The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR, Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR
© Hughes and Jones; licensee BioMed Central Ltd. 2007
Received: 30 April 2007
Accepted: 16 October 2007
Published: 16 October 2007
Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples.
To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA.
Using these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult.
The major epigenetic alterations in eukaryotes are DNA methylation and histone acetylation. Promoter methylation has an important role in controlling the binding of transcription factors and other proteins to the DNA, which in turn modulate the association of methyl-DNA-binding proteins and histone deacetylases to the transcription start sites. This modulation is critical in regulating the switch between transcriptionally active euchromatin (unmethylated) and transcriptionally silent heterochromatin (methylated) and in turn gene expression [1, 2]. The most common methylation event is the addition of a methyl group to the cytosine present in the CpG (cytosine-guanine) dinucleotide . These dinucleotides exist as either CpG islands or as sparsely distributed CpG motifs within the promoter regions of many genes. Hypermethylation (methylation) of these islands or motifs results in transcriptional silencing , whilst hypomethylation (demethylation), either global or gene specific, induces expression .
PCR-based techniques can be used to investigate the methylation status of CpG islands or motifs with the available methods being categorised based on the requirement for bisulfite treatment prior to PCR (or sequencing). Bisulfite treatment converts all unmethylated cytosine to uracil/thymine, while methylated cytosines are retained. MS-PCR, PSQ or BS can then be used to measure cytosine conversion or retention and thus distinguish methylated from unmethylated residues [6, 7]. As an alternative to bisulfite-based approaches, methylation-sensitive restriction endonucleases, which contain one or more CpG motifs within their recognition site, can be employed [8, 9]. These enzymes will only cut the DNA if the cytosine within the CpG motif is unmethylated. For this assay, the DNA (non bisulfite treated) is first digested and then subjected to amplification by PCR (MSRE-PCR) using primers flanking the site of interest. If the CpG is methylated, then a PCR product will be generated, however, if there is no methylation, no product will be generated as the site will have been cut.
When designing methylation detection assays using MS-PCR, BS, PSQ or MSRE-PCR optimisation of the amplification conditions, including primer design, magnesium chloride concentration and annealing temperature, is essential to ensure correct interpretation of results. To enable this, suitable control DNA samples are required that correspond to fully CpG unmethylated and fully CpG methylated DNA.
In this paper, we describe an adaptation of the approach described by Weisenberger and colleagues . The methods presented here use a combination of whole genome amplification (WGA) using the multiple displacement amplification (MDA) approach  with or without subsequent treatment with the CpG methylating enzyme SssI-methylase (M.SssI) to generate matched DNA samples differing in only their CpG methylation. The DNA samples generated using this method can be used as CpG methylation control samples for optimising PCR-based assays, as well as internal controls for all of the steps involved in a methylation detection experiment.
Results and Discussion
Alterations in DNA methylation status can modulate gene expression in the absence of DNA base changes. Although several PCR-based approaches can be implemented to measure methylation, before these can be reliably used to study patient samples it is first essential to optimise assay conditions. Furthermore, as PCR amplification is often the end point measurement in methylation analysis, it is important to have amplification controls as a way of monitoring the whole experimental process, to ensure each step and treatment has worked optimally. While commercially available universally methylated and unmethylated DNA can be used as controls, these have not always been reliable in our assays. As a consequence, we have developed a procedure for generating CpG methylated and CpG unmethylated DNA from the same source DNA using MDA and M.SssI treatment.
Primer sequences for MS-PCR, PSQ, BS and MSRE-PCR
Primer Sequence (5' – 3')
Methylated (m)/Unmethylated (u)
Base pair location
5001854 – 5001830
5001697 – 5001673
5001854 – 5001830
5001697 – 5001673
9127002 – 9127021
9127086 – 9127104
9127002 – 9127021
9127086 – 9127104
5001871 – 5001850
5001675 – 5001656
5001859 – 5001841
5001614 – 5001641
5001795 – 5001821
5001651 – 5001670
4305511 – 4305533
4305741 – 4305765
6233232 – 6233214
6233000 – 6232982
6277588 – 6277566
6277355 – 6277333
The results demonstrate that the combination of MDA and M.SssI treatment generate DNAs that differ only in their CpG methylation status. The examples described herein illustrate uDNA and mDNA can act as methylation-status specific controls for both assay optimisation and as internal controls for methylation experiments. This is important for several reasons; (i) it enables primer and reaction optimisation, (ii) it allows for an experimental checkpoint, for instance ensuring bisulfite conversion is complete, as PCR products will be generated from mDNA and uDNA with both sets of methylation status detection primers if the conversion is incomplete, or for MSRE-PCR to ensure complete digestion and (iii) if mDNA and uDNA controls are processed along side test samples when the controls give expected results then the results obtained from the test samples should be more reliable.
Genomic DNA was extracted using the Qiagen DNA Mini-kit (Qiagen, Crawley, UK) from normal breast tissue obtained following breast reduction surgery, following ethics approval from the North East London LREC. DNA was also obtained from the cell lines HFFF2, MDA-MB231 and MDA-MB468, using the same technique. DNA concentration was determined using the Nano-drop spectrophotometer (NanoDrop Technologies, Wilmington, USA). Both procedures were performed following manufacturer's instructions.
The process of primer design for MS-PCR and BS is critical when using these techniques and it is highly recommended to use specialised software as standard approaches and programs will not be sufficient. For this study, we used either Methyl Primer Express version 1.0 (Applied Biosystems, Foster City, USA) for primer design (MMP-2 and MMP-14) or utilised primers reported previously [BRCA-1 ]. Primer design for pyrosequencing was performed using the PSQ assay design software version 1.0.6 (Biotage, Uppsala, Sweden), whilst primer design for MSRE-PCR can be performed using standard primer design programs. All primers were obtained from Sigma-Aldrich (Gillingham, UK)
Whole Genome Amplification
DNA was amplified using the GenomiPhi Amplification Kit (Amersham Biosciences, Little Chalfont, UK) according to manufacturer's instructions. Briefly, amplification was carried out in two individual steps. The step 1 reaction mixture contained 5–10 ng of DNA in 1 μl of sterile water and 9 μl of Sample Buffer. This mixture was heated at 95°C for 3 minutes and then chilled on ice. Step 1 results in denaturation of the genomic DNA template. The step 2 reaction (amplification) mixture contained 9 μl of Reaction Buffer, 1 μl of Enzyme Mix and the 10 μl from Step 1. The amplification reaction was incubated at 30°C for 16–18 hours. Step 2 allows for binding of the exonuclease resistant random hexamers and subsequent isothermal amplification. The enzyme was inactivated by heating at 65°C for 10 minutes, followed by cooling to 4°C.
Assessment of amplification and purification
Five microlitres of each amplification reaction was electrophoresed through a 1% agarose gel and stained with ethidium bromide in order to assess product yield and product length. Amplification products were purified using the QIAquick PCR Purification Kit (Qiagen) and DNA concentration was determined using a Nano-drop spectrophotometer.
CpG motifs within the WGA DNA were methylated using the CpG Methylase, M.SssI (New England Biolabs, Hitchin, UK) according to manufacturer's instructions. Briefly, 1.5 μg of WGA DNA was combined with 2 μl of 10x NEBuffer 2, 0.1 μl of S-adenosylmethionine (SAM), 5 units of M.SssI and sterile water up to a final volume of 20 μl. The reaction was incubated at 37°C for 1 hour, before being purified using the QIAquick PCR Purification Kit and the DNA concentration determined using a Nano-drop spectrophotometer.
DNA was bisulfite treated using the EpiTect Bisulfite Kit (Qiagen) according to manufacturer's instructions. Briefly, 1 μg of either CpG methylated WGA DNA, unmethylated WGA DNA or cell line DNA in 20 μl of water was combined with 85 μl of Bisulfite mix and 35 μl of DNA protect buffer. The bisulfite DNA conversion was performed using the following conditions; denaturation 5 min 99°C, incubation 25 min 60°C, denaturation 5 min 99°C, incubation 85 min 60°C, denaturation 5 min 99°C, incubation 175 min 60°C, hold 20°C. The bisulfite converted DNA was purified following manufacturer's instructions. Briefly, the bisulfite reaction was mixed with 560 μl of Buffer BL, applied to the spin column and centrifuged at 12,000 rpm for 1 min. The flow through was discarded and the column washed with 500 μl of Buffer BW. Buffer BD (500 μl) was applied to the column and incubated at room temperature for 15 min. The column was centrifuged to remove Buffer BD and then washed twice with Buffer BW (500 μl). Residual BW buffer was removed by an additional spin (12,000 rpm, 1 min). Buffer EB (20 μl) was added to the column to elute the DNA. The DNA concentration was determined using a Nano-drop spectrophotometer.
PCR was carried out in a 25 μl volume containing 25 ng of either CpG methylated and bisulfite treated WGA DNA (fully methylated), bisulfite treated WGA DNA (fully unmethylated) or cell line DNA, 1 μl of each primer (Table 1) (2 mM stock) for either BRCA-1 (methylated or unmethylated) or MMP-2 (methylated or unmethylated), 2 μl of 2.5 mM dNTP mix (Invitrogen, Carlsbad, USA), 2.5 μl of 10x PCR buffer, 1.25 μl of 50 mM MgCl2, 0.1 μl of Platinum Taq DNA polymerase (5 U/μl) (Invitrogen) and sterile H2O up to a final volume of 25 μl.
Amplification was performed using a "Touchdown PCR" approach, conditions were as follows: initial denaturation for 2 min at 95°C; 20 cycles of denaturing for 30 sec at 94°C, annealing for 30 sec starting at 65°C and decreasing by 0.5°C/cycle and elongation for 30 sec at 72°C; followed by 15 cycles of denaturing for 30 sec at 94°C, annealing for 30 sec at 55°C and elongation for 30 sec at 72°C; then 10 min at 72°C. Five microlitres of each amplification reaction was electrophoresed through a 1% agarose gel and stained with ethidium bromide in order to analyse results.
PCR was carried out in a 25 μl volume containing 25 ng of either CpG methylated and bisulfite treated WGA DNA (fully methylated) or bisulfite treated WGA DNA (fully unmethylated) or cell line DNA, 1 μl of each primer (PSQ-PCR; Table 1) (2 mM stock) for BRCA-1, 2 μl of 2.5 mM dNTP mix (Invitrogen), 2.5 μl of 10x PCR buffer, 2.5 μl of 50 mM MgCl2, 0.1 μl of Platinum Taq DNA polymerase (5 U/μl) (Invitrogen) and sterile H2O up to a final volume of 25 μl.
Amplification was performed using the following conditions: initial denaturation for 2 min at 95°C; 45 cycles of denaturing for 30 sec at 94°C, annealing for 30 sec at 58°C and elongation for 30 sec at 72°C; then 10 min at 72°C. Five microlitres of each amplification reaction was electrophoresed through a 1% agarose gel and stained with ethidium bromide in order to analyse results. Using the PCR products as template, PSQ reactions were performed using the BRCA-1 PSQ-S primers (Table 1) and the SQA reagent kit (Biotage, Uppsala, Sweden), following manufacturer's instructions. The results were analyzed using a Biotage PSQ 96MA pyrosequencing system with dedicated Pyro Q-CpG software (Biotage).
PCR was carried out in a 25 μl volume containing 25 ng of either CpG methylated and bisulfite treated WGA DNA (fully methylated), bisulfite treated WGA DNA (fully unmethylated) or non-amplified and untreated genomic DNA, 1 μl of each primer (Table 1) (2 mM stock) for MMP-14, 2 μl of 2.5 mM dNTP mix (Invitrogen), 2.5 μl of 10x PCR buffer, 1.25 μl of 50 mM MgCl2, 0.1 μl of Platinum Taq DNA polymerase (5 U/μl) (Invitrogen) and sterile H2O up to a final volume of 25 μl.
Amplification was performed as described above with the PCR products being purified using the QIAquick PCR Purification Kit. Using the PCR products as template, cycle sequencing reactions were performed using the MMP-14 Forward and reverse primers and the BigDye Terminator Version 3.1 Kit (Applied Biosystems) following manufacturer's instructions. The results were analyzed using an ABI Prism 3130XL Applied Biosystems DNA sequencer.
Methylation-Sensitive Restriction Enzyme PCR
CpG methylated WGA DNA, unmethylated WGA DNA or cell line DNA was digested with HpyCH4IV, following manufacturer's instructions. PCR was carried out in a 25 μl volume containing 25 ng of digested DNA (or undigested DNA as control), 1 μl of each primer pair (2 mM stock) for either MMP-1 or MMP-3, 2 μl of 2.5 mM dNTP mix (Invitrogen), 2.5 μl of 10x PCR buffer, 1.25 μl of 50 mM MgCl2, 0.1 μl of Platinum Taq DNA polymerase (5 U/μl) (Invitrogen) and sterile H2O up to a final volume of 25 μl.
Amplification was performed as described above and 5 μl of each amplification reaction was electrophoresed through a 1% agarose gel and stained with ethidium bromide in order to analyse results.
We would like to acknowledge the researchers at the Cancer Research UK Clinical Centre and Queen Mary's School of Medicine and Dentistry for their assistance. We would in particular like to thank Dr Charles Mein and Miss Christina Fleischmann for carrying out the sequencing and pyrosequencing presented in this paper.
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