Higher DNA Yield for Epidemiological Studies: A Better Method for DNA Extraction from Blood Clot.

BACKGROUND: Blood clots can be used to extract DNA, but they are not as widely used as whole blood or buffy coats. This is due not only because of the relatively low DNA yields and quality obtained from blood clots, but also because sampling prior to DNA extraction is more difficult. METHODS: To solve these problems, we compared several clot liquefaction methods, determined the four most feasible methods, and subsequently performed a comparative analysis among them. We compared the yields and optical density ratios of the resulting DNA samples and assessed their integrity using agarose gel electrophoresis, polymerase chain reaction, and next-generation sequencing (NGS). RESULTS: Each of the four methods has advantages and disadvantages. But in general, higher yields of DNA with better quality and integrity were obtained using the high-shear homogenization method than using the other three methods. Additionally, this method is cost-effective and feasible at large operational scales. The DNA yields and A260/280 ratios were optimal and stable, the operation time and labor costs were acceptable, and the success rate of NGS applications was 99.74%. Furthermore, we developed a simple and rapid method for cleaning the homogenizer head to remove residual samples. According to our experimental results, our cleaning method effectively eliminated the risk of cross-contamination caused by the homogenizer head. CONCLUSION: We recommend high-shear homogenization as a superior method for clot liquefaction. We believe that this method is worthy of large-scale application as it can improve the efficiency of DNA extraction from clots, thus reducing labor and economic costs.

Quality Analysis of DNA from Cord Blood Buffy Coat: The Best Neonatal DNA Source for Epidemiological Studies?

BACKGROUND: Umbilical cord blood is an economical and easy to obtain source of high-quality neonatal genomic DNA. However, although large numbers of cord blood samples have been collected, information on the yield and quality of the DNA extracted from cord blood is scarce. Moreover, considerable doubt still exists on the utility of the buffy coat instead of whole blood as a DNA source. METHODS: We compared the sample storage and DNA extraction costs for whole blood, buffy coat, and all-cell pellet. We evaluated three different DNA purification kits and selected the most suitable one to purify 1011 buffy coat samples. We determined the DNA yield and optical density (OD) ratios and analyzed 48 single-nucleotide polymorphisms using time-of-flight mass spectrometry (TOF MS). We also analyzed eight possible preanalytical variables that may correlate with DNA yield or quality. RESULTS: Buffy coat was the most economical and least labor-intensive source for sample storage and DNA extraction. The average yield of genomic DNA from 200 µL of buffy coat sample was 16.01 ± 8.00 µg, which is sufficient for analytic experiments. The mean A260/A280 ratio and the mean A260/A230 ratio were 1.89 ± 0.09 and 1.95 ± 0.66, respectively. More than 99.5% of DNA samples passed the TOF MS test. Only hemolysis showed a strong correlation with OD ratios of DNA, but not with yield. CONCLUSION: Our findings show that cord blood buffy coat yields high-quality DNA in sufficient quantities to meet the requirements of experiments. Buffy coat was also found to be the most economic, efficient, and stable source of genomic DNA.