Rapid, Simple Alkaline Extraction of Human Genomic DNA from Whole Blood, Buccal Epithelial Cells, Semen and Forensic Stains for PCR

Various protocols have been devised for the extraction of genomic DNA from human tissues and forensic stains for polymerase chain reaction (PCR) depending on such treatments as boiling, hydrolysis with proteinases, exposure to ultrasound waves, detergents or alkali (5,7,9–13,15). Alkaline extraction is among the simplest strategies. It is an efficient means for the solubilization of proteins (e.g., membrane proteins) due to the ionization of aspartic, glutamic, cysteic and tyrosine residues (4), and the primary structure of DNA is relatively stable in alkaline solution (2). Alkaline lysis at 65° or 95°C has been devised for extraction of single cells and for viral DNA in human tissues (7,13), but to our knowledge, alkaline extraction of genomic human DNA has not achieved any widespread usage. Here, we show that efficient extraction of DNA from whole blood can be performed with alkaline treatment at room temperature in a time frame as short as 1 min. Alkaline extraction of buccal epithelial cells, and blood and semen stains, can be accomplished in 5–6 min at 75°C, whereas liquid semen requires 30 min at 75°C. Initially, the solubilizing effect of sodium hydroxide on biological material was assessed at different concentrations and temperatures using heat-denatured pellets of whole blood (5 μL of blood were dried at 90°C for 5 min). It was found that 20 μL ≥0.1 M NaOH at ≥70°C completely dissolved the pellet in 5 min, whereas water or 0.02 M NaOH had no effect even after incubation for 24 h. The extracts were neutralized with 0.02 M Tris-HCl, pH 7.5, and PCR showed that genomic DNA was liberated and accessible as a template (results not shown). Based on these results, 0.1 and 0.2 M NaOH were used as solvents throughout. Aliquots (5 μL) of pooled whole blood from 5 individuals were incubated with 20 μL NaOH (0.1 or 0.2 M) at temperatures ranging from ambient to 90°C for 1–60 min. The reaction was stopped by the addition of 180 μL TrisHCl, pH 7.5 (0.02 or 0.04 M, respectively) to bring the pH from 13.0 to 8.5. The quantity of the liberated DNA in the supernatant was assessed by slotblotting and hybridization using the ACES 2.0+ Human DNA Quantitation System (Life Technologies, Gaithersburg, MD, USA). Further, the quantity/quality of the DNA was estimated from the ability to yield PCR products and from the amount of product generated by amplification (28–35 cycles) of 414and 1600-bp genomic segments at the human ACP1 locus (1,3,6). The 1600-bp segment (in intron 3S/exon 4) was amplified using the following primers, 5′-GGGAAAGTCTAGTTGTTAATAGCATG-3′ (sense) and 5′-GATTACCTATTCAAATCTCTG-3′ (antisense), denaturation at 94°C for 30 s, annealing at 65°C for 30 s and extension at 72°C for 3 min. All extractions were carried out in duplicate or more. The extraction of whole blood was complete after incubation with 0.2 M NaOH for 1 min at room temperature; a longer extraction time or higher temperature did not increase the amount of liberated DNA (Figure 1A). The yield was about 65%, assuming 30 ng of nuclear DNA per microliter of whole blood (5), and the liberated DNA was of high molecular weight, as evidenced by the electrophoretic appearance (Figure 2). After a prolonged (>30 min) incubation at 75°C, the amount of PCR product that could be obtained with the extracted DNA as template decreased, indicating that the quality of the DNA was reduced [Figure 1A, lanes 4–6; and lanes 7–10 (blood stains)]. Introduction of a brief washing step (i.e., suspension of the blood in water, followed by centrifugation and before extraction with NaOH) removed 85% of the proteins in the crude blood extracts, as estimated from an elemental analysis of the content of organic oxygen, nitrogen and carbon in crude and washed extracts. Usually, 5 μL of extract were adequate in 50 μL of PCR; however, the volume of crude, unwashed extract could be increased to 50% of the PCR without significantly impairing the reaction. Extracts were stable for at least a month at 4°C as evi-