Question

Given below is a single stranded sequence of DNA:
5' ACTAGAATTCGCCA 3'
Which restriction enzyme would cleave this sequence in double stranded form? Illustrate the fragments that will be generated upon digestion by this restriction enzyme.

Hint:

EcoRI cuts specifically between G and A in the palindromic sequence $5'\text{--GAATTC--}3'$. The resulting sticky ends can hydrogen-bond with any other DNA fragment cut by the same enzyme, which is then sealed covalently by DNA ligase.

Answer 1

Step 1: Sequence Recognition Analysis

Let us inspect the given single-stranded DNA sequence:
$$ \texttt{5' ACTA-GAATTC-GCCA 3'} $$ We identify the internal 6-base pair palindromic sequence: 5' GAATTC 3'. This is the specific recognition and cleavage site of the Type II restriction endonuclease EcoRI (isolated from Escherichia coli RY13).

Step 2: Double-Stranded Cleavage Pattern

EcoRI cleaves the phosphodiester backbone between the G and A residues on both strands, leaving single-stranded complementary cohesive sticky ends:
$$ \begin{array}{lccccc} \text{Uncut dsDNA:} & & \texttt{5'--A-C-T-A-G} & \downarrow & \texttt{A-A-T-T-C-G-C-C-A--3'} \\ & & \texttt{3'--T-G-A-T-C} & \uparrow & \texttt{T-T-A-A-G-C-G-G-T--5'} \end{array} $$ 
Step 3: Illustration of Digested Fragments

The endonuclease digestion cleaves the DNA into two distinct fragments with 4-base cohesive sticky overhangs:

$$ \begin{array}{l} \textbf{Left Fragment:} \\ \texttt{5'--A-C-T-A-G--3'} \\ \texttt{3'--T-G-A-T-C--5'} \\ \\ \textbf{Right Fragment:} \\ \texttt{5'--A-A-T-T-C-G-C-C-A--3'} \\ \texttt{3'--T-T-A-A-G-C-G-G-T--5'} \end{array} $$ 
Final Answer: The sequence is cleaved by EcoRI. Digestion generates two fragments with complementary single-stranded sticky ends (5'-AATT-3').