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Technical Briefs |
1 Department of Medicine, Division of Nephrology, University Clinic, Würzburg, Germany
2 Center for Cardiovascular Research/Institute of Pharmacology and Toxicology, University Clinic Charité, Berlin, Germany
3 Division of Nephrology and Hypertension, Inselspital, University of Berne, Berne, Switzerland
4 Department of Obstetrics and Gynecology, University Clinic Charité, Berlin, Germany
5 Department of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Würzburg, Germany
aaddress correspondence to this author at: Division of Nephrology, Department of Medicine, University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany; fax 49-931-201-36502, e-mail tom.lindner{at}mail.uni-wuerzburg.de
Complex disorders such as type 2 diabetes or coronary heart disease are of major public interest because they affect millions of individuals. To unravel the genetic background of those traits, genome-wide linkage scans with microsatellite markers have been performed, followed by association studies of single-nucleotide polymorphisms (SNPs) in the identified candidate regions. High-throughput SNP typing technologies such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (1), pyrosequencing (2), TaqMan-based allelic discrimination (3), and others (4)(5)(6)(7)(8) can be used for that approach. However, many smaller and medium-throughput laboratories have no access to rapid, reliable, universal, and cost-effective SNP typing methods. The establishment of such a technique therefore has a high priority.
We have developed a simple and inexpensive two-step method for SNP detection (PCR followed by modified restriction digestion), called double restriction mutagenesis primer PCR (DRMP-PCR). Our approach is based on the introduction of two restriction sites in one of the two PCR primers (mutagenesis reverse primer). This method appears to represent an improvement over older techniques such as PCR–primer-introduced restriction analysis (PIRA) (9) with respect to both specificity and reliability. In addition, we have generated a PERL-based computer program that provides the restriction enzymes and necessary mutagenesis sequences for primer design.
Our method allows detection of SNPs by use of fluorescence scanning sequencers and automated automatic allele calling. The forward primer must therefore have a 5' fluorescence label (hexachloro-6-carboxyfluorescein, 6-carboxyfluorescein, or others) but does not undergo any additional changes. The 3' end of the reverse primer is positioned next to the SNP and forms a restriction site together with one of the two SNP alleles (Fig. 1A
). In most cases, one or two bases of the 3' end of the primer must be changed to generate a restriction site. For prevention of mistyping events and evaluation of the completeness of cleavage, we introduced an additional identical restriction site near the 5' end of the reverse primer (5' control restriction site). These artificial restriction sites should be generated with as few sequence changes as possible.
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The restriction enzyme always recognizes the 5' control restriction site; thus, the entire product will be cut at least once. However, only one allele of the SNP undergoes an additional restriction at the 3' SNP restriction site. A third band would indicate undigested PCR product and can be easily recognized by its larger size (Fig. 1
, A and B, in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue12/).
With the exception of two (ATAT and TATA), all possible 4-bp palindromes are cleavable by restriction enzymes, can be generated by one or two nucleotide substitutions, and can be designed in virtually all sequences. These properties make our method universally applicable. Our computer program, DRMP_V0.9.pl, aids in primer design. It is freely available, and detailed information is given on our website (http://www.uniwuerzburg.de/nephrologie/molecular_genetics/molecular_genetics.htm).
To evaluate our method, we tested two SNPs within the calpain-10 gene (UCSNP43/63) in 625 extensively described (10) type 2 diabetic patients on hemodialysis and a control group of 211 randomly selected nondiabetic blood donors. In addition, we investigated three SNPs located within the mineralocorticoid receptor gene [(hMR SNPs): hMRcod1 (NCBI refSNP ID: rs5522), hMRcod4 (refSNP ID: rs5525), and hMR3UT2 (refSNP ID: rs5534)] in 149 patients with gestational hypertension and 157 women with normal blood pressure during pregnancy. The women delivered at the Department of Obstetrics, Charité Mitte, Humboldt University, Berlin, Germany.
The primer sequences are shown in Table 1
of the online Data Supplemental. PCR was performed in a 10-µL reaction volume with 5 ng of genomic DNA and forward and reverse primers at 250 and 500 nM, respectively. We performed 12 initial touchdown PCR cycles (annealing temperatures between 56 and 66 °C; 
T, –0.5 °C/cycle) followed by 32 PCR cycles at 50–60 °C. We added 1 µL of the PCR products to 5 µL of 1x restriction buffer containing 1 U of restriction enzyme: for UCSNP43, RsaI, Y+/TangoTM; for UCSNP63, Hin6I, Y+/Tango; for hMRcod1, HpaII, Y+/Tango; for hMRcod4, TaiI, R+; for hMR3UT2, Csp6I, Y+/Tango (buffers and enzymes from MBI Fermentas). The reaction was incubated for 6 h at 37 °C. Restriction products of UCSNPs and the hMR SNPs were pooled, and a 6-carboxytetramethylrhodamine-labeled size calibrator was added. PCR products were separated on an ABI 377 DNA sequencer. Genotypes were analyzed with Genotyper® software, Ver. 3.7 (Applied Biosystems). The separation of digested PCR products on a 3.5% agarose gel is shown in Fig. 2 of the online Data Supplement. We established a universal restriction protocol (5 µL of 1x restriction buffer containing 1 U of restriction enzyme and 1.0 µL of PCR product) using the following restriction enzymes: TasI, TaiI, Hsp92II, MspI/HpaII, Bsh1236I, Bsp143I, Hin6I, and Csp6I/RsaI.
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PCR amplification was stable and specific despite the introduction of up to three mismatches in the mutagenesis reverse primers. Even the introduction of two mismatches near the 3' end of the reverse UCSNP43 primer (Fig. 1B
) did not affect amplification. The allele detection rate was >97% (<3% PCR blanks). The ratio between undigested PCR product and allele-specific fragments was <1:10. For demonstration purposes, Fig. 1A in the online Data Supplement shows completely cleaved PCR products, whereas Fig. 1B
shows incomplete digestion.
We validated our method by sequencing each of the five SNPs in 15 randomly selected individuals (5 homozygous for each allele and 5 heterozygous; total of 150 control chromosomes). In addition, the genotypes for UCSNP43 obtained by sequencing in a second control group of 42 individuals from the calpain-10 study (11) were reanalyzed by DRMP-PCR (24 with GG, 14 with GA, and 4 with AA genotypes). We found no differences between the genotype frequencies obtained by DRMP-PCR and sequencing.
DRMP-PCR provides several advantages over conventional restriction applications for low- to medium-throughput SNP genotyping. SNPs can be pooled/multiplexed and analyzed on fluorescence scanning sequencers, enabling automatic allele detection. Mistyping is prevented by an additional control restriction site. Restriction enzymes covering all cleavable 4-bp palindromes can be used. Our computer program aids in primer design and selection of the enzyme.
The introduction of a 3' SNP restriction site represents the PCR-PIRA methodology. Only one of the two SNP alleles forms a full recognition sequence together with the 3' SNP restriction site and will be cleaved. However, in PCR-PIRA, discrimination between a true heterozygote and an incompletely digested PCR product is impossible. The novelty of our method is the introduction of the artificial 5' control restriction site, which monitors the completeness of digestion and thus detects potential mistyping. We recommend generating the 5' control restriction site in the middle of the reverse primer because this position favors good separation of allele-specific fragments and at the same time ensures complete digestion at both restriction sites. We propose using the restriction enzymes with a 4-bp palindromic recognition sequence. The sequence is easy to generate, the enzymes are inexpensive, and only a few changes to the primer sequence will usually be required. For optimum annealing conditions, we propose a length of 30–35 nucleotides for the mutagenesis reverse primer. The labeled forward primer contributes mostly to the specificity of the PCR.
Limitations of our method include possible incompatibilities of the sequence near the SNP with the primer design. Additional SNPs within the potential primer-binding site could lead to allelic drop-out. PCR products must not contain additional restriction sites for the enzymes used (information provided by our program DRMP_V0.9.pl). Finally, some restriction enzymes might be incompatible with the PCR buffer or not active close to the ends of the products.
The manual workflow time for a 96-well plate using electronic single- and multichannel pipettes was 80 min (20-min PCR setup, 15-min restriction setup, 45-min ABI 377 Sequencer setup). The use of pipetting robots with needles, capillary sequencers, and multiplex panels and a further reduction in total PCR reaction volume might substantially reduce the costs. One SNP in 2500 samples would cost approximately US $0.30/genotype (Table 1
). DRMP-PCR provides fast and reliable SNP genotyping at a price comparable to or lower than other techniques (12).
Acknowledgments
T.H.L. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; Li768/3-1, Li768/3-3, Li768/4-1, and Li768/4-2). M.B. was supported by a grant from the Interdisziplinäres Zentrum für Klinische Forschung Würzburg IZKF-E9.
References
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