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Terminal enzyme catalyses the addition of one or more deoxyribonucleotides to the 3' terminus of the DNA molecule.

The enzyme can work on both double stranded as well as single stranded DNA molecules
without the need of any primers.

The enzyme is obtained from calf thymus tissue.

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This group of enzymes removes the phosphate group from 5' terminus of the DNA molecule.

It is active at alkaline pH, hence the name ‘alkaline phosphatase’.

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PNK catalyzes the transfer of a phosphate group from ATP to the 5' terminus of the DNA molecule proteins, DNA and RNA.

It uses ATP as cofactor and adds a phosphate by breaking the ATP into ADP and pyrophosphate.

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Amazon Rainforest, Codajás - State of Amazonas, Brazil

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Types of polymerase involved in genetic engineering

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Types of polymerase involved in genetic engineering

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1. E. coli DNA Polymerase I
2. Klenow Fragment
3. Thermostable DNA Polymerase
4. Reverse Transciptase

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DNA polymerases are enzymes that catalyse the synthesis of a new DNA strand from a pre-existing strand.

The enzyme adds deoxyribonucleotides to the free 3’-OH of the chain undergoing elongation.

The direction of synthesis is 5’-3’.

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Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction.

Example: exonuclease I, exonuclease II etc.

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Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule.

Example: EcoRI, Hind III, BamHI etc

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Poluostrov Kanin, Nenets Autonomous Okrug, Russia

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Cleaves only normal and modified DNA.

Recognition sequences have not been well defined.

Cleavage takes place ~30 bp away from one of the sites.

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These enzymes recognize  and methylate the same DNA sequence but cleave 24–26 bp away.

It requires ATP to cut DNA

It cuts DNA about 25bp away from restriction site.

eg. EcoPI

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These enzymes are used to recognize rotationally symmetrical sequence which is often referred as palindromic sequence.

They require only Mg2+ as a cofactor and ATP is not needed for their activity.

They are composed of only one subunit.

eg. EcoRI, Hind III

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These enzymes have both restriction and modification activities.

Cleavage occurs approximately 1000 bp away from the recognition site.

They require S-adenosylmethionine (SAM), ATP, and magnesium ions (Mg2+) for activity.

eg. EcoKI

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Nucleases are a group of enzymes which cleave or cut the genetic material.

Nucleases which act on or cut the DNA are classified as DNases, whereas those which act on the RNA are called as RNases.

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Restriction endonuclease enzyme recognizes and cuts the DNA strand at specific sequence.

It helps bacteria to defend against bacteriophages bye cutting the invading phage DNA into many pieces.

Also called as biological scissors.

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Reverse transcriptase is used to synthesize complementary strand (cDNA) from mRNA template.

It is also known as RNA dependent DNA polymerase.

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Ligases are enzymes that join the nucleic acid molecules together.

DNA ligase is isolated from E.coli and Bacteriophage commercially and used in recombinant DNA technology.