RESTRICTION ENZYMES

ByMuhammad Adnan Qadar

Also termed as Restriction Endonucleases ORREases OR REFunctions as Molecular Scissiors/DNA cutters

RESTRICTION/DIGESTION

The process of restricting OR breaking down theDNA into pieces

RESTRICTION DIGEST

The product (Fragments of Broken DNA)obtained from restriction

Methyl-Transferase/Methylase OR Mtase

An enzyme which attaches -CH3 group to hostDNA to protect it from REase digestion

TERMS TO KNOW

Also termed as Restriction Endonucleases ORREases OR REFunctions as Molecular Scissiors/DNA cutters

RESTRICTION/DIGESTION

The process of restricting OR breaking down theDNA into pieces

RESTRICTION DIGEST

The product (Fragments of Broken DNA)obtained from restriction

Methyl-Transferase/Methylase OR Mtase

An enzyme which attaches -CH3 group to host

DNA to protect it from REase digestion

TERMS TO KNOW

NOBLE PRIZE

Were awarded

ACTION OF REases

REases can produce Sticky and Blunt ends

REases can be Iso-schizo-mersOnly Type II restriction endonucleases are

the are mostly usedMethylated DNA cannot be digested byReasesSome REases also have dual nature (i.g., Reases

and Mtases)

CHARACTERISTICS OF REase

REases have specific recognitionsequence at which it cuts a DNAmoleculeFor example, the RE called PvuI(isolated from Proteus vulgaris) cutsDNA only at the hexanucleotide5´-CGATCG-3´PvuII, cuts at a different hexanucleotide in this case5´-CAGCTG-3´

Many RE are Isoschizomers (DifferentRE but same target sites)REases cleaves Phosphodiestr bonds of

Nucleotides

SPECIFICITY OF REasesFrequency of Recognition

Sequence

It is calculated

mathematically i.e., a

tetranucleotide appears

every 44=256nt and

Hexanucleotide repeated

every 46=4096nt

Size of restricted DNA

fragment

Depends upon frequency nt

sequence appearance

• Four(four cutters), Five(five cutters), Eight(eight cutters), or even longer nucleotide sequences cutting REases are also available e.g., Sau3A (from Staphylococcus aureus

strain 3A) recognizes GATC and AlwNI cuts CAGNNNCTG

DEGENERATE RECOGNITION SEQUENCES• Some REases cut DNA at any one of a family of related sites, e.g., HinfI recognizes GANTC, so cuts at GAATC, GATTC, GAGTC, and GACTC.

MECHANISM OF RESTRICTONBlunt Ends or Flush End cut

Make a simple double-stranded cut in the middle of the recognition sequenceHindII, PvuII and AluI are examples of blunt end cutters

Reases Cut at exactly the same position Usually by two or four nucleotides As bp can stick together again So these are called sticky ends

Sticky Ends or Cohesive Ends

REases with different recognition sequences may produce the same sticky endsBamHI (recognition sequence GGATCC) and BglII (AGATCT) are examples—bothproduce GATC sticky ends

Measure 2 µg of DNA in 16 µg of the sampleAdd required REase (BglII) pure in the mixtureMaintain pH 7.4 with Tris-HCl (500mM)Mg+2 (100mM) and Nacl (500mM) for proper activity of REAdd dithiothreitol (DTT) 10mM to stablize and preventing inactivation of RE

PERFORMING DIGESTION IN LAB

Maintain 37°C for 1hThen deactivate REase by exposing it for short time to 70°C or by adding EDTA which binds to Mg+2 of REase and deactivates its function