A handmade DNA extraction kit using laundry powder; insights on simplicity, cost-efficiency, rapidity, safety and the quality of purified DNA.

Here we describe an in-house kit for high throughput DNA extraction using laundry detergent. A simplified lysis buffer made only from 0.08 M EDTA, 0.1 M Tris, and laundry powder is the core of our protocol. We extracted genomic DNA from 150 µL of whole blood collected from different farm animals and compared the performance to both the DNeasy Blood & Tissue Kit (Qiagen) and the widely used salting-out procedure. An evaluation of the concentration and quality of the extracted DNA was then assessed by the NanoDrop absorption spectra, agarose gel migration, amplification in PCR and the Sanger sequencing. The in-house kit successfully extracted clean DNA from all blood samples, and discernably outperformed the commercial kits and the original salting-out procedure in the sense of the simplicity, cost-efficiency, quantity, and the quality of purified DNA. Apart from replacing proteinase K and the sodium dodecyl sulfate treatment by the laundry detergent, our protocol instructs a lysis buffer that eliminates sucrose, Triton X-100, MgCl2, NH4Cl, and KCl. Our handmade kit might be of interest for laboratories in underdeveloped countries with a budget shortage or applications in difficult field conditions, for example, when fridge storage for proteinase K cannot be ensured.

A low-cost efficient multiplex PCR for prenatal sex determination in bovine fetus using free fetal DNA in maternal plasma.

BACKGROUND: In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction (PCR) analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. MATERIALS AND METHODS: In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 µl maternal plasma. Two primer pairs for bovine amelogenin gene (bAML) and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. RESULTS: The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. CONCLUSION: The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses.