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What are restriction enzymes?

Class 12 BiologyNCERT chapter: Biotechnology: Principles and Processesइसे हिंदी में पढ़ें

Exam-ready answer

Restriction enzymes, or restriction endonucleases, are 'molecular scissors' that cut DNA at specific sites. Each one recognises a specific palindromic nucleotide sequence, called the recognition sequence, binds to the DNA there and cuts each of the two strands at specific points in the sugar-phosphate backbone. Many leave sticky ends. Examples are EcoRI and Hind II.

Explanation

Restriction enzymes belong to a larger class of enzymes called nucleases. Exonucleases remove nucleotides from the ends of the DNA, whereas endonucleases make cuts at specific positions within the DNA. In bacteria, these enzymes restrict the growth of bacteriophages by cutting the viral DNA. The first restriction endonuclease, Hind II, always cut DNA at a particular point by recognising a specific sequence of six base pairs.

The recognition sites are palindromic: the sequence of base pairs reads the same on the two strands when the orientation of reading is kept the same. For EcoRI it is 5'-GAATTC-3' on one strand and 3'-CTTAAG-5' on the other. Restriction enzymes cut a little away from the centre of the palindrome, but between the same two bases on opposite strands. This leaves single-stranded overhanging stretches called sticky ends.

Sticky ends form hydrogen bonds with their complementary cut counterparts. So when a vector and the source DNA are cut with the same enzyme, their ends pair up and DNA ligase seals them, which is how recombinant DNA is made. Naming follows a rule: in EcoRI, 'E' comes from the genus Escherichia, 'co' from the species coli, 'R' from the strain RY 13, and 'I' shows it was the first enzyme isolated from that strain.

Points to remember

  1. Restriction endonucleases cut DNA at specific recognition sequences and are called molecular scissors.
  2. Exonucleases remove nucleotides from the ends of DNA; endonucleases cut at specific positions within DNA.
  3. Recognition sequences are palindromic, e.g. 5'-GAATTC-3' / 3'-CTTAAG-5' for EcoRI.
  4. Sticky ends are single-stranded overhangs that pair with complementary ends; DNA ligase joins them.
  5. Naming: EcoRI = Escherichia coli, strain RY 13, first enzyme isolated from it.

Exam tip

Write the EcoRI palindrome with 5' and 3' ends on both strands, mark the cut between G and A on each strand, and label the sticky ends that result.

Questions students also ask

Why are they called restriction enzymes?
They were found in bacteria, where they restrict the growth of bacteriophages. The enzyme cuts the DNA of the infecting virus at specific sites so the virus cannot multiply. Another enzyme of the same system adds methyl groups to the bacterium's own DNA.
How are DNA fragments cut by restriction enzymes separated?
They are separated by agarose gel electrophoresis. DNA is negatively charged, so fragments move towards the anode in an electric field, and smaller fragments move farther. The DNA is stained with ethidium bromide and seen as bright orange bands under UV light.
What is a palindromic sequence in DNA?
It is a sequence of base pairs that reads the same on the two strands when the orientation of reading is kept the same. For example, 5'-GAATTC-3' on one strand also reads 5'-GAATTC-3' on the complementary strand.

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