10 min readConcept sheet1-mark MCQ on naming or palindromes, and a 2 or 3-mark question on sticky ends; also a NEET favourite

Restriction enzymes: the molecular scissors

Restriction endonucleases cut DNA at specific palindromic recognition sequences. Many cut off-centre and leave sticky ends that DNA ligase can join to matching ends.

First restriction endonucleaseHind II; it cuts at a specific recognition sequence of six base pairs

How many knownOver 900 restriction enzymes, from over 230 strains of bacteria

EcoRI site5′ GAATTC 3′ paired with 3′ CTTAAG 5′; cuts between G and A on each strand

Naming EcoRIE = genus Escherichia, co = species coli, R = strain RY13, I = first enzyme isolated from that strain

Discovery

In 1963, two enzymes were isolated from E. coli that restricted the growth of bacteriophages (viruses that infect bacteria). One added methyl groups to DNA. The other cut DNA: it was called a restriction endonuclease. Five years later, Hind II was isolated: the first restriction endonuclease whose action depended on a specific base sequence. It always cut DNA at the same point, where it found a particular sequence of six base pairs. Such a sequence is called the recognition sequence. Each restriction enzyme has its own recognition sequence.

Exonuclease vs endonuclease

ExonucleaseRemoves nucleotides from the ends of a DNA molecule, one after another.

EndonucleaseMakes cuts at specific positions within the DNA. Restriction enzymes are endonucleases.

Both belong to the larger class of enzymes called nucleases.

How a restriction enzyme is named

Part of the name	Comes from	In EcoRI
First letter (capital)	Genus of the bacterium	E: Escherichia
Next two letters	Species	co: coli
Next letter	Strain	R: strain RY13
Roman number	Order in which enzymes were isolated from that strain	I: the first

Palindromes and sticky ends: how EcoRI works

1
The enzyme moves along the DNA and 'inspects' its sequence until it finds its recognition sequence.
2
The site is a palindrome: both strands read the same when read in the same direction (5′ to 3′). For EcoRI: 5′ GAATTC 3′ on one strand and 5′ GAATTC 3′ on the other.
3
It binds and cuts the sugar-phosphate backbone of each strand, between G and A: 5′ G↓AATTC 3′.
EcoRI
4
The cuts lie a little away from the centre of the palindrome, but between the same two bases on both strands. Each end is left with a single-stranded AATT overhang.
5★
These overhangs are sticky ends: they form hydrogen bonds with complementary sticky ends cut by the same enzyme.
6★
DNA ligase seals the backbones. If the vector and the foreign DNA were cut with the same enzyme, the result is recombinant DNA.
DNA ligase

★ key step

How to draw the action of EcoRI

Write the double strand: 5′ G A A T T C 3′ on top and 3′ C T T A A G 5′ below, with short lines for base pairs.
Draw a small arrow between G and A on the top strand.
On the bottom strand, draw the arrow between A and G at the right end. Read 5′ to 3′, this cut is also between G and A.
Draw the two fragments pulled apart. On the right fragment the top strand now starts 5′ AATTC; on the left fragment the bottom strand ends in the matching tail (3′ CTTAA 5′). Label both single-stranded AATT tails sticky ends.
Draw a foreign DNA cut the same way, with the same sticky ends.
Show the ends paired and write DNA ligase over the joins; label the product recombinant DNA.

Counting fragments

Problem

A linear DNA molecule has 3 recognition sites for EcoRI. A circular plasmid also has 3 sites for EcoRI. How many fragments does each give after complete digestion?

Working

In a linear molecule, each cut adds one piece: fragments = sites + 1.
Linear DNA: 3 + 1 = 4 fragments.
A circle must be cut once just to open it into one piece: fragments = number of sites.
Circular plasmid: 3 fragments.

Answer

Linear DNA gives 4 fragments; the circular plasmid gives 3. This is why a vector should have only one site for the chosen enzyme: one cut simply opens it.

In real life

Restriction enzymes are the 'scissors' behind every recombinant product, from human insulin to Bt cotton. Forensic labs in India also cut DNA with them during DNA fingerprinting.

Exam angle

2 marks: “What is a palindromic nucleotide sequence? Write the sequence recognised by EcoRI.” A base sequence that reads the same on both strands when read in the same direction (5′ to 3′). EcoRI: 5′ GAATTC 3′ / 3′ CTTAAG 5′.

3 marks: “How do restriction endonucleases produce sticky ends? Why are they so called, and how do they help to make recombinant DNA?” They cut each strand a little away from the centre of the palindrome, between the same two bases, leaving single-stranded overhangs. These form hydrogen bonds with complementary ends. Vector and source DNA cut by the same enzyme have matching ends, which DNA ligase joins.

1 marks: “What do the letters in EcoRI stand for?” E: Escherichia (genus); co: coli (species); R: strain RY13; I: first enzyme isolated from that strain.

Remember it

Name a restriction enzyme like an address: Genus, species, strain, serial number (E-co-R-I).