You cut a plasmid vector with Sal I and want to clone a gene into it. Shown belo

Question

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

Our DNA is made up of nucleotides with 4 possible bases {2 Purines labelled Adenine (A) and Guanine (G), & 2 Pyrimidines labelled Cytosine (C) and Thymine (T)}. These 4 nucleotides are arranged sequentially across two strands that come together to form a ladder with the bases as its rungs. One strand goes from 5’ to 3’ direction and the other strand is anti-parallel to this going from the 3’ to 5’ direction.
Thus there end up being sequences in the middle that are palindromic, that is, read in the same sequence of bases on both strands when reading from the 5’ to 3’ direction. Example:

5’ – AGTTAACA – 3’
3’ – TCAATTGT – 5’

which is read as 5’ – GTTAAC – 3’ on both strands.

Certain enzymes called restriction enzymes were made by bacteria to counter genetic material insertions by viruses. These restriction enzymes treated these palindromic regions in the DNA as restriction sites and were able to cut the DNA there, leading to either a blunt cut:

5’ – AGTT ||| AACA – 3’
3’ – TCAA ||| TTGT – 5’

or a staggered/cohesive cut:

5’ – AGT ||| TAACA – 3’
3’ – TCAAT ||| TGT – 5’

If a linear stretch of double-stranded DNA (dsDNA) is cut at one restriction site, it will produce two end products. Similarly, ‘n’ number of such cuts will produce ‘n+1’ number of final fragments.

Isolate YFG from the genome using Enzyme Aat II
This is because, Aat II leaves compatible staggered ends for annealing the other fragment

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