Q19
2 marksVery Short AnswerSection B

Study the sequence of bases in a DNA molecule that is shown below and answer the questions.

5' – GAATTC – 3'

3' – CTTAAG – 5'

(a) What are such sequences called? Name the enzyme used that recognises such nucleotide sequences. [1]

(b) How do these enzymes function? [1]

Biotechnology: Principles and Processes
Restriction enzymes and palindromic sequences
Official Answer

The given sequence reads the same on both strands in the 5'→3' direction, which is the defining feature here.


(a) Nature of the sequence and enzyme:

  • Such sequences are called palindromic sequences (palindromes) — the base sequence reads the same 5'→3' on both the strands.
  • They are recognised by restriction endonucleases (here, specifically EcoRI).

(b) How these enzymes function:

  • The enzyme scans/inspects the length of the DNA until it finds its specific recognition (palindromic) sequence.
  • It then binds and cuts the two strands of the sugar–phosphate backbone at specific points between the same two bases on opposite strands (between G and A here), a little away from the centre.
  • This staggered cut leaves single-stranded, overhanging 'sticky ends' that can pair with complementary ends of other DNA.
palindromic sequencerestriction endonucleaseEcoRIrecognition sitesticky endsstaggered cutsugar-phosphate backbonemolecular scissors

Marking Scheme

  • 1½ mark: naming the sequence as palindromic sequence/palindrome.
  • 2½ mark: naming restriction endonuclease (EcoRI acceptable).
  • 3½ mark: enzyme recognises specific sequence and makes a cut in the sugar-phosphate backbone of both strands.
  • 4½ mark: staggered/away-from-centre cut producing single-stranded sticky (cohesive) ends.

Hint

Reads the same 5'→3' on both strands — think 'molecular scissors' of E. coli that leave overhangs.

Quick Oral Answer

This is a palindromic recognition sequence; the restriction endonuclease EcoRI binds it and makes a staggered cut in the backbone between G and A, leaving complementary sticky ends used in recombinant DNA technology.

Analysis & Explanation

Concept:

A palindrome in DNA is a sequence of base pairs that reads the same on the two strands when read in the same orientation (5'→3'). Read the top strand left-to-right (GAATTC) and the bottom strand right-to-left in 5'→3' (GAATTC) — identical. This symmetry is what allows a restriction endonuclease to bind and cut both strands.


The enzyme:

  • EcoRI is a restriction endonuclease isolated from Escherichia coli RY13. Naming: E (genus), co (species), R (strain), I (order of discovery).
  • It cuts between G and A on each strand, producing complementary single-stranded overhangs (AATT) called sticky ends.

Exam trap:

  • Do not confuse exonucleases (remove nucleotides from ends) with endonucleases (cut at specific internal sites). Restriction enzymes are endonucleases.
  • Only name the sequence a 'palindrome' — not a 'template' or 'primer'.

Real-world application:

Sticky ends produced by the same enzyme on the vector and the foreign DNA are complementary, so DNA ligase can seal them — this is the very basis of making recombinant DNA and gene cloning.

Common Mistakes

  1. 1Writing 'complementary sequence' or 'template sequence' instead of 'palindromic sequence' — the specific term is required for the mark.
  2. 2Saying the enzyme cuts exactly at the centre — restriction enzymes cut a little away from the centre, leaving overhangs, not blunt cuts, in EcoRI.
  3. 3Confusing restriction endonuclease with ligase; ligase joins, endonuclease cuts.

Interesting Facts

EcoRI was one of the first restriction enzymes discovered (Herbert Boyer, 1970) and its sticky ends made the first recombinant DNA experiments possible in 1972-73.

Over 900 restriction enzymes have been isolated from more than 230 bacterial strains, each recognising a different sequence, giving genetic engineers a huge toolkit of 'molecular scissors'.

Bacteria use restriction enzymes as a defence system to cut up invading viral (phage) DNA, while protecting their own DNA by methylation.

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Frequently Asked Questions

Why is GAATTC called a palindrome?

Because reading the top strand 5'→3' (GAATTC) and the bottom strand 5'→3' (also GAATTC) gives the identical sequence. This rotational symmetry lets the restriction enzyme recognise and cut both strands at equivalent positions.

What are sticky ends and why are they useful?

Sticky ends are the short single-stranded overhangs left after a staggered cut (AATT here). They are 'sticky' because they can base-pair with complementary overhangs on any other DNA cut by the same enzyme, allowing DNA ligase to join a foreign gene into a vector.

Is EcoRI an exonuclease or endonuclease?

It is an endonuclease — it cuts at a specific internal recognition site within the DNA, not at the ends. Exonucleases, by contrast, remove nucleotides one by one from the free ends.