DHPLC-Based Method for DNA Methylation Analysis of Differential Methylated Regions from Imprinted Genes

Abstract

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of fingerprint, revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.

Rapid and Quantitative Method of Allele-Specific DNA Methylation Analysis

“…However, bisulfite genomic sequencing requires the cloning of PCR-amplified products and direct sequencing of multiple individual clones that each represents one parental allele from a single cell. Recent high-throughput and quantitative methylation methodologies determine average methylation for either a few CpG sites [denaturing high-performance liquid chromatography (13)] or at each individual CpG site [hairpin bisulfite PCR (27,28), pyrosequencing (8,16), matrix-assisted laser desorption/ionization time-of-flight mass spectometry (MALDI-TOF MS) (15,29), methylation-specific PCR followed by denaturing high-performance liquid chromatography (MSP-DHPLC) (30), and single nucleotide primer extension denaturing high-performance liquid chromatography (SNuPE-DHPLC) (14)]. None, however, approaches the ease and allele-specific resolution of our allele-specific methylation measurement method.…”

Seeking the 5th Base of DNA Using Chromatographic Methods of Analysis

“…Several studies on the detection of gene-specific methylation using LC ( Table 2), involve the detection of methylation in genomic sites, which are normally deactivated by methylation (SNURF-SNRPN) [111][112][113][114], associated with neurological disorders (Angelman/ Prader-Willi syndromes) or other genetic loci (H19 [112,113], LIT1 locus [112][113][114]), or genes (MEST) [112]. Several other studies involve cancer detection and tumour suppressor genes (p16 [115][116][117][118][119] and RASSF1A [118]), genes involved in apoptosis (death-associated protein kinase (DAPK) [118]), genes involved in DNA repair (mismatch repair gene, hMLH1 [120,121]), or other cancer-associated genes such as TSSC5 [112] and WT1 [112].…”

“…Several other studies involve cancer detection and tumour suppressor genes (p16 [115][116][117][118][119] and RASSF1A [118]), genes involved in apoptosis (death-associated protein kinase (DAPK) [118]), genes involved in DNA repair (mismatch repair gene, hMLH1 [120,121]), or other cancer-associated genes such as TSSC5 [112] and WT1 [112]. Other genes studied include those involved in oxidative stress, such as glutathione S-transferase P (GSTP1) [122] and inflammation, such as cycloxygenase-2 (Cox-2) [120], or the regulation of metabolism, such as insulin-like growth factor II precursor (IGF2) [112]. In addition, the number of epigenetically inactivated genes is very extended (galectin-3 (LGALS3) [122], SMAD [122], Gammasynuclein (SNCG) [123], dihydropyrimidine dehydrogenase [NADP+] precursor [124] and H1 histone [47]) and is expected to further increase.…”

“…This ratio was estimated in X-174, calf thymus, [111][112][113][114] LIT1, LIT1 (alias KCNQ1OT1) [112][113][114] H19 [111][112][113] hMLH1, [120,121] MEST/PEG1, IGF2, GSTP1, LGALS3, SMAD4, SNCG, DPYD, TSSC5, WT1, H1 histone, RASSF1A, and DAPK [47,112,118,122,123] salmon sperm, and several mouse tissues [128]. Another more sensitive assay for the detection of 5mC in calf thymus, plasmid pBR322, and bacteriophage X-174 DNA [131], was later developed, using stable isotope dilution.…”

Molecular diagnosis utility of multiplex ligation-dependent probe amplification

“…After subsequent PCR amplification, all uracil was converted to thymidine, which shows different DNA sequences when compared with the corresponding alleles protected by methylation. This sequence-difference can be analyzed by different downstream techniques such as restriction digestion (COBRA) [54,55] , denaturing HPLC [56][57][58][59][60][61] , bidirectional sequencing [62][63][64][65][66][67][68][69] , melting curve analysis [20,70,71] , nucleotide extension assay (MS-SnuPE) [72][73][74] and microarraybased assays [75][76][77][78][79][80] . However, most of these techniques are labor-intensive and allow only a few loci at a time to be studied for methylation status [20,21] .…”