Microarray-based resequencing by apyrase-mediated allele-specific extension

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

DNA methylation-regulated microRNA pathways in ovarian serous cystadenocarcinoma: A meta-analysis

Epigenetic regulation has been linked to the initiation and progression of cancer. Aberrant expression of microRNAs (miRNAs) is one such mechanism that can activate or silence oncogenes (OCGs) and tumor suppressor genes (TSGs) in cells. A growing number of studies suggest that miRNA expression can be regulated by methylation modification, thus triggering cancer development. However, there is no comprehensive in silico study concerning miRNA regulation by direct DNA methylation in cancer. Ovarian serous cystadenocarcinoma (OSC) was therefore chosen as a tumor model for the present work.Twelve batches of OSC data, with at least 35 patient samples in each batch, were obtained from The Cancer Genome Atlas (TCGA) database. The Spearman rank correlation coefficient (SRCC) was used to quantify the correlation between the CpG DNA methylation level and miRNA expression level. Meta-analysis was performed to reduce the effects of biological heterogeneity among different batches. MiRNA-target interactions were also inferred by computing SRCC and meta-analysis to assess the correlation between miRNA expression and cancer-associated gene expression and the interactions were further validated by a query against the miRTarBase database.A total of 26 potential epigenetic-regulated miRNA genes that can target OCGs or TSGs in OSC were found to show biological relevance between DNA methylation and miRNA gene expression. Furthermore, some of the identified DNA-methylated miRNA genes; for instance, the miR-200 family, were previously identified as epigenetic-regulated miRNAs and correlated with poor survival of ovarian cancer. We also found that several miRNA target genes, BTG3, NDN, HTRA3, CDC25A, and HMGA2 were also related to the poor outcomes in ovarian cancer.The present study proposed a systematic strategy to construct highly confident epigenetic-regulated miRNA pathways for OSC. The findings are validated and are in line with the literature. The inclusion of direct DNA methylated miRNA events may offer another layer of explanation that along with genetics can give a better understanding of the carcinogenesis process.     

Quantitative Detection of Gene Methylated Level of Stool Samples Based on Invader Assay Coupled with Real-time Polymerase Chain Reaction and Its Application in Non-invasive Screening of Colorectal Cancer

Methylation of bone morphogenetic protein 3 (BMP3) in stool DNA is an effective biomarker for non-invasive screening of colorectal cancer. However, a highly sensitive and specific detection method is required. Here, a quantification method for BMP3 methylation was developed by combining real-time polymerase chain reaction (PCR) with invader assay using Beta-actin (ACTB) as a reference. Amplification efficiencies of BMP3 and ACTB were close to 100% after optimizing the concentration of detection probes, FEN1 enzyme and Taq polymerase, and the relative quantification of BMP3 methylation was achieved accurately by ΔCT algorithms. Ten copies and 0.01% of BMP3 methylation level could be successfully detected and non-specific signal was generated from non-methylated template, indicating that the method was highly sensitive and specific. The method was successfully applied to detect BMP3 methylation in fecal DNA from 16 colorectal cancer patients, 7 adenoma patients and 19 healthy volunteers. The results indicated that BMP3 methylation occurred in 5 of 16 cancer patients and 2 of 7 adenoma patients, but was not observed in 19 of healthy volunteers. Therefore, this method could be used to quantify methylation of gene in stool samples, providing an effective technique for non-invasive screening of colorectal cancer.     

Fluorescent conjugated polyelectrolyte as an indicator for convenient detection of DNA methylation

A convenient, sensitive, and label-free method to determine the DNA methylation status of CpG sites of plasmid and human colon cancer cell has been developed. The system relies on highly selective single base extension reaction and significant optical amplification of cationic conjugated polyelectrolytes (CCP-1). The higher fluorescence resonance energy transfer efficiency between CCP-1 and fluorescein-labeled dGTP (dGTP-Fl) is correlated to the incorporation of dGTP-Fl into the probe DNA by single base extension reaction when the target/probe pair is complementary at the methylation site. As low as 1% methylation status can be determined by this new assay method. Because of the optical amplification property of CCP-1, the method exhibited high sensitivity with a concentration of analyte DNA at the picomolar level. The CCP-1 can form a complex with negatively charged DNA through electrostatic interactions, avoiding labeling the DNA target and probe by covalent linking. The isolation steps employed in other typical assays were avoided to simplify operations and increase repeatability. These features make the system promising for future use for early cancer diagnosis.     

Evaluation of 15 polymerases and phosphorothioate primer modification for detection of UV-induced C:G to T:A mutations by allele-specific PCR

Allele-specific polymerase chain reaction is based on polymerase extension from primers that contain a 3' end base that is complementary to a specific mutation and inhibition of extension with wild-type DNA due to a 3' end mismatch. Taq polymerase is commonly used for this assay, but because of the high rate of nucleotide extension from primer 3' base mismatches documented for Taq polymerase, high sensitivity is difficult to achieve. To determine whether other polymerases might improve assay sensitivity, 15 polymerases were tested with mutation-specific primers for two ultraviolet-induced mutations in the human 5S ribosomal RNA genes. Of the 15 polymerases tested, six were capable of discriminating these mutations at levels equivalent to or better than Taq polymerase. All primers were phosphorothioate modified on the 3' end to block removal of the critical 3' mutation-specific base by polymerases containing 3' --> 5' exonuclease "proofreading" activity. The effectiveness of phosphorothioate modification was measured in mock polymerase chain reaction reactions and a time course. All six enzymes containing this exonuclease activity showed some ability to digest phosphorothioate-modified primers and could be divided into two groups, showing fast and slow digestion kinetics. Of the three enzymes that showed slow digestion kinetics, two also showed significantly slower digestion kinetics of unmodified primers.     

Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer‐adjacent tissues collected from 30 CRC patients and then bisulfite‐converted. The exon regions of 5 targeted genes (CNRIP1 , HIC1 , RUNX3 , p15 , and SFRP2 ) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC‐MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC‐MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer‐adjacent individuals, while that of HIC1 , p15 , and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC‐MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.     

Direct Sensing of 5‐Methylcytosine by Polymerase Chain Reaction

The epigenetic control of genes by the methylation of cytosine resulting in 5‐methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′‐mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation‐specific PCR approach directly from untreated human genomic DNA.     

A preamplification approach to GMO detection in processed foods

DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.     

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.     

UVB-induced Cyclobutane Pyrimidine Dimer Frequency Correlates with Skin Cancer Mutational Hotspots in p53

Abstract— Ultraviolet light has been identified as the major carcinogen in skin cancer and the p53 tumor suppressor gene is a major target for UV-induced mutations. The mutations are probably caused by unrepaired UV-induced cyclobutane pyrimidine dimers (CPD) and possibly by the less frequent pyrimidine (6-4) pyrimidone photoproducts. While hot spots for p53 mutations in human nonmela-noma skin tumors correspond quite well to slow spots for CPD repair in cultured cells irradiated with the model mutagen 254 nm UVC (which is not present in terrestrial sunlight), they do not all coincide with sequences that are initially frequently damaged by 254 nm UVC. Using LMPCR (ligation-mediated polymerase chain reaction), we show that environmentally relevant UVB light induces CPD at CC and PyrmC positions much more frequently than does UVC light, and that all eight skin cancer hot spots in p53 are also hot spots for UVB-induced CPD. Our results show that methylation of dipyrimidine sites (PyrmCpG) is associated with an increase rate of CPD formation upon UVB irradiation. Consequently, DNA methylation may increase the mutagenic potential of UVB and explains that several p53 mutation hot spots are found at PyrmCpG. The distribution patterns of CPD formation and the photofootprint patterns found along exons 5 and 6 of p53 gene are suggestive of DNA folding into nucleosomes.     

Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray

Aberrant DNA methylation of CpG sites has been confirmed to be closely associated with carcinogenesis.Based on the hyperbranched rolling circle amplification(HRCA) and microarray techniques,a new method for qualitative detection of methylation was developed.In the present study,padlock probes hybridize the sample DNA at the methylation site to form a probe-DNA complex which is ligated and digested simultaneously by methylation specific enzymes.Only at the methylated CpG site is the padlock probe ligated successfully to form a circle template for the HRCA reaction.Utilizing the method of 3-dimensional polyacrylamide gel-based microarray,the HRCA product will be immobilized on the slide to form a DNA microarray,which can universally hybridize the Cy3-labeled oligonucleotide probe to detect the methylation status of CpG sites.To control the false positive signals,DNA ligase and temperature of ligation/digestion are optimized.Methylation status of four CpG sites located in P15,Ecadherin,hMLH1 and MGMT genes were analyzed successfully with this method and all the results were compatible with that of methylation-specific PCR.Our research proves that this method is simple and inexpensive,and could be applied as a high-throughput tool to qualitatively determine the methylation status of CpG sites.     

The role of DNA hypermethylation in human neoplasia

Cancer development and progression is dictated by a series of alterations in genes such as oncogenes, tumor suppressor genes, DNA repair genes, and others. DNA methylation is an epigenetic modification that is profoundly altered in most cancers. Recently, hypermethylation of CpG-rich areas located in the promoter of genes (CpG islands) has been shown to be commonly implicated in silencing tumor suppressor genes in cancer. By cloning and characterizing a large number of such CpG islands hypermethylated in colon cancer, we found that two processes explain most of these events. Age-related CpG island methylation in a subset of cells in normal tissues, followed by intensification of methylation in cancer cells explains the majority of hypermethylation events in colon cancer and may provide a mechanistic link between aging and cancer formation. Most of the other CpG islands methylated in colon cancer can be explained by a newly described phenotype, the CpG island methylator phenotype (CIMP) which results in multiple methylation events in a subset of cancers. CIMP accounts for the majority of sporadic colon cancers characterized by microsatellite instability, as well as most tumors with k-ras mutations. Understanding further the factors that lead to, and modulate, aberrant methylation in cancer may provide novel avenues for prevention and treatment of this disease.     

Methylation signature genes identification of cancers occurrence and pattern recognition

In order to identify the signature genes of tumorigenesis, the pattern-recognition method was used to analyze the gene methylation (ME) data which included only normal and cancer samples and was collected from the TCGA (The Cancer Genome Atlas) database. Here, we analyzed the DNA methylation profiles of the six types of cancer and the ME signature genes for each cancer were selected by means of a combination of correlation, student's t-test and Elastic Net. Modeling by support vector machine, the accuracy of ME signature genes can be as high as 98 % for training set and as high as 97 % for the independent test set, the recognition accuracy of stage I is more than 97 % for training set and more than 98 % for test set. Then, the common signature genes and common pathways emerging in multiple cancers were obtained. A functional analysis of these signature genes indicates that the identified signatures have direct relationship with tumorigenesis and is very important for understanding the pathogenesis of cancer and the early therapy.     

Advent of the cancer methylome

DNA hypermethylation of CpG islands plays an important role in gene regulation during cancer development. Many techniques have been developed to detect global DNA methylation in cancer cells compared to normal tissues. This knowledge helps us to better understand cancer progression and also aids in the development of new biomarker for early cancer detection. New prognostic tools for monitoring drug efficacy during cancer treatment can also be developed. In this review, we will examine the different techniques that have been used to study DNA methylation, as well as the emerging high resolution, high throughput techniques for identification of methylated regions to defining cancer related genes in the cancer methylome.     

Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem

PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3' end were carried out in order to develop allele-specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3' end were used. In particular, 3' terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases. While less PCR product was observed for primers forming three terminal mismatch pairings, target DNA sequences were efficiently amplified by primers forming two mismatch pairings next to the terminal G/C base pairing. These results indicate that selecting a primer having a 3' terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele-specific primer that eliminates the pseudopositive problem. Trials with the human ABO genes demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.