(b) Restriction enzymes and DNA
◆PCR or polymerase chain reaction is a technique to amplify a gene or a DNA fragment in order to get multiple copies.
◆ It is used in the gene manipulation process.
◆ Phenomena involves the in vitro synthesis of sequences with the help of a template strand, a primer and a thermostable DNA polymerase enzyme produced by a bacterium called as Thermus aquaticus.
◆ enzyme uses the building blocks deoxynucleotides (dNTPs) in order to extend the primer.
◆ Three steps in PCR-
1)Firstly , the double stranded DNA molecules are heated to a high temperature to separate the two strands into a single stranded DNA molecule and a process is called denaturation.
2)●This DNA molecule is then used as a template strand to synthesize a new strand by the DNA polymerase enzyme and this process is called annealing
● leads to the replication of the original DNA molecule, and the process is carried out for multiple cycles to obtain multiple copies of the rDNA fragment.
3) Primer is extended by Taq DNA polymerase isolated from Thermits aquatics.
2.Restriction enzymes and DNA
◆restriction enzymes are molecular scissors use to cut DNA sequences from a particular site.
◆It has a critical role to play in the gene manipulation process. ◆These enzymes identify a particular six-base pair sequence referred to as the recognition sequence and snip the sequence at specific sites.
◆Restriction enzyme are two types are –
(i) Endonuclease – enzyme that cuts within the DNA at specific sites. It serves as a significant tool in genetic engineering. It is used to make a snip in the sequence to get DNA fragments possessing sticky ends. These ends are later fused by the enzyme DNA ligase.
(ii) Exonuclease – enzyme removes the nucleotides either from 3’ or 5’ ends of the DNA molecule.
enzyme that is used for degradation of chitin, that forms the main component of the cell wall of fungi. To isolate the DNA enveloped within the cell membrane of the fungus, the Chitinase enzyme is used to break the cell to release its genetic material.