Heat treatment of bacteria: a simple method of DNA extraction for molecular techniques

To evaluate the efficacy of two simple methods involving use of heat for extraction of bacterial deoxyribonucleic acid [DNA] be used in molecular techniques like polymerase chain reaction [PCR], restriction fragments length polymorphism [RFLP] and DNA sequencing and compare them with DNA extraction using commercial kits. DNA extraction by improved alternative methods and commercial kit. Microbiology Research Laboratory, Faculty of Allied Health Sciences, Kuwait University, Kuwait. Forty isolates of Klebsiella pneumoniae. DNA was extracted from isolates by either boiling for 10 minutes or microwave irradiation for 10 seconds. For comparison, DNA was also extracted using a commercial kit. All extracted DNA samples were analyzed by PCR, RFLP and / or DNA sequencing of TEM and SHV genes of the bacteria. Successful extraction of DNA. PCR, RFLP and DNA sequencing gave the expected results in all the DNA samples extracted by all the three methods [boiling, microwave irradiation and the commercial kit]. The results were qualitatively equivalent in all methods. Heat may be used to extract DNA from K. pneumoniae which can be utilized successfully in performing PCR, RFL and DNA sequencing

Whole-blood polymerase chain reaction and restriction fragment length polymorphism: a simplified method by microwave irradiation

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. 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. PCR/RFLP results using the presented method were qualitatively similar to those obtained by DNA extracted using the other three methods. The presented method proved to be a simpler and cheaper way of processing whole-blood samples for PCR and RFLP analyses

Factor V Kuwait: a novel mutation in the coagulation factor V gene discovered in Kuwait

It was the aim of this study to report a new point mutation in the clotting factor V gene in the general Arab population. The HR2 haplotype was tested in 288 Arabs living in Kuwait - 188 patients with venous thromboembolic disorders [VTE] and 100 healthy subjects - using polymerase chain reaction and restriction fragment length polymorphism techniques. The presence of the new mutation was verified by DNA sequencing. Two [1.06%] VTE patients had guanine instead of the wild-type adenine at nucleotide number 3935 [A3935G] of the factor V gene. This mutation caused a histidine to arginine change in amino acid number 1254 of the factor V molecule. The new mutation is termed 'factor V Kuwait' [His1254Arg] and was absent in the 100 healthy subjects. It appears that factor V Kuwait could be a risk factor for developing VTE in Arabs. A larger study is needed to confirm this observation