Recombinant DNA technology Q2. Restriction enzymes are extensively used in molec

Question

Recombinant DNA technology

Q2. Restriction enzymes are extensively used in molecular biology. The restriction enzymes Sal Iand Xho I cut their recognition sequences as shown below, where the * indicates where it is cut

CUTSITE (Enzyme Sal I)

5’----G*T C G A C----- 3’

3’----C A G C T*G-----5’

CUTSITE (Enzyme Xho I)

5’--- C*T C G A G ---3’

3’---G A G C T*C---5’

2a-2pt) Indicate the 5’ and 3’ ends of the cut molecules below [Both strands and both ends]

Enzyme Sal 1

----G                           T C G A C----

----C A G C T                            G----

Enzyme Xho I

----C                             T C G A G----

----G A G C T                             C----

You cut a plasmid vector with Sal I and want to clone a gene into it. Shown below is what the cut vector would look like

You want to clone your favorite genes (YFG) into this vector.

Shown below is the restriction map and 2 options you have to cut the

gene before you can ligate it with your cut vector.

The Enzyme Xho I cut site is shown in 2a). The enzyme AatII cut site is shown below

CUT SITE Enzyme AatII

5’ ---G A C G T*C ---3’

3’---C*T G C A G---5’

2c-1pt) Cutting the genomic DNA with Xho would release a fragment that looks like this:

T C G A C ------------------------------------------------------C

             G-------------------------------------------------------G A G C T

Cutting the genomic DNA with Aat II would look like this

             C ----------------------------------------------G A C G T

T G C A G ----------------------------------------------C

2c-1pt) Which of the enzymes would give you generate a successful recombinant molecule?

(Write answer below)

Isolate YFG from the genome using Enzyme _____________

Explanation / Answer

Enzyme Sal I:

5' - G - 3' ...... 5' - T C G A C - 3'

3'- C A G C T - 5' ...... 3' - G - 5'

Enzyme XHO I:

5' - C - 3' ..... 5' - T C G A G - 3'

3'- G A G C T - 5' ...... 3'- C - 5'

When a sequence is cut, it keeps the same direction that it already had, and the complementary chain is always going in the opposite direction.

The best enzyme for the YGF gene is the Aat II because the sequence of the gene is not lost or duplicated in any of the parts. The sequence resulting in the cut with Xho I looks like a different sequence, some nucleotides are lost in the process.

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