Genetic Engineering : Cutting & joining DNA

 Genetic Engineering : Cutting & joining DNA 

The science in molecular biology flourished in full throttle in
the 1970s with the discovery that DNA sequences can be cut and
religated at specific target sequences by a group of enzymes
known as restriction endonuclease, for which W. Artier, D. Nathans
and H. Smith received Nobel Prize in 1978. It was observed in
1950s that certain bacteria can exhibit immunity to phage infection.
For example if bacteriophage infects E.coli strain K, it will cause

lysis of that bacterial strain. The progeny phages infect the same
strain with higher efficiency. However, if the progeny phages
grown on strain K are used to infect another E. coli strain B, growth
of phage population is restricted. This phenomenon, known as

‘host controlled restriction modification’ is observed due to presence of restriction enzymes in bacterial cells. When phage A attacks strain B, a site specific restriction enzyme EcoB cuts A DNA as its recognition sequences are present on A DNA, thereby restricting infection. To protect its own DNA from restriction, the bacterium methylates its own DNA by using a methylase enzyme. During this process a small number of phage DNA also gets methylated and avoids restriction. These progeny phages will infect E. coli strain B with much higher efficiency, as they have been modified by host enzyme so that further phage DNA restriction in strain B does not take place. However, if these phage particles attack another strain K that contains a different restriction enzyme

recognizing another sequence, the phage DNA in strain K will be

restricted.

      The restriction endonucleases are classified into three major

groups depending on their sequence specificity, nature of

restriction and structural differences (Table 1). Type I and type III

restriction endonucleases have limited use in DNA cloning and

genetic engineering. Type II restriction endonucleases (Type II R.

E.) are widely used for their sequence specificity, cleaving of

dsDNA within recognition sequence, separate methylation and

no additional requirement of energy.The recognition and restriction sequences of type Ii R. E. are very precise and do not match with one another. The enzymes may further be classified as tetracutter, hexacutter, octacutter etc. depending on the size of the recognition sequence (4, 6 and 8 respectively). For genetic manipulation, hexacufters and tetracutfers are most widely used. Besides, depending on the nature of cut produced by these enzymes. two broad groups may be identified. Some of the restriction enzymes cut exactly at the mid point of the dyad symmetry recognition sequence producing two fragments sequence and cuts at the midpoint to produce two blunt ends with no free nucleotide for hydrogen bonding. For this reason these enzymes are termed as blunt end cutters. Sticky end cutters, on the other hand produce 5’ or 3’ overhangs as the cleavage site 

• The nomenclature of restriction enzymes uses a three letter in DNA: E. coli, HpaII , MspI
• Restriction enzymes can cut DNA
• Cutting a DNA fragment
• Restriction digestion is an enzymatic reaction, by DNA  molecule