LITERATURE REVIEW
1.5 Mapping of Genome-wide DNA Methylation
1.5.2 Methylation-Sensitive Restriction Enzyme PCR
The classic tool of DNA Methylation analysis, Methylation-Sensitive Restriction Enzymes employ the use of restriction enzymes that recognize short pieces of DNA and cleave the DNA at distinct sites within or adjacent to these sequences. Some enzymes are sensitive to methylation and will not cleave the DNA if a cytosine residue is methylated, whilst other enzymes are insensitive to methylation and will specifically digest methylated DNA (Bird, 1986). An example of an enzyme that is insensitive to methylation is McrBC which is an Escherichia coli endonuclease that cleaves methylated DNA on one or both strands. Essentially, restriction enzyme-based methods either enrich for methylated DNA or
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unmethylated DNA. Comparisons are made in one of the following ways; between a sample treated with an enzyme or a combination of enzymes and an untreated control; between a sample treated with a methylation-sensitive enzyme compared with a control treated with a methylation-insensitive isoschizomer or finally, between two test samples, such as two tissue types both digested with the same enzyme. Enrichment of unmethylated DNA, by digesting methylated DNA or by isolating smaller fragments generated by methylation-inhibited enzymes, is particularly useful for analysis of large, heavily methylated genomes. In the human genome, about 60-90% of CpG sites are methylated (Kader and Ghai, 2015), and hence enriching unmethylated DNA significantly reduces the complexity of the sample. The approach is robust, simple and does not require large quantities of DNA. However, efficient digestion of template DNA is absolutely vital, or else spurious results may be obtained. High quality DNA is necessary for efficient analysis and the main drawback of the method is dependence of the availability of recognition sequences that flank the sequences of interest.
Frequently employed enzymes are the isoschizomers HpaII and MspI, both of which recognize the sequence CCGG. Whereas MspI is blocked only by methylation of the outer cytosine, HpaII is blocked by methylation of either cytosine. Since in mammalian genomes, methylation occurs chiefly in CpG sites HpaII is inhibited and MspI is not (Goll and Bestor, 2005). Another useful enzyme employed frequently in genomic studies is McrBC, an E.coli endonuclease that cleaves between two methylated cytosines in the context (G/A) metC, (Lippman et al., 2006; Rollins et al., 2006; Schumacher et al., 2006; Sutherland et al., 1992).
The two sites can be separated by up to 3 kb, but the optimal separation is 55-100 bp (Gowher et al., 2000; Zhou et al., 2002). For this reason, McrBC is an excellent tool for the removal of densely methylated DNA. Although less of an issue with McrBC, sequence polymorphisms between samples can mimic methylation differences if they affect the enzyme recognition site. Therefore, it is safest to use restriction enzymes to compare samples that have no or little polymorphism, such as different tissues from the same organism.
Extensive digestion of genomic DNA by means of the restriction enzyme may be followed by multiplex PCR amplification (Figure 1.3) of user-defined genes via gene-specific primers that flank the recognition site of the enzyme in use, in an amplification reaction termed Methylation-Sensitive Restriction Enzyme Polymerase Chain Reaction (MSRE-PCR) (Choi et al., 2014; Melnikov et al., 2005).
MSRE combined with PCR may be followed with methylation analysis employing standard capillary electrophoresis platforms (An et al., 2013; Choi et al., 2014; Melnikov et
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al., 2005). MSRE-PCR is ideal for rapid DNA methylation analysis in a user defined set of genes, which is based on extensive digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR amplification of surviving fragments. A fragment will be designated ‘unmethylated’ if no PCR product is observed after digestion. Alternatively, the fragment will be called ‘methylated’ if it can be amplified after digestion.
Figure 1.3: Methylation-Sensitive Restriction Enzyme Polymerase Chain Reaction (MSRE-PCR). Genomic DNA is cleaved by the endonuclease, for example HhaI which cleaves at recognition sites (GCGC). This is followed by PCR amplification of surviving fragments. If a fragment may be amplified after PCR, it will be termed ‘methylated’. If a PCR product is not detected after digestion, the fragment is called ‘unmethylated’.
MSRE-PCR allows simple multiplexing and avoids some of the problems inherent in bisulfite conversion, in particular the poorly controlled efficiency of modification, which can be incomplete due to incomplete denaturation or partial renaturation of DNA during treatment (Rein et al., 1997); comprehensive modification of unmethylated cytosines is required for correct readout, which can be influenced by various factors including DNA apurinization during bisulfite treatment (Harrison et al., 1998; Reeben and Pryds, 1994;
Stirzaker et al., 1997) and downstream differentiation of the methylated versus unmethylated sequence in many bisulfite-based methods requires two pairs of primers and two PCR reactions for each potentially methylated fragment which reduces the throughput of MSP and similar techniques, making screening of clinical samples more labour-intensive in bisulfite conversions. Finally, the yield of each product depends on the quality of the corresponding primers and can result in biased PCR if the amplification efficiency is different (Melnikov et al., 2005; Stirzaker et al., 1997). MSRE-PCR is a major detection tool that provides many pros. Since analysis may be performed using a universally known method of electrophoresis special training is not necessary. Multiple DNA templates may be analysed in a single assay (An et al., 2013; Choi et al., 2014). An important feature of the MSRE-PCR assay is its
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ability to detect promoter methylation in heterogeneous samples, even when methylated sequences represent a small fraction of the overall specimen (Melnikov et al., 2005).
1.5.2.1 Capillary Electrophoresis
The primary methodology used for separating and detecting short tandem repeat (STR) alleles, capillary electrophoresis is applied to numerous fields of research, especially in forensic DNA typing. To achieve trustworthy STR typing, three conditions must be met.
Spatial resolution is needed to separate STR alleles that may differ in size by a single nucleotide; spectral resolution is needed to separate fluorescent dye colours from one another so that PCR products from loci labelled with different dyes can be resolved; and third, DNA sizing precision from run to run must be consistent enough so that samples can be related to allelic ladders that are run for calibration purposes (Butler, 2012). These specific requirements have been met with a variety of CE systems; and numerous studies have validated this with excellent findings. When capillary electrophoresis was applied to analyse 53 promoters of breast cancer cell lines (MCF-7, MDA-MB-231 and T47D) that were subjected to MSRE-PCR using Hin61 enzyme, the MSRE-PCR followed by the CE system rectified the methylation status of genes analysed by other techniques (Melnikov et al., 2005).
MSRE-PCR and electrophoresis is often used in the analysis of tDMRs to differentiate body fluids that may be located at crime scenes. When using tDMRs as markers, primers are designed to specifically target the methylated region within the tDMR. This is achieved by designing primers that flank the recognition site of the enzyme that will be used (An et al., 2013; Melnikov et al., 2005). Frumkin and colleagues (2011) subjected 50 DNA samples from blood, saliva, semen, and skin epidermis to digestion by HhaI, followed by multiplex amplification of specific genomic targets with fluorescent-labelled primers, capillary electrophoresis of amplification products, and automatic signal analysis by dedicated software (Figure 1.4). The investigation profitably yielded the source tissue of the samples. The system was described as fully automatable, provided operator-independent results, and allowed combining tissue identification with profiling in a single procedure which is quite favourable for forensic applications. Detection of semen and DNA profiling were combined into one assay and the ability to detect mixtures of semen and saliva in various ratios was demonstrated. The calculated percentage of semen was comparable to the fraction of semen in the samples. The same enzyme, HhaI was employed by An and colleagues (2013) and Choi et al. (2014); both studies successfully differentiated between various body fluids by use of MSRE-PCR in combination with capillary electrophoresis.
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1.5.2.2 Restriction Landmark Genomic Scanning
Based on the notion that within any genome, restriction enzyme sites may signify landmarks, Restriction Landmark Genomic Scanning (RLGS) is a quantitative method (Costello et al., 2002; Song et al., 2005) that allows for high resolution two-dimensional display of direct radio-labelled genomic DNA digested fragments. It enables recognition of high amounts; possibly over two thousand restriction landmarks in just a single assay. The method may be employed to determine epigenetic alterations in tissues, tumours as well as cancer cell lines (Ando and Hayashizaki, 2006; Rush and Plass, 2002). DNA is restricted with a rare-cutting enzyme, as methylation sensitivity of the endonuclease activity of the particular enzyme serves as a basis for detection of differential methylation patterns.
Figure 1.4: Detection of sample is performed automatically by measuring time span from sample injection to sample detection with a laser placed near the capillary end. Laser light is shone on the capillary where a window is burned into the coating of the capillary.
DNA fragments are illuminated upon passing the window. Smaller DNA molecules are detected before larger molecules in order of migration speed which correlates with length or number of base pairs. Data from CE separations are plotted as a function of the relative fluorescence intensity observed from fluorescence emission of dyes passing the detector. The fluorescent emission signals from dyes attached to DNA molecules can then be used to detect and quantify the DNA molecules passing the detector (Butler, 2012).
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Although the system is applicable to all organisms and demonstrates high scanning extensibility by use of a combination of enzymes, it is disadvantageous in terms of efficiency;
as results may be obtained between five days and two weeks. Small amounts of DNA samples cannot be analysed as the method requires a few good quality micrograms (Ho and Tang, 2007; Smiraglia et al., 2007). Additionally the technique requires use of specific software such as Virtual Image-RLGS (VI-RLGS), expensive high-efficiency scanning capacity instruments and advanced image analysis systems such as a Fuji BAS2500 system (Costello et al., 2002; Ho and Tang, 2007; Okuizumi et al., 2010).