amplifying DNA fragments in vivo

Question 1

why/when does amplifying take place?

Answer 1

once you have isolated the DNA fragment using one of the techniques eg RE enzymes, you need to amplify it so there is a sufficient quantity to work with

Question 2

what is in vivo cloning?

Answer 2

where copies of DNA fragment are made INSIDE a living organism and this involves transforming host cells

Question 3

step 1 of in vivo cloning

Answer 3

1. DNA frag is inserted into vector
2. Vector is cut open using same REs used to isolate the DNA fragment containing target gene
3. sticky ends of vector are complementary to the sticky ends of fragment
4. vector DNA and DNA frag are mixed together with DNA ligase
5. this joins the sticky ends of both- LIGATION. the new combination of bases in the DNA is called recombinant DNA

Question 4

what is a vector?

Answer 4

something that’s used to transfer DNA into a cell. They can be plasmids, bacteriophages etc.

Question 5

step 2 of in vivo cloning

Answer 5

1. vector with recombinant DNA is used to transfer the gene into host cells. The host cells that take up the vectors are said to be transformed.

Question 6

stage 3 of in vivo cloning

Answer 6

1. only 5% of host cells will take up the vector so marker genes are used to identify these cells.
2. marker genes are inserted into vectors at same time as the gene to be cloned.
3. host cells are grown on agar plates. each cell replicates and divides creating a colony of cloned cells. transformed cells will produce colonies where all cells contain marker gene.
4. marker gene can code for antibiotic resistance and because the cells are grown on agar plates with a specific antibiotic, only transformed cells will survive and grow
5. when agar plate is placed under UV light, only transformed will flouresce

Question 7

stage 4 of in vivo cloning
promoter and terminators

Answer 7

to produce proteins, you need promoter and terminator regions.
promotor regions are DNA sequences that tell RNA polymerase when to start producing RNA, terminators tell it when to stop. they may be present in the vector DNA or may have to be added. without the right promoter region, the fragment wont be transcribed so no protein will be made