recombinant DNA technology

Question 1

what are the two types of gene libraries?

Answer 1

genomic

cDNA

Question 2

what are the basics of construction a gene library?

Answer 2

clone mixture of DNA pieces into vector
creates a mixture of recombinant vector molecules as each vector takes up one DNA fragment
transformation into bacteria
each bacterium takes up one vector molecule
mixture of bacterial clones carrying particular DNA fragments is the library
can then identify the clones containing the DNA of interest

Question 3

how do you stop the vector self ligating?

Answer 3

treat the cut vector with alkaline phosphatase
removes the 5’ phosphate
DNA ligase cannot religate the cut ends of the vector
genomic DNA is not treated so they can be ligated in

Question 4

how do you construct cDNA libraries?

Answer 4

isolate mRNA from cells or tissue of interest
convert to cDNA using reverse transcriptase
ligate cDNA molecules into vector
transform vector molecules into host cells

Question 5

how do you screen for a clone of interest from a genomic/ cDNA library

Answer 5

hybridisation screening

immunological screening

Question 6

what is hybridisation screening?

Answer 6

screening for a known DNA sequence using probes

Question 7

what is immunological screening?

Answer 7

screen for production of the correct protein from clone using antibodies

Question 8

what are colony/plaque blots?

Answer 8

library of clones represented by colonies of bacteria or bacteriophage plaques on agar plates
blot colonies/plaques onto nitrocellulose or nylon membrane
denature DNA to single stranded form
bind DNA to membrane

Question 9

what are the steps of hybridisation reactions?

Answer 9

prehybridisation blocks non-specific DNA binding sites on membrane
hybridisation with labelled probe
wash away unbound probe and detect bound probes
identify colonies or plaques which bind probe

Question 10

what are the steps involved in immunological screening?

Answer 10

colonies/plaques transferred to membrane
incubated with specific antibody
detect the antibodies bound to clones by using chromogenic substrate for enzyme label

Question 11

what are the three stages of PCR?

Answer 11

denaturation
annealment
extension

Question 12

what temperature do the three stages of PCR occur at?

Answer 12

denaturation= 95°C
anneallment= 60°C
extension= 72°C

Question 13

what is taq polymerase?

Answer 13

a heat stable DNA polymerase derived from Thermus aquaticus used in PCR reactions

Question 14

how many cycles is PCR repeated for?

Answer 14

30

Question 15

what are the uses of PCR?

Answer 15

identification of species
identification of individuals (forensics and paternity testing)
analysis of gene expression
testing for mutations