Question

What are type II restriction endonucleases (RE)? Give an example of a type II RE that generates flush ends and the sequence recognized by it. Mention two other enzymes and their utility in cloning experiment.

Answer 1

Type II restriction endonucleases (RE) are enzymes that recognize and cleave DNA at specific nucleotide sequences. These enzymes are used extensively in recombinant DNA technology for DNA cloning, gene expression analysis, and genome editing. Type II REs are named as such because they cleave the DNA strand within or close to their recognition sequences, which are typically 4-8 base pairs in length and palindromic.

One example of a type II RE that generates flush ends is EcoRI, which recognizes the sequence 5'-GAATTC-3' and cleaves between the G and the A, generating 5'-overhangs. Two other enzymes that are commonly used in cloning experiments are:

1. T4 DNA ligase: This enzyme catalyzes the formation of a phosphodiester bond between the 5' phosphate and 3' hydroxyl ends of DNA strands. T4 DNA ligase is used to join DNA fragments together during cloning experiments.
2. Taq DNA polymerase: This enzyme is a thermostable DNA polymerase that is commonly used in PCR (polymerase chain reaction) amplification of DNA sequences. Taq DNA polymerase has a relatively high error rate, which can result in mutations in the amplified DNA sequence.