Molecular Biology Enzymes: Enzymes used in Genetic Engineering and Gene Manipulation.
These include EcoRI, BamHI
Restriction enzymes cleave DNA at specific sequence sites. The sites are palindromic in sequence. These enzymes are used for mapping, cloning and DNA engineering as DNA may be cut and put subsequently into vectors, such as plasmids.
DNA polymerase is the DNA replication enzyme. It incorporates nucleotides through attching them to the 3′ end of an existing DNA single strand. The reverse DNA strand is used as a template to dictate the sequence through complimentarity. DNA polymerase may be used for DNA sequencing and DNA synthesis.
A polynucleotide kinase transfers phosphate groups from dATP to the 3′ end of DNA molecules. Polynucleotide kinase can label DNA strands with 32P, radioactive molecules, or fluorescence phosphates groups. This enzyme has the advantage of not needing a template to add phosphate groups. Therefore, it keeps adding these phosphates until it is removed.
Reverse transcriptase is an enzyme that forms DNA strands from an RNA template. Found in certain viruses, such as HIV, it can be used in molecular biology to synthesize DNA from expressed sequences, such as transcripts that encode proteins. cDNA denotes complementary DNA generated from an RNA template often.
DNA ligase is a molecular enzyme that circularizes DNA through joining the 3′ end of a DNA strand to a 5′ end of another or the same DNA molecule. Very useful in cloning to make circular vectors.
DNA nucleases digest DNA completey. These enzymes cleave DNA internally, as well as from the ends.
These enzymes digest DNA from either end, the 3′ or 5′ ends. They cleave only from outside of the DNA strands.
These enzymes digest RNA completey, similarly to the DNA nuclease, except for RNA. Used to clear DNA samples of contamination by RNA.
Terminal transferases add nucleotides to the 3′ ends of DNA molecules without the need of a template strand. Can label DNA with 32P or a fluorescent group. Mainly used in cloning.
DNA methylase adds a methyl group to nucleotides. Used in order to prevent restriction enzyme cleavage of sequences. Acts to block the site from cleavage.