Notes on Restriction enzymes and DNA Ligases microbiologystudy

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`

                                                                         3`—CTTAAG—5` 

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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