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Technical Briefs |
Departments of
1
Clinical Chemistry and
2
Haematology, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands
aauthor for correspondence: fax 31-43-3874692, e-mail jdvr{at}klinchem.azm.nl
Current methods for PCR use purified genomic DNA, isolated mostly from leukocytes. To avoid time-consuming, and therefore expensive, procedures to purify genomic DNA, methods have been developed that use whole blood or buffy coats for PCR (1)(2)(3)(4)(5). However, extensive washing procedures become necessary to remove hemoglobin and anticoagulants, which inhibit PCR (3)(6)(7). In addition, heat-cool cycles have to be included to liberate DNA from the nucleus (1)(5). Furthermore, PCR protocols are optimal for genomic DNA, but the salt composition of the PCR mixture needs to be readjusted if PCR is performed with whole blood or buffy coats (5)(8). Finally, digestion of PCR products with restriction enzymes may be severely inhibited by anticoagulants (9)(10).
We developed a simplified method to overcome these drawbacks. Briefly, red blood cells are first lysed in a large volume, which dilutes the hemoglobin and anticoagulants to an extent that PCR and post-PCR treatments are not inhibited. The white blood cells are then resuspended in a Tris-EDTA (0.67 mmol/L Tris-HCl, 0.07 mmol/L EDTA, pH 7.5) buffer commonly used to store genomic DNA; this eliminates readjustment of the salt composition of the PCR mixture. Finally, because one freeze-thaw cycle disrupts white blood cells into cellular fragments more efficiently than several heat-cool cycles, storage at -20 °C of white blood cells resuspended in Tris-EDTA should yield, after thawing, a cell lysate suitable for PCR. This assumption was tested using cell lysates for apolipoprotein E (APOE) genotyping in a PCR protocol optimized for genomic DNA.
Anonymized cell lysates were prepared from whole-blood samples collected in Vacutainer Tubes (Becton Dickinson) by venipuncture. One set of samples was collected from 24 consecutive different patients referred for routine clinical chemistry laboratory investigation; these samples included 12 sodium heparinate- and 12 sodium fluoride-potassium oxalate-anticoagulated whole-blood samples. Samples were also collected from 24 consecutive different patients referred for routine hematologic laboratory investigation; these samples included 12 sodium citrate- and 12 tripotassium EDTA-anticoagulated whole-blood samples.
A volume of 200 µL of whole blood was diluted in 10 mL of ice-cold red blood cell lysis buffer (155 mmol/L NH4Cl, 10 mmol/L KHCO3, 1 mmol/L EDTA, pH 7.4). After 5 min at 20 °C, the solution was centrifuged for 3 min (750g at 20 °C in a Hettich Rotanta P centrifuge). The supernatant was removed, and the pellet was resuspended in 200 µL of Tris-EDTA and stored at -20 °C.
APOE genotyping was performed with the primer set F6/F4 (Amersham/Pharmacia Biotech Inc.), which amplifies part of exon 4 of the APOE gene, including the coding sequence for amino acid residues 112 and 158 (11). The PCR mixture contained 67 mM Tris-HCl (pH 8.4), 17 mM (NH4)2SO4, 6.7 mM MgCl2, 0.1 g/L gelatin, 1 g/L Triton X-100, 0.4 µM F6/F4, 80 µM PCR-grade dNTPs (Roche Molecular Biochemicals), 2 µL of dimethyl sulfoxide (Merck), and 1 µL of thawed cell lysate. PCR was performed with a PTC-200 DNA Engine thermocycler (MJ Research). The PCR protocol included the following steps: (a) denaturation for 5 min at 96 °C; (b) addition of 0.7 U of Taq polymerase (Amersham/Pharmacia Biotech Inc.) at 72 °C, taking the volume of the PCR mixture to 25 µL; (c) denaturation for 1 min at 92 °C; (d) 30 cycles of 65 °C for 1 min, 72 °C for 1.5 min, and 92 °C for 1 min; (e) extension for 5 min at 72 °C; and finally (f) fast cooling to 20 °C.
The restriction enzyme HhaI (5 U; Amersham/Pharmacia Biotech Inc.) was added to 9.5 µL of PCR mixture and incubated for 2 h at 37 °C. Gel-loading buffer (2.5 µL containing 400 g/L sucrose with 2 g/L orange G) was then added, and 10 µL of this mixture was loaded on a 3% MS-agarose gel (Roche Molecular Biochemicals) containing ethidium bromide (0.3 mg/L). After 30 min of electrophoresis (10.7 V/cm), the gel was photographed. The pattern of restriction fragments obtained depends on the presence of either Arg or Cys at positions 112 and 158, respectively and determines the APOE genotype (12).
All 48 cell lysates subjected to this PCR protocol for APOE yielded one specific PCR product with the expected size of 244 bp (Fig. 1A
). PCR with 4 µL of cell lysate rather than 1 µL only marginally affected the yield of PCR products, whereas 35 cycles instead of 30 cycles increased the amount of PCR products (Fig. 1A
). PCR products were readily digested with HhaI, and the APOE genotypes could be discriminated easily (Fig. 1B
). Thus, PCR- and restriction enzyme-inhibiting substances were effectively removed, and one freeze-thaw cycle was sufficient to obtain cell lysates suitable for PCR.
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We next tested three other primer sets that amplified exon 10 of the coagulation factor V and exons 29 and 35 of the type IV collagen 
5-chain gene. The protocol differed from the APOE PCR protocol as follows: for exon 10 of the coagulation factor V gene (13), the annealing temperature was 55.0 °C, whereas for exons 29 and 35 of the type IV collagen 5-chain gene, the PCR mixture contained 10 mM ß-mercaptoethanol instead of 0.1 g/L gelatin and 1 g/L Triton X-100 and the annealing temperatures were 55.5 and 55.0 °C, respectively (Dr. B. Smeets, personal communication). All cell lysates subjected to these modified APOE PCR protocols yielded specific PCR products (Fig. 1C
). Hence, it seemed likely that cell lysates could substitute for purified genomic DNA in PCR protocols without reoptimization.
We analyzed the feasibility of long-term storage of cell lysates at -20 °C by pipetting 1-µL aliquots of cell lysates into PCR reaction vessels directly after preparation. After 6 months, cell lysates were subjected to the APOE PCR protocol. All cell lysates except one yielded specific PCR products (Fig. 1D
). Prior aliquoting would be necessary for long-term storage if additional freeze-thaw cycles diminished the quality of the cell lysates for PCR. Hence, we subjected 50-µL aliquots from the cell lysates to six freeze-thaw cycles, followed by PCR with the APOE protocol. All cell lysates yielded specific PCR products, although the amount of PCR product from 4 of 48 cell lysates was decreased. Repeated PCR on these four cell lysates with the APOE PCR protocol after careful homogenization produced two high and two low yields of PCR product. These results indicate that long-term storage of cell lysates is possible and that aliquoting is not necessary.
Finally, we investigated whether cell lysates were as reliable as genomic DNA for APOE genotyping. No genomic DNA was available from the cell lysates collected as described above; we therefore prepared cell lysates and isolated genomic DNA from tripotassium EDTA-anticoagulated whole blood from 48 consecutive patients referred to the clinical chemistry department for APOE genotyping. We found that the APOE genotypes determined with cell lysates completely matched the APOE genotypes established with genomic DNA. Thus, PCR with cell lysates generated PCR products identical to the PCR products obtained with genomic DNA.
In conclusion, cell lysates prepared as described can be used as an alternative for purified genomic DNA in PCR using PCR protocols designed for genomic DNA. The preparation of cell lysates is fast and based on simple concepts, and long-term storage does not affect the quality of cell lysates for PCR. This method opens up the possibility of using every type of anticoagulated whole-blood sample as a starting source for PCR, circumventing the need to isolate and purify genomic DNA.
References
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