Question

You've identified and purified a novel closed circular viral DNA, and want to treat it with a type II restriction enzyme used in BCH370. This DNA has a single restriction site contained within the following sequence: 5' GGGTTATGAATTCTACA 3' 3, CCCAATACTTAAGATGT 5, a) Indicate the probable region identified by the restriction enzyme, and explain how you can identify the recognition sequence based on certain criteria. (2 marks) b) You then attempt to identify whether or not the viral vector contains an ampicillin resistance gene. How could you use PCR to confirm this, and what are the necessary steps involved? (3 marks) What is a primer-dimer, why is it significant, and how is it formed? (3 marks) c)

Answer 1

(a) The restriction enzyme will cut between A and T. Specific region being that it can be easily hydrolysed as it contains double bond between them.

(b) Once the ampicillin gene is recognized we may use ampicillin gene primers and multiply the gene to many times and then blue white selection can be done to identify if the gene is ampicllin resistance gene or not.

(c) primer dimer is when our gene of interest and the primer we selected fits perfectly that is they are complementary to each other. It is formed when the sequence of the primer and the gene of interest are complementary to each other and it is significant because with better complementarity the multiplication of gene is also efficient.