Year: 2007
Type: dissertation
Abstract: Polymerase chain reaction (PCR) using DNA from various biospecimens, such as blood, plant, tissue, etc, is a valuable tool in the field of molecular biology and medical diagnostics.However, DNA isolation from biospecimens is a laborious and sample-consuming step, and hampers the automation of PCR for large-scale studies.And attempt to PCR from biospecimens without DNA isolation is not achieved, since numerous endogenous and exogenous constituents may inhibit PCR.We used a novel buffer system, 'AnyDirect' that conserves the enzymatic activity of DNA polymerases for effective use in direct PCR from biospecimens under various conditions Using AnyDirect, DNA amplification was achieved from whole blood with a variety of thermostable DNA polymerases.Amplification occurred regardless of target size (up to 1.7 kb), presence of various known PCR inhibitors, and high target GC content.Importantly, low copy number DNA targets were effectively amplified ix from whole blood.The downstream procedures from direct PCR using AnyDirect were examined with various apolipoprotein E (apo E) genotyping methods such as sequencing, SNaPshot assay, PCR-restriction fragment length polymorphism (RFLP), and allele-specific (AS)-PCR.In this study, we have also developed a new genotyping method for apo E. Our multiplex tetra-primer amplification refractory mutation system (multiplex T-ARMS) PCR was performed in a single reaction tube and gave definitive electropherograms at each genotypes.Using AnyDirect solution, multiplex T-ARMS PCR for apo E genotyping from whole blood is a simple, rapid, and accurate method that requires only a single PCR reaction without DNA extraction and any another treatments or expensive instrumentation.The extraction of PCR-compatible genomic DNA from higher plants requires complicated and tedious work because plant cells have rigid cell walls and contain various endogenous PCR inhibitors such as polyphenolic compounds.Therefore, we developed a simple lysis system that can give rise to an appropriate template for direct PCR with AnyDirect solution, making the direct amplification of DNA fragments from plant leaves possible.Our experimental procedure provides a simple, convenient, non-hazardous, non-expensive, and rapid process for DNA amplification from various biospecimens.And our AnyDirect solution allows direct PCR from whole blood and may facilitate detection of genetic diseases or infections by eliminating the time and effort for DNA extraction.The use of AnyDirect could facilitate the development of highthroughput PCR for large-scale diagnostic screening or investigation of various medical conditions.And this method may be applicable for use with lab-on-a-chip x and bio-MEMS technology.
Author: 양영근
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3007612771