8.4.1 Recombinant DNA Flashcards
What are the three ways in producing DNA fragments
- Restriction endonucleases
- Reverse transcriptase
- Gene machine
Describe how restriction endonucleases can be used to produce DNA fragments
- Cuts DNA at specific point to obtain specific gene
- To produce sticky ends
- Produces fragment with introns
Why could using reverse transcriptase enzyme instead of restriction nuclease be advantageous
Reverse transcriptase produces fragment without introns - bacteria cannot splice introns
Describe how reverse transcriptase enzyme can be used to produce DNA fragments
- Reverse transcriptase forms cDNA from mRNA
- DNA polymerase produces a double-stranded fragment
Why is the gene machine a quicker way to produce DNA fragments
Not an enzyme-catalysed reaction
How to amplify DNA fragments in VITRO
PCR
How to amplify fragments in VIVO
Transform host cells
State the conditons for PCR at each stage
- Add DNA polymerase, primers and nucleotides to orginal DNA fragment
- Heat to 95c
- Cool to 55c
- Heat to 72c
- Cycle repeated
Describe why sample heated to 95c in PCR test
Break hydrogen bonds to seperate DNA strands
Why is the sample cooled to 55c
- Allow primers to bind to DNA
- Nucelotides bind to exposed DNA bases by complementary base pairing
Why is PCR re-heated to 72c
Allow DNA polymerase to join nucleotides together
Describe and explain restriction nucleases role in transforming host cells
- Cut specific gene from specimin using restriction endonuclease
- Restriction endonuclease will produce sticky ends
- Use same restriction endonuclease to cut plasmid open to produce sticky ends
- Ensure marker gene is included
Describe DNA ligase, and final steps in transforming a host cell
- DNA ligase joins sticky ends of desitred gene and plasmid
- Inject plasmid into bacterial cell using micropipette
- Add Ca2+ and use electric shocks to get cell to take up plasmid
How to identifiy a transgenic cell
Marker genes
