Optimized Protocol For DNA Extraction from Human Whole Blood.

Abstract

Background/aims: DNA isolation is the initial process in genetic research. The product is used in many PCR reactions (PCR-RFLP, Real-Time PCR, multiplex PCR). That is why it is important to optimize DNA isolation protocol to obtain a good quality of DNA. Our first attempts at isolation, conducted using Purification Kit, did not result in sufficient concentration (6.414 ng*μL^-1) and purity (A-260/280) of 0.764 of isolated DNA.

Methods: We used twice the recommended amount of tissue and cell lysis solution to get more effective cell lysis. We extend the time of vortexing, centrifugation and incubation at critical steps. We manipulated the speed and temperatures of centrifugation. We used cold iso-propanol to get white strands of DNA faster. When rinsing with ethanol we used cold alcohol. We tested efficiency of two methods of drying of ethanol to achieve optimal DNA pureness. We leave the isolated DNA for 20 minutes to evaporate the ethanol and then resuspend nucleic acid in TE Buffer.

Results: Our modifications resulted in the improvement of isolation efficiency. After optimization we achieved DNA concentration (in range of 50-150 ng*μL^-1) and purity (A 260/280) of 1.735. Similar results for DNA parameters were achieved from the whole blood frozen for 2-3 months (concentration in the range of 125.762 ng*μL^-1, pureness: 1.761) and from blood frozen for 18 months (117.94 ng*μL^-1 and 1.7194, respectively). We performed electrophoresis after each isolation to confirm the effectiveness of optimized procedure. The refinements we used in DNA isolation are more efficient than those recommended in DNA Purification Kits.

Conclusion: Our results confirm that optimized DNA protocol fulfills the conditions of good extraction technique: it is relatively fast and easy to perform yet it guarantees a high reproducibility, specificity and sensitivity. There are also no dangerous or harmful steps. Our paper demonstrates innovative and effective approach. It confirms a high effectiveness of method regardless of duration of sample freezing, as well as introduce important modifications (timing, temperature conditions, drying details, absence of K-proteinase) that make overall procedure more productive and relatively fast.