- Technical Note
- Open Access
Automated amplicon design suitable for analysis of DNA variants by melting techniques
© Ekstrom et al. 2015
- Received: 18 November 2014
- Accepted: 26 October 2015
- Published: 11 November 2015
The technological development of DNA analysis has had tremendous development in recent years, and the present deep sequencing techniques present unprecedented opportunities for detailed and high-throughput DNA variant detection. Although DNA sequencing has had an exponential decrease in cost per base pair analyzed, focused and target-specific methods are however still much in use for analysis of DNA variants. With increasing capacity in the analytical procedures, an equal demand in automated amplicon and primer design has emerged.
We have constructed a web-based tool that is able to batch design DNA variant assay suitable for analysis by denaturing gel/capillary electrophoresis and high resolution melting. The tool is developed as a computational workflow that implements one of the most widely used primer design tools, followed by validation of primer specificity, as well as calculation and visualization of the melting properties of the resulting amplicon, with or without an artificial high melting domain attached. The tool will be useful for scientists applying DNA melting techniques in analysis of DNA variations. The tool is freely available at http://meltprimer.ous-research.no/.
Herein, we demonstrate a novel tool with respect to covering the whole amplicon design workflow necessary for groups that use melting equilibrium techniques to separate DNA variants.
- Amplicon design
- DNA variation
- High resolution melting
- Capillary electrophoresis
Friedrich Miescher discovered the nucleic acid in 1868–69 (reviewed by R. Dahm) [1, 2]. This may be defined as the starting point of a research field that has expanded into a huge area of medical and biological research. Many important methodological advances have been made in order to facilitate analysis of DNA. The polymerase chain reaction (PCR), first published by Kleppe et al. , which opened for mass amplification of DNA amplicons, is still a key method in modern molecular analysis of DNA. PCR amplification has the ability to amplify specific target sequences, as well as whole genomes up to a factor of 1011. In 1977, a DNA sequencing approach introduced by Sanger used dideoxy nucleotides to terminate enzymatic amplification of single stranded DNA . This method was refined and eventually used to sequence almost the entire human genome. PCR amplification and Sanger sequencing, and variations of these core techniques, are still very important parts in present day DNA research. Following the development of these and other molecular methods, techniques enabling separation and visualization of amplified DNA have taken place, from the starting point of using radioactively labeled DNA and gel electrophoresis, through laser-induced fluorescence capillary electrophoresis, until various high throughput-sequencing platforms with the capacity to determine up to 5G bases/day.
One set of methods that was developed to detect unknown DNA variants was based on differential migration velocities of mutant single-stranded sequences or wild type/mutant heteroduplexes drawn through a macromolecular matrix by an electric field [5–7]. Of these, denaturing gel electrophoresis when performed in capillaries (constant denaturing capillary electrophoresis, CDCE) under optimized conditions has been demonstrated to comprehensively detect any point mutation, including single base insertions and deletions, in target sequences of ~70–140 bp [8, 9]. Methods employing CDCE have been reported with sensitivities to detect and identify mitochondrial and nuclear point mutations at levels at or above 2 × 10−6 mutations per gene copy in human cells, tissues and pooled blood samples [10–12]. Under a given concentration of chemical denaturant, such as urea, multiple capillary runs were required to define the generally narrow temperature range (~0.1 °C) that would separate heteroduplexes containing any of a wide variety of single deletion or single substitution mutants from wild type homoduplexes. CDCE has been adapted to commercial multi-capillary instruments, thus increasing the throughput of the method [13, 14]. A second improvement of the method was the introduction of oscillating temperature. By rapidly changing the denaturing condition in the capillaries, multiple DNA target sequences could be scanned simultaneously in the same run [13, 15–17]. One limitation of melting gel techniques results from DNA sequences rich on GC content, which can lose the resolution power due to complete strand dissociation at elevated temperatures. However, data from the complete melting map of the human genome indicates that the melting gel method can be applied to about 90 % of the human genome . Because melting gel techniques are still much in use (more than 700 articles published in 2013), we have created a computational workflow that selects PCR primers , validates primer specificity in the genome of interest , and computes the DNA melting profile  with an artificial high temperature melting domain . This web application, which is embedded in the Galaxy framework [23–25], will simplify amplicon design and increase the throughput of the method when amplicons are analyzed in multi-capillary instruments. Additionally, the amplicons designed in this web application, are also suitable for high resolution melting. This is a method that emerged after the introduction of thermocycler with fluorescent detection systems enabling visualization of the PCR amplification of DNA in real-time [26–30].
Reference SNP number and primers batch-designed and used in HRM assays
Forward primer (5′-3′)
Reverse primer (5′-3′)
This is the first web application, to our knowledge, that combines the three features of primer selection around a DNA variant, controlling primer pair for specificity, and of computing the melting properties of the amplicon with or without a GC-clamp. Hence, this tool is novel with respect to covering the whole workflow necessary for groups that currently use melting equilibrium techniques to separate DNA variants.
Variant melting profile
The Variant Melting Profile is served as a tool within the Galaxy framework, which is an open source, web-based platform for data intensive biomedical research [23–25]. Galaxy is here used in combination with components from the Genomic Hyperbrowser [36, 37] as well as Google Charts for visualization (https://developers.google.com/chart/).
DNA variants (single nucleotide variants) can be given either as genomic coordinates (chromosome:position:reference_allele:alternative_allele), or as dbSNP reference IDs (e.g. rs9648696). User-specified parameters for selection of PCR primers (e.g. optimum primer sizes and primer melting temperatures etc.), as given by the Primer3 program  is used for finding suitable primers. The top candidate amplicon within the list of candidate primers is checked for specificity in the human genome using UCSC In-Silico PCR . If the amplicon maps uniquely, a GC clamp can be added to the PCR amplicon, and the DNA melting profiles for the nucleotide variants are visualized in an interactive fashion. The melting profiles are generated using the thermodynamic model provided by Blossey and Carlon .
DNA extraction, amplification and CTCE
Genomic DNA was extracted from anonymous blood donor samples by use of GenoVision M48 extraction robot (Biorobot M48 station, Qiagen, Norway), following standard procedures as given by the instrument manufacture.
A 42 base pair sequence of dGTP and dCTP, also known as a GC-clamp, labeled with 6-FAM, was incorporated at one end of the amplified target using a set of three primers in the PCR setup. An amplicon was designed for analysis of DNA variation identified by NCBI SNP references number, rs2252586.
Reverse ½ GC-lamp tailed
The PCR reaction mixture contained approximately 5 ng/µl genomic DNA, 0.4 mM dNTPs (0.1 mM of each dNTP) (VWR, Oslo, Norway), 1X Taq buffer, 0.075unit Taq/µl, 0.15 µM each of labeled GC clamp and the 1/2 GC-clamp tailed primer, while 0.3 µM of the “forward” primer (Integrated DNA Technologies, Leuven, Belgium) and 3 mM MgCl in a total reaction volume of 10 µl. Amplification was performed in a Eppendorf Mastercycler ep gradient S (Eppendorf, Hamburg, Germany) cycling 35 time between the temperatures of; cycles of denaturation for 10 s at 94 °C, annealing at 55.7 °C for 20 s and elongation at 72 °C for 30 s.
Six-fam labeled PCR products were analyzed with a 96-capillary DNA analyzer, i.e. the MegaBACE™ 1000 DNA Analysis System (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The instrument was modified to allow for elevated temperatures up to 65 °C. For detailed information about the modification, please contact the author P.O. Ekstrøm. Standard sequencing polyacrylamide (MegaBACE LPA) containing urea was replaced in the capillaries prior to each run. Samples were loaded into the capillaries from 96-well plates by electrokinetic injection at 161 V/cm for 15 s. The electrophoresis was carried out at a constant field of 145 V/cm. Laser-induced fluorescence was used with excitation at 488 nm (blue laser) and detection of emission at 520 nm (FAM channel). The scan rate was 1.75 Hz. The optimal separation temperature proposed by these programs was adjusted based on the urea concentration in the matrix. For each molar increment of urea, the temperature was lowered approximately 3 K (Kelvin) [40, 41].
The denaturing temperature in the capillary chamber, the cycling temperature, was programmed in the macro.ini file used by the Instrument Control Manager (ICM) software package (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Data were converted to text files by MegaBACE Sequence Analyzer View and Edit software.
Real time-PCR and high resolution melting (HRM)
A 25 µl Real time-PCR reaction was made up of 12.5 µl 2xMIX (PerfeCTa® SYBR® Green SuperMix, Quanta Biosciences, Gaithersburg, USA) 0.75 µl forward and 0.75 µl reveres primer with a concentration of 0.3 µM each. 2 µl DNA template and 9 µl H2O. The mixture was cycled 50 times with the following temperature, 95 °C for 15 s and 60 °C for 30 s. The fluorescence was read for each cycle, in a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories AB, Oslo, Norway).
High resolution melting was performed after the temperature cycling by slowly increasing the temperature from 65–95 °C in increments of 0.1 °C. Fluorescent signal was recorded for 5 s after each 0.1 °C increment.
Primers used for the real-time PCR and HRM were selected by the Variant Melting Profile tool, checked for specificity by ePCR ordered from IDTDNA, with the following base composition: “Reverse” primer 5′CAACACGTTTCACCAGTGCA3′ and “forward” primer 5′TGCAAGATTGTACCTTCCTTGGT3′.
HRM curve data were normalized to the pre-melt (initial fluorescence) and post-melt (final fluorescence) signals. Thus, all samples were set to uniform, relative values from 100–0 %. The temperature axis of the normalized melting curves was shifted to the point where the entire double-stranded DNA was completely denatured. Differences in melting curve shape were further analyzed by subtracting the curves from a reference curve. We used a heterozygote sample as reference.
Following the initial test of HRM, 12 fragments were automatically designed on SNPs with references number given in Table 1. The fragments were subjected to Real time-PCR and HRM as described above, save for the annealing temperature which was set to 57 °C.
Project name: MeltPrimer
Project home page: http://meltprimer.ous-research.no/
Operating system(s): Linux
Programming language: HyperBrowser/Galaxy framework, SQLite, Blat/gfServer/gfPcr, Primer3
Any restrictions to use by non-academics: no licenses needed.
POE specified the underlying software components and was responsible for testing of the Variant Melting Profile tool. POE amplified DNA by PCR and separated variants. SN and MJ implemented the computational workflow underlying Variant Melting Profiles. EH participated in the design of the study. All authors contributed equally in the writing of the manuscript and have read and approved the final version. All authors read and approved the final manuscript.
This study was supported by Research Council of Norway Grants 221580 and 218241, and Grant 71220 -PR-2006-0433 from The Norwegian Cancer Society.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Dahm R. Discovering DNA: Friedrich Miescher and the early years of nucleic acid research. Hum Genet. 2008;122:565–81.View ArticlePubMedGoogle Scholar
- Dahm R. Friedrich Miescher and the discovery of DNA. Dev Biol. 2005;278:274–88.View ArticlePubMedGoogle Scholar
- Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG. Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA’s as catalyzed by DNA polymerases. J Mol Biol. 1971;56:341–61.View ArticlePubMedGoogle Scholar
- Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA. 1977;74:5463–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Andersen PS, Jespersgaard C, Vuust J, Christiansen M, Larsen LA. Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening. Hum Mutat. 2003;21:455–65.View ArticlePubMedGoogle Scholar
- Ganguly A. An update on conformation sensitive gel electrophoresis. Hum Mutat. 2002;19:334–42.View ArticlePubMedGoogle Scholar
- Li Q, Liu Z, Monroe H, Culiat CT. Integrated platform for detection of DNA sequence variants using capillary array electrophoresis. Electrophoresis. 2002;23:1499–511.View ArticlePubMedGoogle Scholar
- Khrapko K, Hanekamp JS, Thilly WG, Belenkii A, Foret F, Karger BL. Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis. Nucleic Acids Res. 1994;22:364–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Khrapko K, Andre P, Cha R, Hu G, Thilly WG. Mutational spectrometry: means and ends. Prog Nucleic Acid Res Mol Biol. 1994;49:285–312.View ArticlePubMedGoogle Scholar
- Andre P, Kim A, Khrapko K, Thilly WG. Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence. Genome Res. 1997;7:843–52.PubMed CentralPubMedGoogle Scholar
- Khrapko K, Coller H, Andre P, Li XC, Foret F, Belenky A, et al. Mutational spectrometry without phenotypic selection: human mitochondrial DNA. Nucleic Acids Res. 1997;25:685–93.PubMed CentralView ArticlePubMedGoogle Scholar
- Li-Sucholeiki XC, Thilly WG. A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA. Nucleic Acids Res. 2000;28:E44.PubMed CentralView ArticlePubMedGoogle Scholar
- Ekstrom PO, Bjorheim J, Gaudernack G, Giercksky KE. Population screening of single-nucleotide polymorphisms exemplified by analysis of 8000 alleles. J Biomol Screen. 2002;7:501–6.View ArticlePubMedGoogle Scholar
- Bjorheim J, Minarik M, Gaudernack G, Ekstrom PO. Mutation detection in KRAS Exon 1 by constant denaturant capillary electrophoresis in 96 parallel capillaries. Anal Biochem. 2002;304:200–5.View ArticlePubMedGoogle Scholar
- Ekstrom PO, Bjorheim J, Thilly WG. Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE). BMC Genet. 2007;8:54.PubMed CentralView ArticlePubMedGoogle Scholar
- Ekstrom PO, Khrapko K, Li-Sucholeiki XC, Hunter IW, Thilly WG. Analysis of mutational spectra by denaturing capillary electrophoresis. Nat Protoc. 2008;3:1153–66.PubMed CentralView ArticlePubMedGoogle Scholar
- Kristensen AT, Bjorheim J, Wiig J, Giercksky KE, Ekstrom PO. DNA variants in the ATM gene are not associated with sporadic rectal cancer in a Norwegian population-based study. Int J Colorectal Dis. 2004;19:49–54.View ArticlePubMedGoogle Scholar
- Liu F, Tostesen E, Sundet JK, Jenssen TK, Bock C, Jerstad GI, et al. The human genomic melting map. PLoS Comput Biol. 2007;3:e93.PubMed CentralView ArticlePubMedGoogle Scholar
- Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365–86.PubMedGoogle Scholar
- Hinrichs AS, Karolchik D, Baertsch R, Barber GP, Bejerano G, Clawson H, et al. The UCSC genome browser database: update 2006. Nucleic Acids Res. 2006;34:D590–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Tostesen E, Jerstad GI, Hovig E. Stitchprofiles.uio.no: analysis of partly melted DNA conformations using stitch profiles. Nucleic Acids Res. 2005;33:W573–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Myers RM, Fischer SG, Maniatis T, Lerman LS. Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 1985;13:3111–29.PubMed CentralView ArticlePubMedGoogle Scholar
- Goecks J, Nekrutenko A, Taylor J. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010;11:R86.PubMed CentralView ArticlePubMedGoogle Scholar
- Blankenberg D, Von KG, Coraor N, Ananda G, Lazarus R, Mangan M et al.: Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol 2010, Chapter 19: Unit-21.Google Scholar
- Giardine B, Riemer C, Hardison RC, Burhans R, Elnitski L, Shah P, et al. Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005;15:1451–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Hardies SC, Hillen W, Goodman TC, Wells RD. High resolution thermal denaturation analyses of small sequenced DNA restriction fragments containing Escherichia coli lactose genetic control loci. J Biol Chem. 1979;254:10128–34.PubMedGoogle Scholar
- Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003;49:853–60.View ArticlePubMedGoogle Scholar
- Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT. Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem. 2003;49:396–406.View ArticlePubMedGoogle Scholar
- Gundry CN, Bernard PS, Herrmann MG, Reed GH, Wittwer CT. Rapid F508del and F508C assay using fluorescent hybridization probes. Genet Test. 1999;3:365–70.View ArticlePubMedGoogle Scholar
- Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res. 1996;6:995–1001.View ArticlePubMedGoogle Scholar
- Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115.PubMed CentralView ArticlePubMedGoogle Scholar
- Hinselwood DC, Abrahamsen TW, Ekstrom PO. BRAF mutation detection and identification by cycling temperature capillary electrophoresis. Electrophoresis. 2005;26:2553–61.View ArticlePubMedGoogle Scholar
- Bjorheim J, Ekstrom PO, Fossberg E, Borresen-Dale AL, Gaudernack G. Automated constant denaturant capillary electrophoresis applied for detection of KRAS exon 1 mutations. Biotechniques. 2001;30:972–5.PubMedGoogle Scholar
- Bjorheim J, Ekstrom PO. Review of denaturant capillary electrophoresis in DNA variation analysis. Electrophoresis. 2005;26:2520–30.View ArticlePubMedGoogle Scholar
- Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR protocols—a guide to methods and applications. London: Academic press; 1990, p. 482. ISBN: 0-12-372181-4.Google Scholar
- Sandve GK, Gundersen S, Johansen M, Glad IK, Gunathasan K, Holden L, et al. The Genomic HyperBrowser: an analysis web server for genome-scale data. Nucleic Acids Res. 2013;41:W133–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Sandve GK, Gundersen S, Rydbeck H, Glad IK, Holden L, Holden M, et al. The Genomic HyperBrowser: inferential genomics at the sequence level. Genome Biol. 2010;11:R121.PubMed CentralView ArticlePubMedGoogle Scholar
- Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, et al. The human genome browser at UCSC. Genome Res. 2002;12:996–1006.PubMed CentralView ArticlePubMedGoogle Scholar
- Blossey R, Carlon E. Reparametrizing the loop entropy weights: effect on DNA melting curves. Phys Rev E Stat Nonlin Soft Matter Phys. 2003;68:061911.View ArticlePubMedGoogle Scholar
- Hutton JR. Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea. Nucleic Acids Res. 1977;4:3537–55.PubMed CentralView ArticlePubMedGoogle Scholar
- Klump H, Burkart W. Calorimetric measurements of the transition enthalpy of DNA in aqueous urea solutions. Biochim Biophys Acta. 1977;475:601–4.View ArticlePubMedGoogle Scholar