Helper-Dependent Chain Reaction (HDCR) for Selective Amplification of Methylated DNA Sequences.

Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA fragments in a methylation-dependent manner can be combined with a novel method of amplification, Helper Dependent Chain Reaction (HDCR), to selectively amplify these fragments. HDCR uses "Helper" oligonucleotides as templates for extension of the free 3' end of target fragments to incorporate tag sequences at the ends of fragments. These tag sequences are then used for priming of amplification of target fragments. Modifications to the amplification primers (Drivers) and the Helpers ensure that there is selection for the sequences within target fragments with each cycle of amplification. The combination of methylation-dependent enzymes and HDCR allows the sensitive and selective amplification of methylated DNA sequences without the need for bisulfite treatment.

A panel of genes methylated with high frequency in colorectal cancer.

BACKGROUND: The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. METHODS: Combined epigenomic methods - activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment - were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). RESULTS: Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. CONCLUSIONS: This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymes.

We have developed a novel technique for specific amplification of rare methylated DNA fragments in a high background of unmethylated sequences that avoids the need of bisulphite conversion. The methylation-dependent restriction enzyme GlaI is used to selectively cut methylated DNA. Then targeted fragments are tagged using specially designed 'helper' oligonucleotides that are also used to maintain selection in subsequent amplification cycles in a process called 'helper-dependent chain reaction'. The process uses disabled primers called 'drivers' that can only prime on each cycle if the helpers recognize specific sequences within the target amplicon. In this way, selection for the sequence of interest is maintained throughout the amplification, preventing amplification of unwanted sequences. Here we show how the method can be applied to methylated Septin 9, a promising biomarker for early diagnosis of colorectal cancer. The GlaI digestion and subsequent amplification can all be done in a single tube. A detection sensitivity of 0.1% methylated DNA in a background of unmethylated DNA was achieved, which was similar to the well-established Heavy Methyl method that requires bisulphite-treated DNA.

Relative distribution of folate species is associated with global DNA methylation in human colorectal mucosa.

Folate exists as functionally diverse species within cells. Although folate deficiency may contribute to DNA hypomethylation in colorectal cancer, findings on the association between total folate concentration and global DNA methylation have been inconsistent. This study determined global, LINE-1, and Alu DNA methylation in blood and colon of healthy and colorectal cancer patients and their relationship to folate distribution. Blood and normal mucosa from 112 colorectal cancer patients and 114 healthy people were analyzed for global DNA methylation and folate species distribution using liquid chromatography tandem mass spectrometry. Repeat element methylation was determined using end-specific PCR. Colorectal mucosa had lower global and repeat element DNA methylation compared with peripheral blood (P < 0.0001). After adjusting for age, sex and smoking history, global but not repeat element methylation was marginally higher in normal mucosa from colorectal cancer patients compared with healthy individuals. Colorectal mucosa from colorectal cancer subjects had lower 5-methyltetrahydrofolate and higher tetrahydrofolate and formyltetrahydrofolate levels than blood from the same individual. Blood folate levels should not be used as a surrogate for the levels in colorectal mucosa because there are marked differences in folate species distribution between the two tissues. Similarly, repeat element methylation is not a good surrogate measure of global DNA methylation in both blood and colonic mucosa. There was no evidence that mucosal global DNA methylation or folate distribution was related to the presence of cancer per se, suggesting that if abnormalities exist, they are confined to individual cells rather than the entire colon.

Sensitive measurement of unmethylated repeat DNA sequences by end-specific PCR.

We describe a new method that is well-suited for the determination of the methylation level of repetitive sequences such as human Alu elements. We have applied the method to the analysis of cell and tissue DNAs and expect it to have wide utility in studies of DNA methylation in cancer and other disease states, in monitoring response to epigenetic cancer therapies and in epidemiological studies. Only 1 ng DNA is needed for a duplex, one-tube real-time PCR in which methylation level and the amount of input DNA are concurrently measured. The relative cutting by the methylation-sensitive enzyme BstUI is compared with that of the methylation-insensitive enzyme DraI to give a measure of DNA methylation. The method depends upon the use of 5'-tailed, 3'-blocked oligonucleotides called facilitator oligonucleotides (Foligos). Only cut DNAs with specific matching sequences at their 3' ends can copy the tails of the Foligos and thus become tagged and available for subsequent PCR. Both the tagging and PCR are carried out by the same enzyme, Taq DNA polymerase. Because amplification only occurs if suitable ends have been generated in the target DNA, we have called this method end-specific PCR (ESPCR). ESPCR avoids the bisulfite treatment step that is usually required to measure methylation.

Hypomethylation of repeated DNA sequences in cancer.

An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine. Hypomethylation of the genome can affect both single-copy genes, repeat DNA sequences and transposable elements, and is highly variable among and within cancer types. Here, we review our current understanding of genome hypomethylation in cancer, with a particular focus on hypomethylation of the different classes and families of repeat sequences. The emerging data provide insights into the importance of methylation of different repeat families in the maintenance of chromosome structural integrity and the fidelity of normal transcriptional regulation. We also consider the events underlying cancer-associated hypomethylation and the potential for the clinical use of characteristic DNA methylation changes in diagnosis, prognosis or classification of tumors.

Evolutionary evidence suggests that CpG island-associated Alus are frequently unmethylated in human germline.

Most Alu elements are considered to belong to the methylated fraction of the genome that has undergone CpG depletion, whereas CpG islands are characteristically unmethylated. By analysing the CpG content of >12,000 autosomal CpG island-Alu Sx pairs we wanted to study what happens when an Alu is situated close to a CpG island. We have found that many Alus located close to CpG islands have retained a high proportion of CpG sites, which is consistent with these Alus being unmethylated in the human germline.

Bisulphite differential denaturation PCR for analysis of DNA methylation.

Differential denaturation during PCR can be used to selectively amplify unmethylated DNA from a methylated DNA background. The use of differential denaturation in PCR is particularly suited to amplification of undermethylated sequences following treatment with bisulphite, since bisulphite selectively converts cytosines to uracil while methylated cytosines remain unreactive. Thus amplicons derived from unmethylated DNA retain fewer cytosines and their lower G + C content allows for their amplification at the lower melting temperatures, while limiting amplification of the corresponding methylated amplicons (Bisulphite Differential Denaturation PCR, BDD-PCR). Selective amplification of unmethylated DNA of four human genomic regions from three genes, GSTP1, BRCA1 and MAGE-A1, is demonstrated with selectivity observed at a ratio of down to one unmethylated molecule in 10(5) methylated molecules. BDD-PCR has the potential to be used to selectively amplify and detect aberrantly demethylated genes, such as oncogenes, in cancers. Additionally BDD-PCR can be effectively utilized in improving the specificity of methylation specific PCR (MSP) by limiting amplification of DNA that is not fully converted, thus preventing misinterpretation of the methylation versus non-conversion.

Headloop suppression PCR and its application to selective amplification of methylated DNA sequences.

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5' extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure-this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5' extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10(5)-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.