Segregation of partly melted DNA molecules

Segregation of partly melted DNA molecules is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The method stands on the observation that the electrophoretic mobility of partly melted DNA fragments in a denaturing gradient gel is low and that they persist in the gel so long as the remaining helical part is sufficiently resistant to strand dissociation and dissociates slowly. Such features are observed in DNA fragments derived from CpG islands. These DNA fragments are preferentially retained in a denaturing gradient gel after prolonged electric field exposure, permitting the enrichment of DNA fragments derived from CpG islands. The principle and practical application of this method are reviewed.     

The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partlymelted molecules.

We isolated fragments containing parts of CpG islands from human chromosomal regions chosen for expected differences in gene density by segregation of partly melted molecules. Restriction fragments of P1 bacteriophage clones covering a region of 11q13 and those of cosmid clones derived from Xp22 were recovered from bands in denaturing gradient gels that were retained following prolonged exposure to electric field. Forty-five independent fragments derived from 11q13 and five from Xp22 were isolated. Nucleotide sequence analysis revealed that 11 of the 45 fragments from 11q13 contained CpG islands including four derived from known genes in 11q13. None of the five fragments derived from Xp22 resembled CpG islands. The number of CpG island fragments obtained was consistent with the expectation based on the number of Not I restriction endonuclease sites present at these regions. Adjustment of parameters in our quasi-theoretical approach to the rate of fragment dissociation improves the discrimination between retention and non-retention. The results support probable identification of CpG island fragments by their reduced rate of strand dissociation when retarded in a denaturing gradient gel.     

Segregation of partly melted molecules: isolation of CpG islands by polyacrylamide gel electrophoresis

The technique of segregation of partly melted molecules (SPM) is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The approach is conceptually simple and uses denaturant gradient gel electrophoresis to separate DNA molecules digested with restriction endonucleases. The SPM methodology has successfully been applied to the identification of genes from anonymous, unsequenced DNA fragments and CpG islands methylated in human cancer. In this article the theoretical background and practical application of the SPM method is reviewed.     

Quick Identification and Localization of CpG Islands in Large Genomic Fragments by Partial Digestion with Hpa II and Hha I

More than 50% of mammalian genes are associated with CpG islandsand thus they serve as a good gene marker. We have devised asimple method to scan large pieces of native or cloned genomicDNA for CpG islands. The method is based on the presence ofmultiple Hpa II and Hha I sites in CpG islands, at a frequency30 times higher than in the rest of the genome. The steps includecomplete digestion of DNA with a rare-cutting restriction endonuclease(to produce large fragments with defined ends), partial digestionwith Hpa II and Hha I, and subsequent Southern hybridizationwith an end probe. This identifies a CpG island as a clusterof sub-bands and, based on their electrophoretic mobility, onecan immediately locate the island relative to the ends. Formany vectors, universal probes flanking the cloning site areavailable, enabling the simultaneous analysis of a large numberof samples. We demonstrated the usefulness of the method byanalyzing known CpG islands in native genomic DNA and lambda,cosmid and P1 clones, and by isolating two novel transcribedislands from anonymous cosmid clones. Our method is quick, inexpensive,and can detect CpG islands with few or even no rare-cutter sites.     

Alterations in DNA helix stability due to base modifications can be evaluated using denaturing gradient gel electrophoresis

DNA molecules that differ by a single base-pair can be separated by denaturing gradient gel electrophoresis due to the sequence-specific melting properties of DNA. Base modifications such as methylation are also known to affect the melting temperature of DNA. We examined the final position of DNA fragments containing either 5-methyl-cytosine or 6-methyl-adenine in denaturing gradient gels. The presence of a single methylated base within an early melting domain resulted in a well-resolved shift in fragment position relative to the unmethylated sequence. In addition, fragments containing hemimethylated and fully methylated sites could be distinguished, and a proportionally larger shift was observed with an increasing number of methylated bases. Denaturing gradient gel electrophoresis thus provides a sensitive method for analyzing the methylation state of DNA, which is not dependent on the presence of restriction enzyme cleavage sites. We also demonstrate that denaturing gradient gel electrophoresis can be used to obtain a quantitative estimate of the change in helix stability caused by modification of one or two bases in a complex DNA sequence. Such estimates should allow more accurate modeling of melting of natural DNA sequences.     

Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis.

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.     

DNA motifs associated with aberrant CpG island methylation

Epigenetic silencing involving the aberrant methylation of promoter region CpG islands is widely recognized as a tumor suppressor silencing mechanism in cancer. However, the molecular pathways underlying aberrant DNA methylation remain elusive. Recently we showed that, on a genome-wide level, CpG island loci differ in their intrinsic susceptibility to aberrant methylation and that this susceptibility can be predicted based on underlying sequence context. These data suggest that there are sequence/structural features that contribute to the protection from or susceptibility to aberrant methylation. Here we use motif elicitation coupled with classification techniques to identify DNA sequence motifs that selectively define methylation-prone or methylation-resistant CpG islands. Motifs common to 28 methylation-prone or 47 methylation-resistant CpG island-containing genomic fragments were determined using the MEME and MAST algorithms (). The five most discriminatory motifs derived from methylation-prone sequences were found to be associated with CpG islands in general and were nonrandomly distributed throughout the genome. In contrast, the eight most discriminatory motifs derived from the methylation-resistant CpG islands were randomly distributed throughout the genome. Interestingly, this latter group tended to associate with Alu and other repetitive sequences. Used together, the frequency of occurrence of these motifs successfully discriminated methylation-prone and methylation-resistant CpG island groups with an accuracy of 87% after 10-fold cross-validation. The motifs identified here are candidate methylation-targeting or methylation-protection DNA sequences.     

Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes

It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes.     

Alu-PCR Approach to Isolating NotI-Linking Clones from the 3p14-p21 Region Frequently Deleted in Renal Cell Carcinoma

In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating Not I sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.     

A Hybrid Approach for CpG Island Detection in the Human Genome

Background

CpG islands have been demonstrated to influence local chromatin structures and simplify the regulation of gene activity. However, the accurate and rapid determination of CpG islands for whole DNA sequences remains experimentally and computationally challenging.

Methodology/Principal Findings

A novel procedure is proposed to detect CpG islands by combining clustering technology with the sliding-window method (PSO-based). Clustering technology is used to detect the locations of all possible CpG islands and process the data, thus effectively obviating the need for the extensive and unnecessary processing of DNA fragments, and thus improving the efficiency of sliding-window based particle swarm optimization (PSO) search. This proposed approach, named ClusterPSO, provides versatile and highly-sensitive detection of CpG islands in the human genome. In addition, the detection efficiency of ClusterPSO is compared with eight CpG island detection methods in the human genome. Comparison of the detection efficiency for the CpG islands in human genome, including sensitivity, specificity, accuracy, performance coefficient (PC), and correlation coefficient (CC), ClusterPSO revealed superior detection ability among all of the test methods. Moreover, the combination of clustering technology and PSO method can successfully overcome their respective drawbacks while maintaining their advantages. Thus, clustering technology could be hybridized with the optimization algorithm method to optimize CpG island detection.

Conclusion/Significance

The prediction accuracy of ClusterPSO was quite high, indicating the combination of CpGcluster and PSO has several advantages over CpGcluster and PSO alone. In addition, ClusterPSO significantly reduced implementation time.     

Reduced representation bisulfite sequencing design for assessing the methylation of human CpG islands in large samples

The reduced representation bisulfite sequencing (RRBS) method has been developed for the high-throughput analysis of DNA methylation based on the sequencing of genomic libraries treated with sodium bisulfite by next-generation approaches. In contrast to whole-genome sequencing, the RRBS approach elaborates specific endonucleases to prepare libraries in order to produce pools of CpG-rich DNA fragments. The original RRBS technology based on the use of the MspI libraries allows one to increase the relative number of CpG islands in the pools of genomic fragments compared to whole-genome bisulfite sequencing. Nevertheless, this technology is rarely used due to the high cost compared with bisulfite methylation analysis with hybridization microarrays and significant residual amount of data represented by the sequences of genomic repeats that complicates the alignment and is not of particular interest for developing DNA methylation markers, which is often the main goal of biomedical research. We have developed an algorithm for estimating the likelihood that recognition sites of restriction endonucleases will be represented in CpG islands and present a method of reducing the effective size of the RRBS library without a significant loss of the CpG islands based on the use of the XmaI endonuclease for library preparation. In silico analysis demonstrates that the optimum range of the XmaI-RRBS fragment lengths is 110–200 base pairs. The sequencing of this library allows one to assess the methylation status of over 125000 CpG dinucleotides, of which over 90000 belong to CpG islands.

     

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method¹. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 °C during the gel run allows for the separation of unstructured DNA or RNA molecules.In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol ^1,2.     

DDGGE, a simplified denaturing gradient gel electrophoresis method for the detection of sequence variations in PCR fragments

The need for rapid analysis of sequence variations in PCR fragments of the same length is increasing in medical diagnostics and environmental studies. Therefore a modified denaturing gradient gel electrophoresis (DGGE) method was developed in which mixed PCR fragments of 1,500 bp could be analysed on a conventional DNA sequencing gel apparatus. In addition, PCR primers without long GC-clamps could be used to amplify the target genes. © Rapid Science Ltd. 1998     

Development of a polymerase chain reaction-based denaturing gradient gel electrophoresis technique to study nematode species biodiversity using the 18s rDNA gene

A polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) technique was developed to evaluate nematode biodiversity. Amplified fragments (approximately 630 bp) of nematode 18S rDNA could be separated by DGGE at 60 °C for 5.5 h using a gradient of 30–55% denaturant. No DNA from the nematodes’ food source was observed on the gels. The sensitivity of the system was tested using DNA from six species of Steinernema. The system clearly separated PCR fragments from all species except S. tami and S. carpocapsae, which had similar melting behaviour despite being widely separated on the phylogenetic tree.     

Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography

We report here a novel method to simultaneously detect CpG methylation and single nucleotide polymorphisms (SNPs) using denaturing high performance liquid chromatography (DHPLC). PCR products of bisulfite-modified CpG islands were separated using DHPLC. BstUI digestion and DNA sequencing were used in confirmation studies. Consistent with the BstUI digestion assay, the 294 bp PCR product of the modified hMLH1 promoter showed different retention times between the methylated cell lines (RKO and Cla, 6.7 min) and the unmethylated cell lines (PACM82 and MGC803, 6.2 min). No hMLH1 methylation was observed in 13 primary gastric carcinomas and their matched normal tissues. One hMLH1 SNP was detected in gastric cancer patients, in both cancer and normal tissues. DNA sequencing revealed that the SNP is a G→A variation at –93 nt of the hMLH1 promoter. A two-peak chromatogram was also obtained in the 605 bp PCR product of the Cox-2 promoter of the AGS, HEK293 and MKN45 cell lines by DHPLC. Another peak corresponding to methylated CpG islands was observed on the chromatogram of the Cox-2-methylated AGS cell line after bisulfite treatment. In conclusion, methylation in homoallelic and heteroallelic CpG islands could be detected rapidly and reliably by bisulfite–DHPLC. A SNP in the target sequence could also be detected at the same time.     

1.5-Mb YAC Contig in Xq28 Formatted with Sequence-Tagged Sites and Including a Region Unstable in the Clones

A contig of 20 yeast artificial clones (YACs) has been assembled across 1.5 Mb of Xq28 and formatted with nine previously reported probes and nine STSs developed from the sequence of probes and end fragments of YACs. YAC end fragments were obtained by subcloning, Alu-vector PCR, or primer-ligation PCR methods. Eighteen of the YACs were recovered from a library specific for Xq24-q28; two that fill a gap were obtained from a second library made from total human DNA. One region, containing probes pX78c and 2A1.1, was unstable in YACs, but it was possible to generate a self-consistent map of DNA over the entire contig. Overlaps were confirmed by Southern blot analyses of YAC DNAs, and pulsed-field gel electrophoresis confirmed the extent of the contig and identified at least four CpG islands in the region.