Notes on Restriction enzymes and DNA Ligases

What are Restriction Enzymes?

Restriction enzymes are enzymes that cuts double stranded DNA at unique sequence called recognition sequence or restriction site

  • Also called as molecular scissors, molecular knives or molecular scalpels
  • Hind II is the first restriction nuclease isolated from Haemophilus influenzae bacteria

Restriction Enzymes belongs to the class nucleases or nucleic acid degrading enzymes

two types: 1. Restriction endonuclease: makes cuts within the DNA molecule

                  2. Restriction exonuclease: remove nucleotides from the ends

3 types of Restriction endonucleases:

  • Type I, II and III
  • Type II is used in rDNA technology as its makes cuts within recognition site
  • Eg: Eco R1, Hind III
  • Mg2+ is required for cleavage
  • Type I and Type III makes random cuts; therefore, rarely used in rDNA technology

How Restriction Enzymes cut DNA molecule?

How Restriction enzymes works? Eco R1

Example is Eco R1: EcoR1 Restriction site :   5`---GAATTC---3`


Watch this video for better understanding

Characteristics of restriction sites

  • Recognition sites are often 4-8 base pairs
  • They are Palindromic sequences. Palindromic sequence is a DNA sequence of base pairs that reads the same on two strands in 5`-3` & 3`-5`direction.

Cut patterns

1.Staggered cuts or cohesive or sticky ends

  • cuts that generate protruding single stranded ends eg: Eco R1
  • More preferred in rDNA technology

5`---GAATTC---3`                        5`---G         AATTC---3`

3`---CTTAAG---5`    ------>          3`---CTTAA         G---5` 

2.Blunt ends or even cuts

  • cuts both strands at the same site, generating blunt or flush ends eg: AluI

5`---AGCT---3`                                5’---AG     CT---3’  

3’---TCGA---5’        -------->            3’---TC      GA---5’

DNA ligase

Ligases are Joining enzyme that joins DNA fragments by forming phosphodiester bond. The process is called ligation.

How DNA ligase works?

How DNA ligase joins DNA molecule?

  • Phosphodiester bond is between the 5’P of a nucleotide of one DNA fragment and the 3’ OH end of the other
  • Requires ATP or NAD+ for its activity
  • Eg: T4 ligase

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