The identification of thousands of single nucleotide polymorphisms

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1 Utilizing Genomic DNA Purified From Clotted Blood Samples for Single Nucleotide Polymorphism Genotyping Karissa K. Adkins, PhD; Daniel A. Strom, MS; Thomas E. Jacobson, MBA, CQE; Cara R. Seemann, BS; Darin P. O Brien, PhD; Ellen M. Heath, PhD Context. Linking single nucleotide polymorphisms to disease etiology is expected to result in a substantial increase in the number of genetic tests available and performed at clinical laboratories. Whole blood serves as the most common DNA source for these tests. Because the number of blood samples rises with the number of genetic tests performed, alternative DNA sources will become important. One such alternative source is clotted blood, a byproduct of serum extraction. Efficiently using an already procured blood sample would limit the overall number of samples processed by clinical laboratories. Objective. To determine if DNA purified from clotted blood can be effectively used for single nucleotide polymorphism genotyping. Design. DNA was purified from the clotted blood of 15 donors. Single nucleotide polymorphism genotyping for the methylenetetrahydrofolate reductase and factor V Leiden mutations was performed with each DNA sample by 2 independent methods. Results. High-quality DNA was obtained from each of the 15 individual clotted blood samples as demonstrated by UV spectrophotometric analysis, gel electrophoresis, and polymerase chain reaction amplification. The DNA was used successfully to obtain genotype data from both the methylenetetrahydrofolate reductase and factor V single nucleotide polymorphism assays for all samples tested. Conclusions. Clotted blood is a clinically abundant sample type that can be used as a source of high-quality DNA for single nucleotide polymorphism genotyping. (Arch Pathol Lab Med. 2002;126: ) The identification of thousands of single nucleotide polymorphisms (SNPs) in the human genome has prompted a rise in population-based research studies designed to link gene-specific SNPs to complex disease states, pharmacogenetic applications, and individual predisposition and susceptibility to diseases. 1,2 For example, SNPs have been associated with increased risk for vascular disease. These risk factors include single-base mutations in the factor V Leiden (G1691A), prothrombin (G20210A), and methylenetetrahydrofolate reductase (MTHFR, C677T) genes. 3 5 Many molecular diagnostic laboratories have adopted testing for these genetic risk factors, with whole blood being the most common source for DNA. As more SNPs are identified as genetic risk factors, a rise in the number of genetic tests will occur, bringing about a concurrent rise in the number of blood samples entering clinical laboratories for processing. One way of alleviating this resource stress is to use collected blood samples more efficiently. This article demonstrates the feasibility of using clotted blood as a DNA source for SNP genotyping. In clinical laboratories, Accepted for publication October 18, From Gentra Systems Inc, Minneapolis, Minn. Presented at the 10th Annual William Beaumont Annual Seminar on Molecular Pathology, DNA Technology in the Clinical Laboratory, Royal Oak, Mich, March 9, All authors are employees of Gentra Systems Inc and as such have a financial interest in the products described in the manuscript. Reprints: Ellen M. Heath, PhD, Gentra Systems Inc, th Ave N, Suite 120, Minneapolis, MN ( eheath@gentra.com). blood clots are normally discarded from patient blood samples collected for serologic and viral testing, following removal of serum. The use of these blood specimens for genetic testing will help limit the number of blood samples processed by clinical laboratories, provide a valuable alternative source for DNA, and reduce the number of samples required from the patient. This would save time and resources. MATERIALS AND METHODS Sample Collection Whole blood from 15 individuals was collected in four 5-mL clot-activating tubes (catalog No ) and one 10-mL K 3 EDTA tube (catalog No ) available from Becton Dickinson (Franklin Lakes, NJ). Blood clots were allowed to form by incubating the clot-activating tubes at room temperature for 60 minutes. Serum was separated from the clots by centrifugation for 10 minutes at 1300g and removed with a disposable pipette. Each clot was transferred to a 50-mL, polypropylene, conical centrifuge tube and stored at 80 C until use. A white blood cell count was obtained from blood collected in the EDTA tubes using a CBC5 Coulter Counter (Beckman Coulter, Fullerton, Calif). An informed consent form for genetic testing was obtained from each donor. Genomic DNA Purification Genomic DNA was purified using commercially available CLOTSPIN tubes (catalog No. CS-0050, Gentra Systems Inc, Minneapolis, Minn) and the PUREGENE DNA purification kit (catalog No. D-5000, Gentra Systems). Each frozen clot was thawed rapidly at 37 C and placed on ice. The thawed clots were transferred 266 Arch Pathol Lab Med Vol 126, March 2002 Single Nucleotide Polymorphism Genotyping Adkins et al

2 Figure 1. Gel electrophoresis of genomic DNA purified from clotted blood. DNA was electrophoresed on a 0.7% agarose gel. Lanes 1 and 17 contain 200 ng of HindIII-digested lambda DNA. Lanes 2 through 16 contain 100 ng of genomic DNA from each individual blood clot. was vortexed for 10 seconds to resuspend the white blood cell pellet. Following resuspension, 5 ml of cell lysis solution (Gentra Systems) and 25 L of proteinase K (20 mg/ml) (Gentra Systems) were added to each tube to initiate protein digestion. The tube was then placed in a 55 C water bath for 30 minutes or until clumps of cellular material were no longer visible. To facilitate the disruption of the material, each tube was vortexed at high speed 3 times for 10 seconds at regular intervals during the incubation. After incubation, each tube was cooled on ice for 5 minutes, and 1.67 ml of protein precipitation solution (Gentra Systems) was added. Each sample was vortexed at high speed for 20 seconds and then centrifuged at 2000g for 10 minutes. After centrifugation, each tube was incubated on ice for 2 minutes to stabilize the protein pellet. The supernatant was then transferred to a tube containing 5 ml of 100% isopropanol and 9 L of a DNA carrier (glycogen solution, 20 mg/ml; Gentra Systems). The tube was inverted 50 times to mix reagents and centrifuged for 3 minutes at 2000g. The resulting DNA pellet was rinsed with 5 ml of 70% ethanol and centrifuged for 1 minute at 2000g. The 70% ethanol was decanted and the tube inverted on clean absorbent paper for 10 seconds. DNA was allowed to hydrate overnight in 500 L of DNA hydration solution (Gentra Systems) with gentle rocking at room temperature. Figure 2. Polymerase chain reaction amplification of 3 target loci using genomic DNA purified from clotted blood. Three loci were amplified from each DNA sample (lanes 1 to 15) using gene-specific primers and then visualized using gel electrophoresis. The DNA size reference flanking the amplified samples is 250 ng of a 100 base pair DNA ladder (Invitrogen, Carlsbad, Calif). A, Factor V; B, MTHFR;C,CYP2D6. to CLOTSPIN tubes that contain specially designed perforated inserts and centrifuged at 2000g for 5 minutes to disperse the clots. Following clot disruption, 15 ml of red blood cell (RBC) lysis solution (Gentra Systems) was added to the CLOTSPIN insert to rinse the residual clot material. After the RBC rinse, all clot material retained on the insert was transferred to the filtrate using a disposable micropipette tip. The tube was vortexed for 3 seconds to mix and placed on a rotator for 5 minutes to lyse contaminating RBCs. After rotation, the tube was vortexed for 3 seconds and centrifuged for 5 minutes at 2000g. The supernatant was poured off, and a second RBC lysis step was performed with 5 ml of RBC lysis solution to further remove RBC contaminants. Following centrifugation, the supernatant was poured off, leaving behind approximately 100 L of RBC lysis solution. Each tube DNA Yield Analysis DNA concentration was determined using a Beckman DU-64 UV spectrophotometer (Beckman Coulter) after a 1:20 dilution in ultrapure water. To calculate the DNA concentration, the A 320 reading (background) was subtracted from the A 260 reading, and the resulting number multiplied by the DNA extinction coefficient of 50 g/ml. DNA yield was calculated by multiplying the hydration volume and the DNA concentration for each sample. Percent yield was calculated by dividing the observed DNA yield by the theoretical maximum yield. The maximum yield was determined using the following formula: [volume of blood] [white blood cell count] [estimated quantity of DNA in a human diploid cell ( pg)]. The A 260 /A 280 ratio was calculated by subtracting the A 320 value from each reading before division. DNA Quality Analysis The quality of the purified DNA was assessed by using gel electrophoresis to size separate 100 ng of each sample on a 0.7% agarose gel. The DNA was electrophoresed for 1 hour at 80 V with g/ml of ethidium bromide added to the gel and running buffer. The gel was photographed using a Kodak Digital Imaging System EDAS 120 LE (Rochester, NY) photodocumentation system. Polymerase Chain Reaction Amplification and Analysis DNA quality was further evaluated by amplification of 3 target loci: factor V with an amplicon size of 267 base pairs (bp), MTHFR with an amplicon size of 198 bp, and CYP2D6 with an amplicon size of 1501 bp. In all cases, a template quantity of 100 ng was used for each polymerase chain reaction (PCR). Unless otherwise noted, amplification reactions were in a 25- L volume using a master mix containing 1 Taq polymerase buffer (Promega, Madison, Wis), 0.05 U/ L of Taq polymerase (Promega), 0.2 mmol/l deoxynucleotide triphosphate mix (Promega), 1.5 mmol/l MgCl 2 (Promega), and 1 mol/l each of forward and reverse primers (synthesized by Research Genetics, Huntington, Ala). Amplification of the factor V locus was performed using the primers detailed by Ridker et al. 6 The PCR conditions were as follows: 94 C for 5 minutes; 30 cycles of 94 C for 30 seconds, 58 C for 30 seconds, and 72 C for 30 seconds; and 72 C for 7 minutes. Amplification of the MTHFR locus was performed with the primers described by Frosst et al. 5 The PCR conditions for MTHFR were identical to factor V except for an increase in the annealing temperature from 58 C to65 C. Amplification of the cytochrome P450 2D6 (CYP2D6) locus was performed with primers described by Stüven et al. 7 The reaction mix for CYP2D6 was composed of 0.05 U/ L of Taq polymerase (Promega) and Arch Pathol Lab Med Vol 126, March 2002 Single Nucleotide Polymorphism Genotyping Adkins et al 267

3 Figure 3. Single nucleotide polymorphism (SNP) genotype analysis of genomic DNA purified from clotted blood. DNA samples from 15 individuals were genotyped for SNPs located in the factor V (A) and MTHFR (B) loci using the Invader assay. The graphs show the ratio of the wild-type fluorescent signal to the mutant fluorescent signal, corrected for background fluorescence and plotted on a logarithmic scale. Ratios were called wild type if they were 5, heterozygous if they were 3.0 but 0.3, and mutant if they were 0.2. Ratios 5.0 but 3.0 or 0.3 but 0.2 were considered equivocal. 1 FailSafe PCR PreMix E (Epicentre, Madison, Wis). The PCR conditions were as follows: 94 C for 2 minutes; 30 cycles of 94 C for 30 seconds, 63 C for 30 seconds, and 72 C for 2 minutes; and 72 C for 6 minutes. All amplifications were analyzed using 2% agarose gel electrophoresis as described herein. SNP Genotyping The SNP genotyping was performed with the Invader SNP detection kits for factor V and MTHFR (MTHFR catalog No , factor V catalog No ; Third Wave Technologies, Madison, Wis) according to the manufacturer s instructions. For each reaction, 200 ng of DNA was added to the prepared reaction mix. The reactions were carried out in a PE9700 thermal cycler (Applied Biosystems, Foster City, Calif), according to the instructions provided by Third Wave Technologies for Direct Read analysis. Fluorescence generated by the Invader reactions was detected using a CytoFluor Series 4000 fluorimeter (Applied Biosystems). The genotype analysis was performed using software provided by Third Wave Technologies. In all cases, the samples were blinded and randomized to maintain anonymity of the donors. Restriction Fragment Length Polymorphism Analysis Restriction fragment length polymorphism (RFLP) analysis was performed using the PCR amplicons generated from the factor V and MTHFR loci. The 267-bp amplification product from the factor V locus was digested with MnlI (New England Biolabs, Beverly, Mass) at 37 C for 24 hours. The digest mix consisted of 5UofMnlI, 1 NEB restriction enzyme buffer 2 (New England Biolabs), and 100 g/ml of bovine serum albumin (New England Biolabs). The 198-bp amplification product from the MTHFR locus was digested with HinfI (Life Technologies, Grand Island, NY) at 37 C for 24 hours. The digest mix consisted of 5 U of HinfI and 1 React 2 Buffer (Life Technologies). The products of both digestion reactions were analyzed by gel electrophoresis on a 3% Metaphor agarose (FMC, Rockland, Me) gel for 2 hours at 50 V and then 15 minutes at 80 V, with g/ml of ethidium bromide added to both the gel and running buffer. RESULTS The quality of the DNA purified from clotted blood using the CLOTSPIN tube in combination with the PUREGENE 268 Arch Pathol Lab Med Vol 126, March 2002 Single Nucleotide Polymorphism Genotyping Adkins et al

4 Figure 4. Genotype identification of amplified DNA using restriction fragment length polymorphism. A, Amplification products for the factor V loci (Figure 2, A) were digested with MnlI to determine genotype. A wild-type genotype ( / ) was identified if following digestion 3 bands of 163, 67, and 37 base pairs (bp) were observed. A mutant genotype ( / ) was determined if the digestion resulted in 2 bands of 200 and 67 bp. The heterozygous genotype ( ) showed bands of each size found in the mutant and wild-type digestions. B, Amplification products for the MTHFR loci (Figure 2, B) were digested with HinfI to determine genotype. A wild-type genotype was identified if the original amplification band of 198 bp was still present following digestion. A mutant call genotype was determined if a single band of 175 bp was present following digestion. The presence of both bands indicated a heterozygous genotype. The DNA samples tested in lanes 1 to 15 are flanked by 1 g of a 50-bp DNA ladder (Promega, Madison, Wis). DNA purification kit and used for SNP genotyping was evaluated by 3 methods. First, spectrophotometric analysis was used to determine DNA yields and to assess purity of the purified DNA. As shown in the Table, average DNA yields for the 4 tubes of clotted blood taken from each individual ranged from 31 to 158 g of DNA, varying significantly by individual. The percent yield, determined using the white blood cell count, as described in the Materials and Methods section, also varied among individuals, ranging from 20% to 60%. The A 260 /A 280 ratios obtained ranged from 1.77 to 1.88, indicating high-purity DNA. Second, gel electrophoresis showed high-molecularweight DNA without significant degradation (Figure 1). Finally, 3 target loci were amplified by PCR to demonstrate integrity and purity of the DNA. Figure 2 shows the expected amplification products from the factor V, MTHFR, and CYP2D6 loci, detected in all 15 DNA samples. The SNP genotyping was performed with DNA purified from 1 of the 4 replicate samples collected from each individual donor. Each sample was tested for both factor V and MTHFR mutations. The SNP detection results are reported as a ratio of wild type to mutant fluorescent signal, corrected for background fluorescence. Genotype calls are based on these ratios, with the requirement that both the mutant and wild-type fluorescent signals be 20% above the signal of a no-target control. The results of the factor V genotyping, shown in Figure 3, A, demonstrate that 14 wild-type genotypes and 1 heterozygous genotype were identified in the samples tested. Results of the MTHFR genotyping, shown in Figure 3, B, demonstrate that 10 wild-type, 3 heterozygous, and 2 mutant genotypes were identified in the clotted blood samples tested. In addition to the Invader assay, SNP genotypes were determined using RFLP analysis. The RFLP genotyping was performed with 1 of the 4 replicate DNA samples collected from each donor. Genotype results were consistent with those obtained with the fluorescent SNP assay shown in Figure 3. As shown in Figure 4, A, lane 3, one sample amplified for factor V gave a digestion pattern consistent with a heterozygous genotype, whereas all other samples had only the 3 bands expected of a wild-type genotype. Figure 4, B, shows the results obtained from HinfI digestion of the 198-bp MTHFR amplification prod- DNA Yield From 15 Individuals (4 Samples for Each Donor) DNA Yield, g % Yield A 260 /A 280 Donor No Arch Pathol Lab Med Vol 126, March 2002 Single Nucleotide Polymorphism Genotyping Adkins et al 269

5 ucts. In lanes 1 and 2, the smaller band (175 bp) indicates a mutant genotype, and the double bands seen in lanes 3, 11, and 15 indicate a heterozygous genotype. All of the other samples showed the original 198-bp amplification product (Figure 4, B) following digestion, indicating a wild-type genotype. CONCLUSIONS The results of this study demonstrate that DNA of sufficient integrity to perform SNP genotyping can be purified from clotted blood. The DNA yields obtained using the CLOTSPIN tube in combination with the PUREGENE DNA purification kit varied considerably among individuals, and it is suggested that this variation in yield is due to individual differences in clot formation. However, DNA quality was consistently high in each sample as indicated by gel electrophoresis, calculated A 260 /A 280 ratios, and PCR amplification results. Furthermore, all samples gave identical and definitive genotype identification with 2 independent assays, Invader and RFLP, as shown in Figures 3 and 4. Both of these assays are commonly used diagnostic assays in clinical laboratories. The results of this study add support to using clotted blood, a currently underused by-product of clinical testing, for genetic analysis. We are grateful to Melissa Dols for technical assistance and Nicole Osterhaus for drawing blood for this study. References 1. Schork NJ, Fallin D, Lanchbury JS. Single nucleotide polymorphisms and the future of genetic epidemiology. Clin Genet. 2000;58: Riley JH, Allan CJ, Lai E, Roses A. The use of single nucleotide polymorphisms in the isolation of common disease genes. Pharmacogenomics. 2000;1: Bertina RM, Koeleman BPC, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369: Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3 -untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88: Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10: Ridker PM, Hennekens CH, Lindpaintner K, et al. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl J Med. 1995;332: Stüven T, Griese EU, Kroemer HK, et al. Rapid detection of the CYP2D6 null alleles by long distance- and multiplex-polymerase chain reaction. Pharmacogenetics. 1996;6: Arch Pathol Lab Med Vol 126, March 2002 Single Nucleotide Polymorphism Genotyping Adkins et al