Recombinant DNA Technology (Chapter 21) Flashcards
How are genes isolated from human DNA?
Use restricted endonuclease to cut DNA at a specific base sequence
What is the function of reverse transcriptase?
Makes cDNA from RNA
What enzyme produces cDNA from RNA?
Reverse Transcriptase
What is the function of endonuclease?
Enzymes which cut DNA into smaller fragments by cutting DNA at a specific base sequence/breaks phosphodiester bonds.
What is recombinant DNA?
Contains genes/sections of DNA from two species/ 2 types of organism.
What are sticky ends used for?
Join 2 pieces of DNA by complementary base-pairing/binding.
Describe the stages of making recombinant DNA
1) Cut desired DNA from another organism/mRNA fro call/organism
2) Using restriction endonucleases/reverse transcriptase to get DNA (if mRNA)
3) Use restriction endonuclease to produce complimentary sticky ends
4) Use same enzyme to cut plasmids/other gene (at sticky end
5) Mix together and use DNA ligase to join wanted gene to plasmid/other gene (at sticky ends)
6) If a plasmid return to cells and place in solution of Ca+ which makes bacteria cell walls and membranes more permeable, allowing plasmid to be taken up.
What is a vector?
A carrier of DNA/a gene into another cell/organism/host.
Describe the stages of genetic fingerprinting
1) DNA is cut using restriction enzyme
2) Use electrophoresis to separate DNA fragments according to size and shape
3) Southern blotting/ transfer to nylon membrane
4) Make single stranded so probe will bind
5) Apply probe
6) Radioactive/fluorescent
7) Reference to tandem repeats/VNTRs
8) Identify using autoradiography
What does electrophoresis do and how does it work?
Separates DNA pieces
Move towards anode/move because charged. Separates according tp size/charge.
What features are separated by gel electrophoresis?
- Mass/number of amino acids/polypeptides
- Charge
- R group
What is inserted in the plasmid along with the gene to ensure that the gene was only effective in target cells?
Promoter
