Recombinant DNA Technology

Question 1

What is recombinant DNA technology

Answer 1

• the creating of combinations of DNA sequences from at least two different sources
• it often involves the insertion of a DNA fragment into a DNA cloning vector (normally a modified bacterial plasmid) to generate a recombinant DNA molecule that is amplified in E.coli

Question 2

What are some of the applications of recombinant DNA technology

Answer 2

• production of recombinant proteins (human insulin)
• generation of transgenic animals/plants (eg. golden rice)
• studying functions of genes/regulatory regions/proteins
• vaccines

Question 3

What are the general stages of recombinant DNA

Answer 3

• the generation of amplification and recombination DNA molecules involves cutting and joining DNA molecules
• transformation and selection of bacterial cells, and in some cases amplification of DNA by PCR/R-T PCR
• the recombinant DNA molecules can then be analysed in various ways

Question 4

Cutting DNA molecules

Answer 4

• requires the use of restriction enzymes (eg HindIII and BamHI)
  (these are enzymes produced by bacteria that recognise specific PALLINDROMIC sequences)
• the enzymes ‘cut’ both strands of DNA by cleaving the phosphodiester backbone
• some enzymes cut DNA producing single-stranded overhang (cohesive/sticky) end but others cut blunt ends

Question 5

What does annealing allow

Answer 5

annealing allows for recombinant DNA molecules to form by complementary base pairing, however the two strands are not covalently bonded

• DNA ligase seals the ‘nicks’ in the hybridised DNA molecules, it covalently joins the DNA molecules (does both sticky and blunt ends but blunt ends not as go)

Question 6

What is a PCR reaction and what does it require

Answer 6

Polymerase Chain Reaction
- allows amplification of a selected DNA region in vitro
- repetition of three stages

requirements: DNA template, DNA primers, Taq polymerase, buffer solution with Mg2+

DNA primers: act as starting point to synthesise complementary ends

Taq polymerase: from a hot spring so adapted to withstand high temperature of thermocycler

Mg2+ needed for polymerase to work

Question 7

What are the three stages of the PCR reaction

Answer 7

I. Denaturation (95 degrees)
- heating DNA into single strand

II. Annealing (55-65 degrees)
- annealing of DNA primers to template strand

III. Extension (72 degrees)
- Taq polymerase synthesises new DNA by adding nucleotides to 3’ end of primers

Question 8

How does a PCR with sticky ends vary

Answer 8

to facilitate insertion of PCR into plasmid vector, PCR primers can be assigned to their 3’ part which anneals to target sequence whilst 5’ forms restriction site

• product can then be cut with restriction enzymes to form cohesive/sticky ends

Question 9

What is Reverse-Transcription PCR (R-T PCR)

Answer 9

• have to remove introns in order to express eukaryotic proteins
• to obtain cDNA, a viral reverse transcriptase enzyme is used
• mRNA is used as a template to synthesise cDNA
• RNase digests the mRNA and then PCR is carried out as normal on cDNA

Question 10

What are cloning vectors

Answer 10

cloning vectors are DNA molecules that can hold pieces of DNA for replication in an organism

• has polylinker
• contains multiple recognition sites for different restriction enzymes (normally inside lac2 gene)
• this allows the plasmid to be cut open by the enzymes and insert sequences of DNA
• a selectable marker is present in the plasmid allowing only the bacteria with the vector to be selected (usually antibiotic resistance)

Question 11

How does selection occur

Answer 11

Plating on agar with antibiotics
- only some of the bacteria cells will have taken up the plasmid, any that have not will be killed by the antibiotic

Blue/White seleciton
- the lacZ gene codes for beta-galactodsidase, and can also catalyse the reaction of colourless X-gal into product which oxidises blue
- integration of an insert into multiple cloning sites causes white colonies on the plate but plasmids without a vector will appear blue

Question 12

How is the sequence of nucleotides established

Answer 12

DNA sequencing (Sanger sequencing)

requires: single strand DNA template, primers, DNA polymerase, dNTPs, ddNTPS with flurochromes

when ddNTPS are used they stop the synthesis of DNA causing small fragments to form
- these fragments can then be put in gel electrophoresis and compared to expected values to see if the vector has been put into the plasmid