8B- Amplifying DNA Fragments Flashcards
What is in vivo cloning?
Copies of the DNA fragment are made inside living organisms.
How does in vivo cloning work?
The SNA is inserted into a vector, usually plasmids or bacteria.
The vector is cut open using restriction endonuclease.
The sticky ends are complementary so DNA ligand joins the sticky ends to the vector (ligation). This is called recombinant DNA.
The vector is inserted into host cells. Host cells take up the vector containing the gene of interest are said to be transformed.
Marker genes are inserted into vectors. Host cells are grown on agar plates. They divide and create a colony, all the cells should have the marker gene.
If it’s fluorescent it should be under UV. If it’s antibiotic resistance the antibiotic should be added and if they survive it has been passed on.
Why do you need promoter and terminator regions?
The vector needs to contain a specific promoter and terminator regions.
Promoter regions are DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA.
Terminator regions tell it when to stop.
What does in vitro amplification use?
Polymerase chain reaction (PCR).
How does polymerase chain reaction (PCR) work?
A mixture containing the DNA sample, free nucleotides, primers and DNA polymerase is heated to 95c to break the hydrogen bonds between the two strands.
A primer are short pieces of DNA that are complementary to the bases at the start of the fragment.
DNA polymerase is an enzyme that creates new DNA strands.
The mixture is cooled to 50/65c so that the primers can anneal.
It’s then heated to 72c so the DNA polymerase can work. The free nucleotides join.
Two new copies of the DNA are formed in once cycle. One the second cycle four pieces are used as templates.
Every cycle it doubles.
