8.4 Recombinant DNA Flashcards
What is recombinant DNA?
The combination of DNA from different organisms (from one into a transgenic organism)
How can we make DNA fragments?
Restriction endonucleases
Gene machine
Reverse transcriptase
How does reverse transcriptase produce a DNA fragment?
- mRNA of target DNA
- reverse transcriptase used to make DNA
mRNA isolated + free floating DNA nucleotides + reverse transcriptase
How do restriction endonucleases produce a DNA fragment?
- Some sections of DNA are palindromic = recognition sequences
- Cut at complementary recognition sequence
-Produces sticky ends
DNA incubated with specific restriction endonuclease
What are sticky ends?
Short lengths of unpaired bases at each ends of a DNA fragment
How can we amplify DNA fragments?
In vitro or in vivo
How does in vitro DNA amplification work?
DNA sample, free nucleotides, DNA polymerase & primers
1) Heated to break H bonds between DNA strands
2) Cooled to allow primer anneal
3) Heat to polymerase optimum T
Repeat
How does in vivo DNA amplification work?
1) Inserted into vector DNA e.g. plasmid or bacteriophage
- Cut using restriction endonuclease + DNA ligase = recombinant DNA
2) Vector with recombinant DNA used to transfer gene into host cell
- Once taken up = transformed
3) Identify transformed cells
What is ligation?
The process in which DNA helicase synthesises the production of H bonds between the sticky ends of complementary DNA fragments
Why are promoter and terminator regions important in amplifying DNA fragments in vivo?
- Indicate where RNA polymerase starts and ends
- Won’t start or end in the correct spots
Describe how gel electrophoresis works
- Amplified DNA sample and primers that bind to both sides of VNTRs
- Add fluorescent tag
- Add to electrophoresis : wells in gel slab with buffer soln and charge applied
- Samples sort by size
What is genetic counselling?
Advising patients about risk of genetic disorders
What is genetic fingerprinting?
Comparing genomes of different individuals
