Restriction endonucleases are DNA-cleaving enzymes that recognize one or several target sequences and cut DNA at or near these sequences. Lifeasible uses restriction endonucleases as the basis for restriction cleavage of methylated DNA (McrBC) or unmethylated DNA (e.g., NotI and Hpall) with one or more enzymes. We have combined this method with capillary sequencing, microarrays, and other technologies to detect genome-wide methylation levels in various organisms, limited to CpG sites recognized by endonucleases.

Methylation-sensitive amplification polymorphism (MSAP)

MSAP is based on the amplified fragment length polymorphism (AFLP) technique. MSAP technology can detect cytosine methylation changes at CCGG sites on a genome-wide scale compared to other DNA methylation measurement techniques. We can operate directly based on AFLP without knowing the DNA sequence information to be measured. We can use this method to help you with genome-wide methylation status studies of plants and animals.

• Extraction of genomic DNA.
• According to the different sensitivity of MspI and Hpa II enzymes to methylation, genomic DNA was double digested with EcoR I / MspI and EcoR I / Hpa II enzyme combinations, respectively.
• After attaching the junction, PCR amplification (pre-amplification) was performed with primers designed according to the junction sequence plus a selective base.
• The pre-amplified product was diluted, and a second PCR amplification (selective amplification) was performed by adding two more selective base primers.
• The amplified products were denatured and electrophoresed on 6% PAGE gels.
• The PAGE gel was processed by isotope radioautography or silver staining.
• The methylation status of the genomic CpG sites was determined by counting and analyzing the DNA bands.
• The differentially amplified fragments were recovered and sequenced, and the methylation differential sites could be identified by comparison.

McrBC enzymatic cleavage

McrBC is a DNA methylation-specific restriction endonuclease that recognizes the methylated C in the (A/G)C motif and cleaves DNA containing methylated cytosine on one or both strands. We usually combine this technology with microarray technology to detect methylated DNA.

The DNA recognition site of McrBC contains two half-sites of the (G/A) mC form that do not act on unmethylated DNA. The optimal half-site for cleavage has a spacing of 55 bp-103 bp, and this spacing can be up to 3 kb long. McrBC cleavage requires the involvement of guanosine triphosphate (GTP). This enzyme binds specifically to methylated DNA in the presence of a non-hydrolyzable analog of GTP without producing a cleavage reaction. McrBC cleaves on a pair of PumCG sequence elements, and we can use this technique to detect a significant fraction of methylated CpG.

Lifeasible is based on restriction endonucleases, and we will combine this approach with technologies such as capillary sequencing and microarrays, allowing us to detect genome-wide methylation levels in a wide range of organisms. Please feel free to contact our staff for more information.

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For research or industrial use.

Related Services

• Bisulfite-Based DNA Methylation Detection
• Immunoprecipitation of Methylated DNA
• SMRT Sequencing for Direct Detection of DNA Methylation
• Single Molecule Nanopore Dependent Methylation Sequencing
• RNA Methylation
• RNA-Directed DNA Methylation
• DNA Hydroxymethylation