Whole-Blood Polymerase Chain Reaction and Restriction Fragment Length Polymorphism: A Simplified Method by Microwave Irradiation

Abstract

Objectives: The aim of the present study was to develop a simple, quick and cheap method to process whole-blood samples for the molecular techniques polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) without the use of expensive reagents or sophisticated machines. Materials and Methods: Venous whole-blood samples were collected from 40 individuals. The samples were frozen at –80°C, and then rapidly thawed at 37°C. Each sample was incubated with distilled water, then boiled in a microwave and centrifuged. The supernatant was taken directly for PCR and RFLP. For comparison, PCR and RFLP were performed on DNA purified from the same samples using the phenol-chloroform method and two commercial DNA extraction kits. Results: PCR/RFLP results using the presented method were qualitatively similar to those obtained by DNA extracted using the other three methods. Conclusion: The presented method proved to be a simpler and cheaper way of processing whole-blood samples for PCR and RFLP analyses.

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection

“…Another significant feature of our microwave-based lysing approach is the ability to simultaneously prepare and fragment genomic DNA, noting that this is only achieved by a two-step process using current commercial approaches. The DNA fragmentation patterns obtained from our study are similar to previous reported microwave studies (14, 17), but without the need of a sophisticated microwave system, and yet still carried out in seconds instead of several minutes. This is a significant benefit of our approach.…”

“…Our simple one-step procedure involving a 2.45 GHz household microwave can lyse bacteria in a little as 30 seconds. As demonstrated by the present and other studies, the isolated DNA can be successfully used for a variety of molecular approaches including PCR (15, 16), MAMEF (1–6), and next generation sequencing (17). Another significant feature of our microwave-based lysing approach is the ability to simultaneously prepare and fragment genomic DNA, noting that this is only achieved by a two-step process using current commercial approaches.…”

“…Microwave irradiation is primarily known as a tool for sterilization purposes, but most recently it has been used for other purposes including acceleration of chemical reactions and release of genomic DNA. Microwaves have been shown to be effective for the preparation of genomic DNA from a variety of biological systems including bacteria (13, 14), bacteriophage (15), and spores (6), but also for preparation of DNA for real-time PCR analysis (16, 17). More recently, microwave irradiation has been used exclusively for the purpose of DNA fragmentation for various molecular approaches.…”

A simple genotyping procedure without DNA extraction to identify rare blood donors

“…Among the most potent PCR inhibitors reported are haemoglobin/heme and IgG fraction . Two main lines of investigation have been successfully followed, that is pre‐PCR handling of samples (freezing/heating of samples, microwave treatment) and PCR adjustment .…”

A novel method for whole blood PCR without pretreatment

“…Further, some methods entailed washing of blood samples and their suspension in a lysis buffer before subjecting them to PCR (de Vries et al, 2001;Narayan, 1992;Nordvag et al, 1992). Other authors reported the use of heating (McCuskar et al, 1992), hydrogen peroxide (Rudbeck and Dissing, 1998), microwave irradiation (Jadaon et al, 2009), formamide (Alison et al, 2005 and alkali (Victor et al, 2009) for the pretreatment of whole blood samples. Besides this, the use of Tth instead of Taq polymerase was also recommended for amplifying DNA directly from whole blood due to the higher efficiency of the former (Alison et al, 2005;Panacchio and Lew, 1991).…”