L4: Cloning by PCR and Muragenesis

Question 1

cDNA library

Answer 1

Library of expressed genes, all the genes expressed in cell or tissue cloned into host organism

Expressed genes represented as mRNA molecules

Question 2

Reverse transcriptase

Answer 2

RNA dependent DNA polymerase

Copies RNA to DNA

Question 3

Construction of cDNA

Answer 3

Hybridise mRNA with oligo-dT primer -> transcribe RNA into cDNA (reverse transcriptase) -> remove RNA with alkali. Add poly(dG) tail -> hybridise with oligo-dC primer -> synthesise complementary strand (DNA polymerase) -> protect cDNA by methylation at EcoRI sites -> ligate cDNA to restriction site linkers -> cleave with EcoRI. Remove replaceable region vector arms -> vector arms with sticky ends -> ligate to arms -> package in vitro -> recombinant virions -> infect e.coli

Library of expressed genes, all genes expressed in cell or tissue cloned into host (lambda phage)

Question 4

Hybridisation screening

Answer 4

Colonies/plaques transferred to surface of membrane (DNA binds) -> probe hybridises to any complementary DNA on membrane -> nitrocellulose filter is washed and audiographed -> development of film shows position of colonies with DNA complementary to probe -> +ve colonies removed from agar plate and cultured in nutrient broth -> purify insert DNA and confirm identity (sequence the DNA)

Question 5

Probe

Answer 5

Set of oligonucleotides

Designed from AA sequence of protein

Will only hybridise to DNA on membrane where DNA is complementary for probe

Question 6

Some characteristics of PCR

Answer 6

Uses thermostable DNA polymerase

Amt of DNA doubles each cycle

Sensitive (needs tiny amt of DNA template)

Occasionally get copying error (Taq DNA polymerase has no proof reading activity)

Oligonucleotide primer can contain one or more mismatches (except at 3’ end) -> deliberate introduction of a mutation

Question 7

Ligation of PCR products to vectors

Answer 7

PCR products have single deoxyadenosine residue overhanging at 3’ end (because of Taq DNA polymerase

-> use vector with T overhang at 3’ end

Anneal & ligate with DNA ligase