amplifying DNA fragments

Question 1

why do you need to amplify (make lots of copies of) DNA fragment

Answer 1

so there is a sufficient quantity to work with

Question 2

in vivo

Answer 2

transforming host cells

Question 3

in vivo

step 1 - DNA frag inserted into vector

Answer 3

1. vector is something used to transfer DNA into a cell e.g. plasmids or bacteriophages (viruses that infect bacteria)
2. vector DNA cut using same restriction endonuclease that was used to isolate DNA. sticky ends complimentary
3. vector DNA and DNA frag mixed together with DNA ligase (enzyme) which joins the sticky ends
4. new combination of bases = recombinant DNA

Question 4

in vivo

step 2 - vector transfers DNA frag into host cell

Answer 4

1. vector with recombinant DNA used to transfer gene into host cell
2. plasmid vector - host cells have to be persuaded to take vector in
3. bacteriophage vector - infects host cell by injecting DNA into it
4. host cells taken up gene = transformed cells

Question 5

in vivo

step 3 - identifying transformed host cells

Answer 5

1. marker gene inserted into vector at same time as gene
2. host cells grown on agar plates. cells divide and replicate creating colony of clones
3. marker gene can code for antibiotic resistance - grown on agar plate containing antibiotic - only transformed cells with marker gene survive and grow
4. marker gene can code for fluorescence - UV light only transformed cells fluoresce

Question 6

in vivo what is essential

Answer 6

specific promoter and terminator regions
(DNA sequences that tell enzyme DNA polymerase when to start and stop producing mRNA)
may be present in vector DNA or may have to be added

Question 7

in vitro

polymerase chain reaction

Answer 7

1. reaction mixture contains-
   DNA sample,
   free nucleotides,
   primers (short pieces of DNA complimentary to the bases at the start of the fragment you want),
   DNA polymerase (enzyme that creates new DNA strands)
2. heat mixture to 95C break H bonds between 2 strands
3. mixture cooled between 50C and 65C so primers can bind/anneal to strands
4. heat to 72C so DNA polymerase can work
5. DNA polymerase lines up free DNA nucleotides alongside each template strand. specific base pairing = complimentary strands formed
6. 2 new copies of fragment formed and 1 cycle complete.
7. cycle starts again -> all 4 strands used as templates. each cycle doubles amount of DNA