Question

The restriction endonuclease BamH1 has the recognition sequence G/GATCC and Sau3A the recognition sequence /GATC. Genomic DNA (G/C content = 56%) is digested with Sau3A and ligated into a vector digested with BamH1. What percentage of fragments could potentially be ligated into the vector and what is the average fragment length of the insert DNA?

Answer 1

Ans: As given in the question that BamH1 has the recognition sequence 5'-G/GATCC-3' and its complementary sequence is palindromic to the original site, and Sau3A has the recognition sequence 5'-N/GATC-3' where N can be any nucleotide and its complementary sequence is also palindromic to the original sequence.

Now for the ligation part look at the image i enclosed below:

Now from this we can see that the fragement treated by Sau3A is completely ligated to the vector treated with BamH1 because they both produce same sticky ends.

Now genomic DNA is digested with Sau3A and its recognition site is GATC, normally the probability of occuring a single base is 1/4 and so the frequency of occuring of GATC together in the genome is (1/4⁴)which is equal to 1/256 which means that GATC sequence will occur after every 256 base pairs. So the average fragment length of the insert DNA is 256 bp, but it is given that the %GC content in the genomic DNA is 56% which means that %AT content should be 44% and %G content is 28%, %C content is 28%, %A content is 22% and %T content is 22%.

So now for GATC sequence the probability is 0.28*0.22*0.22*0.28 which is equal to 0.00379456 or we can say that once in every ~ 264 base pairs.

So after taking consideration of %Base content in the genome the average length of the fragment produced by the treatment of Sau3A is 264 base pairs.