Question

A linear piece of DNA has the following restrictions sites:

Xbal Noti Pstl EcoRI 2 kb A 4 kb 1 kb 2 kb 3 kb

You decided to set up an experiment where you added one of these restriction enzymes to this DNA. After this DNA was digested with that restriction enzyme, you separated the resulting fragment(s) using agarose electrophoresis. After the gel was stained with ethidium bromide, you observed the following gel (the DNA ladder is your reference standard and is comprised of a series of DNA fragments of known length).

a) Which restriction enzyme would generate the fragment(s) shown on this gel. Explain how you arrived at your answer.

b) Explain how a restriction enzyme recognizes and cuts double-stranded DNA.

Answer 1

Answer A ) As in the result of gel electrophoresis image represent the two lane label as DNA ladder and sample. The lane of sample consist of two band and in reference to the DNA ladder band, the band present in sample lane are of 9 kb and 3 kb size.

If we carefully observe the image of linear DNA with restriction site, EcoRI is the one which will give fragment of 9 kb( 2kb+4kb+1kb +2kb) and 3 kb. Thus from this it can be concluded that EcoRI is the restriction enyme used in the sample.

Answer b ) Restriction enyme are the endonuclease enzyme which recognize the specific sequence which is present within the nucleotide strand and cut over it. theyare highly specific, once they recognize their specific sequence then pricisely cut within the sequence. As in case of EcoRI which cut at specific sequence 5' GAATTC 3'

THE DOUBLE STRAND DNA

5' G*AATTC 3'

3' CTTAA*G 5'

STAR * REPRESENT THE EXACT SITE WHERE THE EcoRI CUT.

There are three categories of restriction enzyme base on there recognition property.

TYPE 1 : These restriction enzyme recognize the specific site but cut randomly at 1000 base pair away from the recognition site. example EcoB RESTRICTION ENZYME

TYPE 2: These restriction enzyme recognize the site and cut within that rcognition site Example : EcoRI

TYPE 3 : These restriction enzyme recognition the specific site but cut site about 25 to 30 base pair away from the site . Example Ecop15