You've identified and purified a novel closed circular viral DNA, and want to treat it with a typ…
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)
(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.