Protocol for Genomic DNA Preparation from Fresh or Frozen Serum for PCR Amplification

Blood is the primary human tissue for clinical diagnosis. Serum may be stored in banks after diagnosis, and most of the specimens may have available corresponding clinical records. These serum samples can be important sources of genomic DNA for human genetic studies in the identification of disease-causing genes or the study of mutations in prenatal diagnosis (10,16), in the detection of tumor DNA from patients with pancreatic (12), lung (3), colorectal (1) and breast cancer (15). However, DNA isolation from serum is difficult because of the low content of DNA in serum and the small amounts of serum that may be available. Martin et al. (11) first described a procedure to isolate DNA for genotype analysis, and others have developed different methods for serum DNA isolation (6,14). Dixon et al. (4) have tested 13 currently available DNA extraction methods. We have also reported a method for DNA extraction from small amounts of serum (9). However, for DNA analysis on a larger scale, a simple, automation-amenable protocol is required. In the present study, we describe a simple protocol for DNA preparation from serum. It includes only two steps, alkaline lysis and neutralization, with two different solutions in one tube. Blood was collected in a red top Vacutainer (no additives) (Becton Dickinson, Franklin Lakes, NJ, USA) with a 22G1 needle and was allowed to clot at room temperature for at least 2 h. The clot was detached from the wall of the tube by a wooden applicator stick and then kept at 4°C overnight. It was centrifuged at 700× g for 5 min; the serum (supernatant) was collected and used for DNA extraction. For alkaline lysis, 10 μL serum were incubated with 1 μL 0.2 M KOH at 37°C for 20 min. During this treatment, various macromolecules in serum may be denatured or hydrolyzed (i.e., RNA) (7). To identify optimal conditions for the alkaline lysis, we tested different KOH concentrations (0.1 or 0.2 M) at different incubation temperatures (55°C, 45°C or 37°C). Ten microliters of serum were used, and the incubation time was 20 min. To neutralize the solution, 5 μL neutralization buffer (9 parts 1 M Tris, pH 8.3:1 part 2 M HCl) were added to the serum lysate and vortexed briefly. The final DNA solution volume was 16 μL, and 3 μL each condition were used for PCR amplification. In this study, the target DNA seBenchmarks