Epigenetic processes, such as acetylation and methylation of DNA, alter the structure of chromatin, making genes accessible or inaccessible to transcription, respectively. Methylation studies often incorporate bisulfite treatment of DNA in order to determine the methylation status of specific CpG sites. However, PCR amplification of bisulfite-treated DNA poses technical challenges for conventional polymerases due to a variety of factors, including the presence of uracil in the treated DNA. Designed for PCR for Combined Bisulfite Restriction Analysis (COBRA)/Bisulfite sequencing, TaKaRa EpiTaq HS (for bisulfite-treated DNA) is a hot-start (HS) DNA polymerase well-suited for methylation analysis of CpG islands and also for DNA amplification of AT-rich and GC-rich templates. This hot-start enzyme is coupled with an anti-Taq antibody that suppresses polymerase activity prior to high-temperature denaturation, preventing non-specific amplification due to mispriming or primer-dimer formation. EpiTaq HS DNA polymerase and included reagents allow for the adjustment of Mg2+ and dNTP concentrations to achieve optimal amplification efficiency and specificity, and to facilitate robust amplification of a wide range of target sizes—even targets that previously failed to amplify.
I was having difficulty amplifying PCR products from bisulfite-treated DNA. Takara EpiTaq was able to successfully amplify products using all of my primer combinations. I was able to amplify a product of ~1,000 bp, whereas with other polymerases I was lucky to get up to 300 bp. —Researcher at the UNIVERSITY OF SASKATCHEWAN
Results
Bisulfite treatment of DNA prior to PCR amplification
Methylation of cytosine at the C-5 position is the predominant covalent epigenetic modification in mammalian cells. Bisulfite treatment is a widely used, effective, and convenient method for DNA methylation analysis (Figure 1). Treatment of DNA with sodium bisulfite starts with the addition of bisulfite to the C-6 position of cytosine, allowing its deamination into 5,6-dihydrouracil-6-sulfonate. Next, the addition of an alkaline solution removes the sulfonate group, creating uracil in the place of cytosine. Methylation blocks the first step in this reaction—preserving the cytosine—while unmodified cytosine is converted to uracil and reads as thymine when assayed.
Figure 1. With bisulfite treatment, unmethylated cytosine is converted to uracil, while methylated cytosine remains intact. EpiTaq HS DNA polymerase cleanly amplifies templates containing uracil; it converts uracil to thymine and methylated cytosine to unmethylated cytosine. Therefore, every remaining cytosine in the PCR product was previously methylated. Unfortunately, bisulfite-treated DNA consists of fragmented, single-stranded molecules that are difficult to amplify with traditional polymerases. Additionally, nonspecific amplification is common in bisulfite-treated DNA due to its high AT content. EpiTaq HS DNA polymerase is optimized for amplification of this low-quality, AT-rich DNA-containing uracil; inclusion of an anti-Taq antibody that inactivates the hot-start enzyme until the initial denaturation step minimizes nonspecific amplification products due to primer dimerization and mispriming. Data in the following sections demonstrate EpiTaq HS DNA polymerase's superiority over other polymerases, which had difficulty amplifying uracil-containing, degraded DNA with a biased base-pair composition.
High amplification efficiency for a wide range of target sizes
A major barrier to successful methylation analysis of bisulfite-treated DNA is the amplification of longer targets, such as BRCA1 (1,017 bp). Other polymerases sufficiently amplify short targets but usually fail with longer targets. Figure 2 demonstrates that EpiTaq HS DNA polymerase is equally efficient at amplifying short (~150 bp) and long (~1,000 bp) targets, increasing success rates. Figure 3 compares EpiTaq HS DNA polymerase with six other polymerases, including one Takara polymerase. EpiTaq DNA polymerase outperforms all other polymerases tested when amplifying both long (BRCA1) and short (CDKN2A) targets.
Figure 2. EpiTaq HS DNA polymerase provides high yields for a broad range of target sizes. All targets, from 153 bp to 1,017 bp, were amplified with EpiTaq HS DNA polymerase using these standard PCR reaction conditions: 40 cycles of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 30 sec (or 1 min for 613-bp and 1,017-bp samples).
Figure 3. Comparison of amplification efficiencies between EpiTaq HS DNA polymerase and five other polymerases. Lanes 1–7 were tested using the manufacturers' recommended protocols. EpiTaq HS DNA polymerase shows excellent yield compared to the other enzymes, especially for the longer target, BRCA1.
I could not get a PCR product using a competitor's polymerase. I got it with EpiTaq HS. Thanks. —Researcher at FOX CHASE CANCER CENTER
Reaction optimization by adjusting Mg2+ concentration
Magnesium is a cofactor for DNA polymerase, and adjusting its concentration is important for obtaining the highest possible amplification efficiency. Lowering the magnesium concentration increases specificity while raising the magnesium concentration improves amplification efficiency and extension. TaKaRa EpiTaq HS (for bisulfite-treated DNA) includes all reagents necessary for PCR, packaged separately to make it easy to optimize reactions for maximum yield: a MgCl2 solution, a dNTP mixture, the EpiTaq enzyme, and a Mg2+-free buffer. Figure 4 demonstrates the importance of adjusting the Mg2+ concentration. The amplification efficiency of CDKN2A is highest at 2.5 mM Mg2+, while the amplification efficiency of MLH1 is highest at 3.0 mM Mg2+ regardless of target length.
Figure 4. Bisulfite treatment of HeLa genomic DNA was performed with the MethylEasy Xceed Rapid DNA Bisulfite Modification Kit. The targets were CpG island regions upstream of CDKN2A or MLH1. Amplification sizes were 316 bp (CDKN2A), 607 bp (MLH1), and 877 bp (MLH1).
Conclusions
Traditionally, several factors impede successful amplification of bisulfite-treated DNA (containing uracil). Bisulfite treatment converts stable, double-stranded DNA into single-stranded, AT-rich fragments that are notoriously difficult to amplify, especially for longer targets. EpiTaq HS DNA polymerase, containing hot-start technology, together with associated reagents for optimization, gives excellent yields and superior amplification over a wide range of target sizes. These attributes reduce overall time expenditure because other polymerases (such as those tested here) may not reliably amplify targets in the upper size range.
