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EPICENTRE Forum 4 (1) Rapid Purification of Genomic DNA from Buffy Coat Using the MasterPure™ Genomic DNA Purification Kit Karolyn J. McMaster and Linda Sabatini, University of Wisconsin Clinical Science Center, Department of Pathology and Laboratory Medicine Introduction In our clinical molecular diagnostic laboratory where a multitude of patient samples are processed, a rapid DNA purification system that consistently provides a good yield of high quality genomic DNA is critical. In our laboratory, the preferred method of processing and storing peripheral whole blood samples is to isolate the buffy coat (white blood cell layer) into aliquots in microcentrifuge tubes, then store the tubes at -70°C until the particular assay for that sample is performed. For some assays, the samples may need to be stored for up to two months before DNA is isolated from the frozen buffy coat. Southern-based assays (eg., Fragile X mutational analysis) require a yield of over 30 µg of high quality genomic DNA. Using the MasterPure™ Genomic DNA Purification Kit (Epicentre Technologies), we report that genomic DNA can be quickly isolated from frozen buffy coats that have been stored for up to three months at -70°C. This isolated genomic DNA is of a quality sufficient for PCR amplification and restriction endonuclease digestion. Methods and Results DNA Purification The MasterPure Genomic DNA Purification Kit was used to process DNA from eight archived buffy coat samples. Five milliliters of whole blood were routinely collected into sodium citrate tubes and the white blood cells were isolated by centrifugation. The white blood cell layer (buffy coat; 500 - 600 µl) was transferred to a 1.5 ml microcentrifuge tube, mixed, and 200 µl were removed into two 1.5 ml microcentrifuge tubes. The buffy coat samples were either used immediately for DNA isolation or stored at -70°C. After storage for approximately three months at -70°C, DNA was purified from two 200 µl samples from each of four donors (A-D). The samples were made anonymous then each sample was processed according to the protocol provided with the kit with the exception that 200 µl of buffy coat were used instead of 300 µl, and the buffy coats were frozen instead of fresh. Processing the eight samples from buffy coat to isolated genomic DNA pellet took approximately 45 minutes. The isolated genomic DNA samples were resuspended to a final volume of 100 µl in TE buffer then solubilized at room temperature for approximately 16 hours. Concentrations and absorbance ratios of OD260/280 were determined by using a Pharmacia Gene Quant® Spectrophotometer (Table 1). The absorbance ratio was satisfactory and the concentration and yield were high enough to test the performance of genomic DNA samples in two of the assays that are routinely performed in our laboratory. The two samples from each patient were combined and the concentration and absorbance ratios were again quantified before the assays were performed.
RFLP and Southern Blot Analysis The DNA samples were then used for Factor V R506Q (FVR506Q) gene mutation analysis by PCR-Restriction Fragment Length Polymorphism (RFLP).1 A point mutation in the Factor V gene at nucleotide position 1691 results in a GtoA base substitution. This mutation generates a restriction fragment length polymorphism (RFLP) for the restriction enzyme Mnl I. The Factor V gene status of the patient can subsequently be determined by direct visualization of digestion fragments after agarose gel electrophoresis. The genomic DNA obtained was amplified using primers specific for a 267 base-pair region spanning nucleotide position 1691 in the Factor V gene. The 267 base-pair amplicon was then digested with the restriction endonuclease Mnl I and the fragments were resolved on a 2.5% NuSieve® 3:1 (FMC) agarose gel (Figure 1). Fragment sizes for a normal Factor V gene generated by Mnl I digestion are: 163 base pairs, 67 base pairs, and 37 base pairs. DNA from a homozygous FVR506Q patient would result in fragments of 200 and 67 base pairs. Heterozygous individuals show a combination of the two patterns. All samples in Figure 1 show the presence of the 163 bp fragment and the absence of the 200 bp fragment, indicating these DNA samples were derived from homozygous normal individuals.
The purified genomic DNA was also used to detect mutations associated with Fragile X syndrome by Southern transfer and hybridization. Fragile X-linked mental retardation is associated with an expansion and hyper-methylation of the CGG repeat region in the FMR 1 gene. Southern blot analysis of digested genomic DNA was used to assess the size and degree of methylation of the CGG repeat region of the FMR 1 gene. The restriction endonuclease Eag I is methylation sensitive and thus will not cut when its recognition sequence has been methylated, as in association with the Fragile X disease or as a normal process of X-chromosome inactivation in females. Purified genomic DNA was digested with EcoR I and Eag I and the fragments resolved in a 1.5% agarose gel. Fragment size was subsequently determined by denaturing and transferring the digested DNA to a nylon membrane, and detected using a 32P-labeled probe. The probe specific for the CGG repeat region of the FMR 1 gene detects a 5.1kilobase band in genomic DNA digested with EcoR I, and detects a 2.8 kilobase band when the genomic DNA is digested with both EcoR I and Eag I.2 Two of the samples shown in Figure 2 (Lanes 3 and 6) contain only the 2.8 kb fragment characteristic of a normal male, while the other two samples (Lanes 4 and 5) contain both the 2.8 kb and 5.1 kb fragments characteristic of a normal female.
Conclusions The MasterPure Genomic DNA Purification Kit consistently produces a reasonable yield of good quality DNA (15-60 µg from 200 µl of buffy coat) that can be used in a wide variety of assays. The protocol proved to be flexible in terms of storage and processing options, and because the purification protocol is rapid, multiple samples can be processed in a short period of time. As a result of this evaluation, we now routinely use the MasterPure Genomic DNA Purification Kit on both frozen and fresh buffy coat samples.
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