Question

The restriction endonuclease BamH1 has the recognition sequence G/GATCC and Sau3A the recognition sequence /GATC. Genomic DNA (G/C content = 65%) 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

The restriction site for BamH1 is G/GATCC i.e. total 6 nucleotides. Thus the average number of recognition sites present in any DNA will be given by 4⁶ or 4096. Hence, the probability of cutting and thus ligating into the DNA would be 4096. Since the DNA contains 65% GC content, the final probability would be given by:

65 * 1/4096 * 100 % or 15.8%.

Similarly, the probability for Sau3A would be given by: 65 * 1/4⁴ * 100 % or 65/100*256 or 25.39%.

The size of fragment length of the insert DNA can be found as below:

For BamH1: 4⁶ OR 4096 bp

For Sau3A: 4⁴ OR 256 bp