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.
KeywordsAmplicon design Primer3 DNA variation Mutation DGGE 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].
Results and discussion
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.
Availability and requirements
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.
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