Genotyping Techniques Research Report Melting Curve Analysis of SNPs (McSNP): A Gel-Free and Inexpensive Approach for SNP Genotyping BioTechniques 30:358-367 (February 2001) J.M. Akey, D. Sosnoski1, INTRODUCTION nostic probe once per PCR cycle (25), E. Parra1, S. Dios1, K. Hiester1, have been applied to SNP genotyping 1 The third-generation genetic map methods (6,10,14). The dsDNA-specif- B. Su, C. Bonilla, L. Jin, and M.D. Shriver1 comprised of single nucleotide poly- ic dye SYBR Green I (Moleclar morphisms (SNPs) is being expeditious- Probes, Eugene, OR, USA) has been University of Texas Houston ly developed (24). SNPs are the most used to analyze the melting curves of Health Science Center, Houston, abundant type of sequence variation in PCR products, which are characterized 1 TX and Penn State University, the human genome and will be useful by a rapid loss of fluorescence as the University Park, PA, USA tools in many diverse applications that temperature is raised through the sam- include disease gene mapping, evolu- ples’ melting temperature (Tm) (17). tion, pharmacogenetics, and forensics Since the Tm is a function of product ABSTRACT (3,11). An impressive SNP resource al- length, sequence composition, and GC ready exists as nearly 300000 have been content, it should be possible to distin- High-throughput methods for assaying deposited into publicly accessible data- guish DNA fragments that differ with DNA variation require two important steps: bases (see http://www.ncbi.nlm.nih.gov/ respect to these parameters by melting (i)discriminating the variation and (ii)de- SNP/, http://snp.cshl.org/, and http://hg- curve analysis (MCA). The general tecting the signal. In this report, we describe base.cgr.ki.se/). However, without paral- principles of MCA were used to devel- a novel SNP genotyping method that we re- lel progress in SNP genotyping technol- op Tm-shift genotyping, an SNP geno- fer to as melting curve analysis of SNPs ogy, their true power and inherent typing assay based on allele-specific (McSNP). McSNP combines a classic ap- benefits will not come to fruition. PCR (6). While this represents an im- proach for discriminating alleles, restriction Novel genotyping methods amen- portant advance in MCA applied to SNP enzyme digestion, with a more recent able to high-throughput analysis should genotyping, it is subject to the inherent method for detecting DNA fragments, melt- ideally be gel-free, robust, inexpensive, limitations of allele-specific PCR, such ing curve analysis. Melting curve analysis is and simple to perform. To this end, as difficulty in reaction optimization. performed by slowly heating DNA frag- these requirements have inspired the In the burgeoning field of SNP geno- ments in the presence of the dsDNA-specific development of a variety of genotyping typing technology, it is easy to forget the fluorescent dye SYBRGreen I. As the sam- assays, including the oligonucleotide elegantly simple and accurate approach ple is heated, fluorescence rapidly decreas- ligation assay (OLA) (12), genetic bit of assaying SNP variation by digesting es when the melting temperature of a partic- analysis (GBA) (15), mass spectro- PCR products with restriction enzymes ular fragment is reached. We show that it is scopy (8), “chip” technology (24), Taq- [i.e., PCR-restriction fragment length possible to determine the composition of Man(14), and dynamic allele specific polymorphism (RFLP) typing]. In this simple mixtures of DNA fragments, such as hybridization (DASH) (10). Although report, we describe how MCA can be those that result from restriction enzyme di- many SNP genotyping methods have applied to classical PCR-RFLP meth- gestions of short PCR products. McSNP is been developed, no single technology ods. Specifically, a simple, inexpensive, well suited for high-throughput genotyping has emerged as being clearly superior and high-throughput assay for SNP because 96 samples can be analyzed and because of limitations such as cost, genotyping that we refer to as MCA of automatically scored in 20 min. Our results complexity, and accuracy. SNPs (McSNP) is presented in which clearly demonstrate that McSNP is a simple, Recently, the principles underlying the melting profiles of restriction en- inexpensive, and accurate means of geno- kinetic PCR (9), which is predicated on zyme-digested PCR products are ana- typing SNP variation. monitoring the fluorescence of a diag- lyzed. In total, we present data on seven 358BioTechniques Vol. 30, No. 2 (2001) SNPs, clearly demonstrating that this ences 7, 5, and 22, respectively. Marker (Hybaid). Other instruments that may method is a practical, robust, and sensi- M89 was obtained from Reference 21. be suitable for MCA include the Gene- tive approach to assaying SNP variation. The samples used in this study repre- Amp5700 and ABI Prism7700 Se- sent a subset of samples that we are us- quence Detection Systems (Applied ing in other studies and are comprised Biosystems), the LightCycler(Roche MATERIALS AND METHODS of African-American, European-Amer- Molecular Biochemicals, Indianapolis, ican, and Hispanic populations. More IN, USA), the iCycler(Bio-Rad Lab- PCR specific details can be found elsewhere oratories, Hercules, CA, USA), the (16,20). Informed consent from each Smart Cycler (Cephied, Sunnyvale, SNPs were amplified by PCR, and subject was obtained for participation CA, USA), and the Sentinel(Strata- most reactions were conducted under a in this study. gene, La Jolla, CA, USA). However, standard set of conditions. PCR was we have not explored the general accu- performed in either a Hybaid Multi- Restriction Enzyme Digestion racy, robustness, and scoring software Block System (Hybaid, Franklin, MA, associated with these machines, which USA) or a GeneAmp 9700 (Applied Restriction enzyme digestions were may differ substantially. Melting curves Biosystems, Foster City, CA, USA) and performed in a final volume of 25 µL, are acquired by ramping the tempera- consisted of the following thermocy- which consisted of 10 µL PCR product, ture from 35°C to 90°C at a rate of cles: 95°C for 5 min, followed by 30 1 U appropriate restriction enzyme, 1× 0.04°C/s and monitoring the change in cycles of 95°C for 30 s, 55°C for 30 s, reaction buffer supplied with the re- fluorescence of SYBR Green I at 520 72°C for 30 s, and a final extension at striction enzyme, and, when recom- nm. The total run time is approximately 72°C for 5 min. PCRs were performed mended by the supplier, 1×bovine 22 min, which can be generally cut in in 25-µL reactions consisting of 25 ng serum albumin (BSA). In particular, half once the Tmis determined and the genomic DNA, 1.5 mM MgCl2, 2.5 µL markers 4019, M89, and 14867 were thermal range adjusted accordingly. It 10×PCR buffer (Life Technologies, digested by the restriction enzyme is during the slow ramp to 90°C that the Rockville, MD, USA), 0.1 mM dNTPs, NlaIII, while markers CKMM, 14319, fluorescence is captured. The raw data 0.04 µM each primer, and 2.5 U Taq FY-null, and LPL were digested by the are first converted to relative fluores- DNA polymerase (Life Technologies). restriction enzymes TaqI, RsaI, StyI, cence by dividing each point by the ini- The exception to these conditions was and PvuII, respectively. We did not find tial fluorescence (i.e., 35°C) (6). Final marker Fy-null, in which the final it necessary to purify the PCR products melting curves are reported as the MgCl2was 2.0 mM. Primer sequences before restriction enzyme digestions, three-point smoothed negative first de- were as follows: CKMM (5′-GCAG- which were performed as recommend- rivative of fluorescence, with respect to GCGCCTACTTCTGG-3′and 5′-AGC- ed by the suppliers for a period of 4–24 temperature versus temperature with a TCATGGTGGAAATGGAG-3′), 4019 h. All restriction enzymes were pur- baseline subtraction. The baseline cor- (5′-CAGGCCAAGAGCGTCCTA-3′ chased from New England Biolabs rection for each data point is calculated and 5′-TGCCACTCTGTGAACAGC- (Beverly, MA, USA). by subtracting the slope from a linear AA-3’), 14319 (5′-CATCTGAGTGC- regression line of the first nine data AAGATAAAAAGGA-3′and 5′-CCC- MCA points. All genotypes were confirmed ACCCCCAAATCATCTAT-3′), FY- by agarose gel electrophoresis. Specifi- null (5′-GCCCAGAACCTGATGGCC- Melting profile reactions were ana- cally, samples were amplified by a sec- CTCATTAGTGCT-3′and 5′-CTGTC- lyzed in a final volume of 50 µL. All re- ond set of PCR primers to produce larg- AGCGCCTGTGCTT-3′), 14867(5′-G- actions contained 5 µL restriction en- er products more suitable for scoring GCAGGACATTCCAAGGCTCTC-3′ zyme product, 5 µL 10×SYBR Green I by agarose gel electrophoresis. These and 5′-CACCCTGGGTTAACACATT- (which comes as a 10000×stock sup- samples were then independently di- CA-3′), LPL (5′-TGCAAGGGTTTT- plied by Molecular Probes), and were gested and scored by agarose gels. GCTTAATTCT-3′and 5′-CAACAA- brought to a final volume of 50 µL in CAAAACCCCACAGC-3′), and M89 double-distilled water. Various destabi- (5′-ACAGAAGGATGCTGCTCAGC- lizing agents were added to these reac- RESULTS TT-3′and 5′-GCAACTCAGGCAAA- tions including formamide, dimethyl GTGAGACAT-3′), in which the bold sulfoxide (DMSO), and urea (all from Restriction Enzyme MCA Profiles denotes the mismatched base to create Fisher Biotech, Fair Lawn, NJ, USA). the putative NlaIII restriction site (see Here, we provide the details of only The experimental protocol of Mc- Results section). SNP markers WI- those additives that had an enhancing SNP is very simple and is outlined in 4019 (dbSNP ID: 2420), WI-14319, effect on MCA. The optimal final con- Figure 1. Figure 2 illustrates typical Mc- and WI-14867 were identified by centrations of these additives were ei- SNP genotype profiles for six autosomal querying the Whitehead Institute ther 10% formamide or 10% DMSO.