Question

(i) EcoR I has played a very important role in rDNA technology.

(I) Explain the convention for naming EcoR I.

(II) Write the recognition site of this endonuclease restriction enzyme.

(ii) Name the overhanging stretches of DNA produced by these restriction enzymes. Describe their role in the formation of rDNA.

Hint:

Name = genus E, species co, strain R, order I; site 5-GAATTC-3; the cut leaves sticky ends that pair and are sealed by DNA ligase.

Answer 1

Step 1: Naming convention of EcoR I:
The first letter comes from the genus. E stands for the genus Escherichia.
The next two letters come from the species. co stands for coli.
The fourth letter R comes from the strain, here RY13.
The Roman numeral I shows the order in which the enzyme was isolated from that strain. EcoR I was the first one isolated.

Step 2: Recognition site of EcoR I:
EcoR I recognises a palindromic sequence of six base pairs.
\[ 5^{\prime}\text{-GAATTC-}3^{\prime} \]

\[ 3^{\prime}\text{-CTTAAG-}5^{\prime} \]

It cuts both strands between G and A, so the two strands are cut at different points.

Step 3: The overhanging stretches:
The cuts leave single-stranded overhangs at each end. They are called sticky ends (cohesive ends).
For EcoR I the overhang is AATT. Such ends can form hydrogen bonds with a matching overhang.

Step 4: Role in making rDNA:
The same enzyme cuts both the vector DNA and the foreign DNA, so both get identical sticky ends.
When mixed, the sticky ends of the foreign DNA pair with those of the vector by hydrogen bonds between complementary bases.
The enzyme DNA ligase then joins the sugar-phosphate backbone and seals the nicks. This gives the recombinant DNA.

Final Answer:
(i)(I) E-co-R-I = genus, species, strain, order of isolation. (II) 5-GAATTC-3 / 3-CTTAAG-5. (ii) Sticky ends join the foreign DNA to the vector, sealed by ligase.