B21- Recombinant DNA Technology Flashcards
(9 cards)
1
Q
What is recombinant DNA technologies
A
- Allows manipulation and transfer of genes between organisms
- Creates transgenic/genetically modified organisms by combining DNA from different sources
2
Q
How do you make proteins
A
- Isolate DNA fragments
- Insert DNA into vector
- Transform into host cells
- Identify transformed cells
- Grow/clone host cells
3
Q
Methods to Produce DNA Fragments:
A
- mRNA to cDNA conversion using reverse transcriptase
- Restriction endonucleases cut DNA at specific sequences
- ‘Gene machine’ process - determines protein structure, designs and synthesizes DNA fragments
4
Q
What is the PCR process
A
- PCR is an automated method for copying DNA fragments
- Uses a thermocycler to control temperature
- Requires DNA template, polymerase, nucleotides, primers, and ATP
5
Q
How does PCR work
A
- Denaturing at 95°C - separates DNA strands
- Annealing at 55°C - primers attach to DNA
- Synthesis at 72°C - new DNA strands form
6
Q
How do you analyse DNA
A
- Electrophoresis separates DNA fragments by size
- DNA fragments can be visualized using Ethidium Bromide
- Fluorescent or radioactive probes can detect specific DNA sequences
- FISH technique can locate specific DNA sequences using fluorescent probes
7
Q
How does IVF work using a vector
A
- Uses restriction enzymes to create sticky ends and DNA ligase to join fragments
- Transformation involves mixing plasmids with bacteria, using calcium ions and heat shock (42°C), with about 1% success rate
8
Q
How can you identify if IVF uses a vector is successful
A
- Using antibiotic resistance genes
- Green fluorescent protein markers
- Enzyme activity detection
9
Q
What are the advantages of IVF using a vector?
A
- Useful for gene therapy
- No contamination risk
- Higher accuracy than PCR
- Can produce large amounts of gene product
