Question

Name the five key tools for accomplishing the tasks of recombinant DNA technology. Also mention the functions of each tool.

Answer 1

Introduction :

The five key tools involved in recombinant DNA technology are as follows:

1.Enzymes involved in DNA manipulation

2.Cloning vectors

3.Gel electrophoresis

4.PCR

5.Host organism

Enzymes involved in DNA manipulation:

Enzymes involved in DNA manipulation are as follows:

1.Restriction enzymes

• These enzymes serve as molecular scissors to cut DNA into defined fragments.
• These enzymes recognize special palindromic nucleotide sequences of 4-8 nucleotides in length in DNA.
• Restriction endomucelases (a type of restriction enzyme) are used to isolate gene of interest and to create sticky ends in plasmid vectors.
• Example - Eco R I

2.Polymerase enzyme

• These enzymes cause extension of DNA strands in the 5’ to 3’ direction by using template DNA.
• E.g., Taq polymerase in PCR

3.Ligase enzyme

• The DNA ligase joins two DNA strands by forming a phosphodiester bond between them.
• It is known as molecular glue.
• It is used to join genes of interest with the cloning vector.
• E.g., T4 DNA ligase is used in rDNA technology.

Cloning vector :

Cloning vehicles that carry the gene of interest into the recipient cell are called cloning vectors.

Different types of cloning vectors are listed below:

• Plasmids and viruses are commonly used as vectors.
• Plasmids are extra chromosomal circular DNA present in prokaryotic organisms such as bacteria, that are capable of replicating themselves.
• The most commonly used vectors are plasmids such as pBR322 and pUC18.
• Ti plasmid obtained from Agrobacterium is the commonly used vector for plants.
• Disarmed retroviral vectors are commonly used for animals.

Gel electrophoresis:

• Gel electrophoresis is a technique used to separate DNA fragments based on their sizes under the influence of an electric field.

PCR:

1. Polymerase Chain Reaction is used to amplify the gene of interest.

• Using PCR even small samples of DNA can be amplified and used in rDNA technology.
• It involves three steps - denaturation, annealing and extension.

Host organism :

• The organism in which the gene of interest (with or without vector) is inserted and multiplied is known as the host organism.
• Escherichia coli is commonly used for multiplication of various genes.