A restriction enzyme, restriction endonuclease, or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.
A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
In biochemistry, the DNA methyltransferase family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor.
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.
Isocaudomers are pairs of restriction enzymes that have slightly different recognition sequences, but upon cleavage of DNA, generate identical overhanging termini sequences. These sequences can be ligated to one another, but then form an asymmetrical sequence that cannot be cleaved by a restriction enzyme.
The HpaII tiny fragment Enrichment by Ligation-mediated PCR Assay is one of several techniques used for determining whether DNA has been methylated. The technique can be adapted to examine DNA methylation within and around individual genes, or it can be expanded to examine methylation in an entire genome.
Neoschizomers are restriction enzymes that recognize the same nucleotide sequence as their prototype but cleave at a different site. In some special applications this is a very helpful feature.
Combined Bisulfite Restriction Analysis is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA. The technique is a variation of bisulfite sequencing, and combines bisulfite conversion based polymerase chain reaction with restriction digestion. Originally developed to reliably handle minute amounts of genomic DNA from microdissected paraffin-embedded tissue samples, the technique has since seen widespread usage in cancer research and epigenetics studies.
Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput technique for analyzing the genome-wide methylation profiles on a single nucleotide level. It combines restriction enzymes and bisulfite sequencing to enrich for areas of the genome with a high CpG content. Due to the high cost and depth of sequencing to analyze methylation status in the entire genome, Meissner et al. developed this technique in 2005 to reduce the amount of nucleotides required to sequence to 1% of the genome. The fragments that comprise the reduced genome still include the majority of promoters, as well as regions such as repeated sequences that are difficult to profile using conventional bisulfite sequencing approaches.
