Genetic technology Flashcards
1
Q
What are three ways of transferring genes?
A
- using restriction endonuclease to extract gene
- making gene from mRNA from reverse transcriptase
- artificial synthesis of gene from nucleotides using computer programming
2
Q
How does restriction endonuclease work?
A
- derived from prokaryotes/bacteria
- cuts DNA at specific sites
- can be palindromic (blunt) or staggered (sticky) ends
- destroys viral DNA in bacteria
- same restriction enzyme used for vector
3
Q
How is DNA made from reverse transcriptase?
A
- add DNA primer to mRNA
- mRNA is a template
- DNA strand synthesized by reverse transcriptase
- cDNA formed
- DNA polymerase uses cDNA to create double strand
4
Q
Why are plasmids good vectors?
A
- small: easily taken up by the cell
- can be cut by restriction endonuclease: has specific sites
- replicate independently from each other: so a large amount of DNA is transcribed
- contain marker genes: such as antibiotic resistance genes, so can be recognized
- circular: makes them stable
5
Q
How are plasmids inserted into bacteria?
A
- transformation
- plasmid and bacteria bathed in ice-cold calcium chloride solution
- incubated at 40 degrees C
- bacteria given electric shocks
- this makes membrane porous
- so plasmids can enter
6
Q
What are the five main enzymes used in gene technology?
A
- DNA ligase
- DNA polymerase
- Restriction enzyme
- Reverse transcriptase
- Cas9
7
Q
What are the three main vectors used in gene technology?
A
- Plasmids
- Viruses
- Liposomes
8
Q
What is a promoter?
A
- length of DNA containing binding site for RNA polymerase
- controls gene expression and transcription
9
Q
Why are promoters needed for gene transfer?
A
- promoters initiate gene expression/transcription
- binding site for RNA polymerase
- otherwise gene has to be inserted near existing promoter
- can disrupt gene expression
- precise position of promoter in eukaryotes
10
Q
What are marker genes?
A
- gene transferred with desired gene
- to identify cells that have been altered and contain rDNA
11
Q
What are some advantages to marker genes over antibiotic resistance genes?
A
- easy to identify/screen
- more economical
- some antibiotics are no longer effective (bacteria will still grow)
- resistance gene can be passed to other bacteria
12
Q
Outline the process of gene editing
A
- form of genetic engineering
- involves deletion, insertion or replacement of gene
- Cas9: cuts DNA at specific points determined by the base sequence of a molecule of guide RNA
13
Q
What is PCR?
A
- amplification of DNA
- only small sample of DNA needed
- rapid
- Denaturation: DNA separated into two strands at 95 degrees
- primer added
- Annealing: 60-65 degrees
- complementary base pairing between primer and strand
- Extension: DNA strand synthesized at 72 degrees
- done by DNA/Taq polymerase (which is stable and can be reused)
14
Q
Why is Taq polymerase used in PCR?
A
- high optimum temperature/heat stable
- does not need replacing each cycle
- process is more efficient/faster than using DNA polymerase
15
Q
Outline the process of gel electrophoresis:
A
- DNA collected from root of hair or saliva
- DNA amplified by PCR
- DNA fragmented by restriction endonuclease
- enzyme will cut close to VNTR regions
- DNA loaded into agarose gel by micropipette
- DNA must be close to negative electrode
- gel submerged in electrolyte/buffer solution
- electrical current applied to tank
- DNA phosphate groups give negative charge
- attracted to anode
