Content
- overview
- Quantification of bisulfite-converted DNA
- Evaluation of the quality of bisulfite-converted DNA
- Bisulfite PCR and primer design
- primer design for MSP
- Bisulfite sequence
- Advanced technology bisulfite sequence
Learn more about bisulfite conversion
Overview of bisulfite conversion
When starting bisulfite conversion, there are a number of issues to consider to ensure optimum performance and subsequent accuracy ofDNA methylationanalysis. Bisulfite treatment is an inherently harsh process that drastically alters the chemical composition and physical properties of DNA. The incoming DNA is transformed from a large, stable double-stranded molecule into a randomly fragmented collection of single-stranded fragments, with almost all cytosines completely changed to uracil. These changes dramatically affect the quantification, quality assessment, amplification, and analysis of bisulfite-converted DNA. These points and the special considerations they require in relation to UV spectrophotometry (NanoDrop), agarose gel electrophoresis and PCR are highlighted below.
Quantification of bisulfite-converted DNA
Converted DNA should be quantified as RNA using a UV spectrophotometer (NanoDrop) with Ab260 nm 1.0 = 40 µg/ml. When determining the recovery of bisulfite-converted DNA, two primary factors must be considered: 1.) The integrity of the starting material and 2.) RNA contamination. The quality of DNA, in terms of size, used as input for bisulfite conversion is the most important factor in assessing recovery. Feedstock degradation will result in increased sample loss during the bisulfite conversion process. In addition, RNA contamination will contribute significantly to Ab260 nm, resulting in an overestimation of the amount of DNA. RNA carried over to conversion is lost, causing yields to appear low compared to input. It is important to note that the conversion is not compromised in any of the above cases.
Evaluation of the quality of bisulfite-converted DNA
Agarose gel electrophoresis (2% gel with 100 bp marker) of the converted DNA can be used to assess recovery and fragmentation. Most of the time, nothing will be visible on the gel when it is first removed from the gel tank. This is normal and results from the DNA being almost entirely single-stranded after conversion. Cooling the gel for several minutes in an ice bath will cause enough base pairing to allow ethidium bromide to intercalate so that the DNA is visible. Converted DNA will be run as a smear, typically >1500 to 100 bp. It is important to load enough material on the gel to allow visualization: usually about 100 ng/well is sufficient.
The challenges of bisulfite-converted DNA- DNA fragmentation and limited base pairing make it difficult to visualize DNA after bisulfite conversion. Bisulfite-converted human genomic DNA samples were analyzed on a 2% agarose gel with a 100 bp marker visible normally (left panel) and after cooling in an ice bath (right panel).
Bisulfite PCR and primer design
Bisulfite PCR is the most common technique used for methylation analysis of bisulfite-converted DNA and also the most accident-prone. Remember that the DNA will be significantly fragmented as the strands will no longer be complementary and will be almost completely devoid of cytosine. Primer design is key to successful bisulfite PCR. Unlike regular PCR, bisulfite PCR primers must be long (generally between 26-30 bases) and the amplicon size must be relatively small (between 150-300 bp). Ideally, primers should not contain CpG sites, however, if needed, locate them at the 5' end of the primer with a mixed base at the cytosine site. It is also important to note that only one clone of the bisulfite-converted template will be amplified from any one set of primers. Only the reverse primer will actually bind to the target DNA, which in turn will create a template for the forward primer pair. Typically, 35 to 40 cycles are required for successful enhancement. Warm-start polymerases are strongly recommended, as non-specific amplification is relatively common with bisulfite-converted DNA because it is AT-rich. Annealing temperatures between 55-60°C usually work well and an annealing temperature gradient should be performed with each new set of primers to ensure optimal amplification of the specific target.
Annealing step for primers used for bisulfite PCR.Bisulfite PCR replicates were incubated in a gradient thermocycler using an annealing temperature gradient from 55 to 65 oC (left to right). Equal volumes of each reaction were run on a 2% agarose gel with a 100 bp DNA probe.
Design of primers for methylation-specific PCR (MSP)
Methylation-specific PCR (MSP) relies on amplification to assess methylation status at specific CpG sites. Success with this system depends on differential template amplification using methylated (M) and unmethylated (U) primer sets. While most of the considerations for primer design are identical to those for bisulfite PCR, the treatment of CpG sites within the primer is completely different. For MSP it is necessary to locate CpG sites at the 3' end of the primers with cytosines in the methylated primers (M) and thymines in the unmethylated primers (U).
Flowchart of primer design for bisulfite PCR and methylation-specific PCR (MSP).(A) After bisulfite treatment, the two transformed DNA template strands are no longer complementary. It is important to note that only one clone will be amplified with any one set of primers. This is illustrated by the following examples: (B) Bisulfite PCR primers are designed for subsequent sequencing and analysis of cytosines within the amplicon. CpG sites within the primers should be avoided or at the 5' end with a mixed base at the cytosine position (Y= C/T, R= G/A). Sequence data are usually represented by a "lollipop" plot, where closed circles represent methylated cytosine sites and open circles represent unmethylated ones. (C) Methylation-Specific (MSP) PCR primers are designed to target and assess methylation status at specific CpG sites. CpG sites within the primers should be at the 3' end to increase their specificity for methylated (M) or unmethylated (U) templates. Fully methylated or unmethylated templates will generate a single amplicon only from their representative set of primers after MSP. Samples with mixed methylation will be amplified by both sets of primers.
Bisulfite sequence
Bisulfite sequencing remains one of the most common techniques used to analyze bisulfite-converted DNA and provides single-base analysis of the entire amplicon. Cloning followed by sequencing with vector-specific primers is recommended to obtain the best sequencing results for methylation quantification. Direct sequencing of bisulfite PCR products is not recommended as it generally produces poor sequencing quality and will confound the quantification of partially methylated sites. Pyrosequencing can be used as an alternative to direct sequencing.
Advanced technology bisulfite sequence
Next-generation sequencing-based applications, such as reduced-representation bisulfite sequencing (RRBS) and whole-genome bisulfite sequencing (WGBS), are increasingly being used to achieve genome-wide single-base methylation analyzes and to identify potential candidates of biomarkers. In both workflows, conversion efficiency is a primary concern, as even relatively small variations in performance can affect thousands of individual sites. If library preparation is performed before bisulfite conversion, it is important that the adapters added to the fragmented DNA are methylated to preserve their sequence. The use of appropriate control DNAs is also recommended if non-CpG methylation is a factor in samples (e.g., plants) and to account for the inability of bisulfite conversion to differentiate between methylation (5-mC) and hydroxymethylation (5-mC ). - hmC).
Reduced representation bisulfite sequence (RRBS) methylation diagram.Reduced representation bisulfite sequence (RRBS) methylation diagram. The data show the relative methylation rate at individual CpG sites in mouse DNA. The methylation rate occurs in a ∼3 Mb region of mouse chromosome 19. Bisulfite sequencing libraries were prepared using mouse genomic DNA prepared with (D4010, D4011 - Zymo Research) and converted to bisulfite usingEZ DNA methylationtechnology before next-generation sequencing.