermostable DNA polymerases necessary for successful DNA amplification by PCR.[1] SmartAmp2, short for Smart Amplification Process Version 2, is an isothermal DNA amplification method for rapidly detecting single nucleotide polymorphisms (SNPs) and mutations with high sensitivity and specificity.[2,3] Contrary to regular PCR, which requires thermal cycling to denature, anneal and extend DNA strands; Aac polymerase, a proprietary enzyme with strong strand displacement activity, is
used in SmartAmp2 to catalyze DNA amplification at a uniform temperature. Optimized SmartAmp2 primer design achieves complete background suppression of mismatch amplific
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