Gene Technology Flashcards
(28 cards)
describe how restriction endonucleases produce sticky ends on DNA fragments
- Restriction endonucleases cuts DNA as palindromic restriction sites
- leaving sticky ends
- this can be done around a target gene
how does reverse transcriptase work?
why can it be useful?
- reverse transcriptase converts mRNA to cDNA (complementary DNA)
- it can be useful because cells only have 2 copies of each gene in the nucleus which is relatively hard to access
how does artificial synthesis (the gene machine) work?
- you design a protein by deciding the aa sequence
- the machine joins up to 25 nucleotides together
- to form an oligonucleotide
- the oligonucleotides can be joined together to make the whole synthetic gene
name 3 ways to isolate target genes
- gene machine
- reverse transcriptase
- restriction endonucleases
what do you need to add to isolated target genes?
- promoter and terminator regions
- marker gene
- (sticky ends if not made by restriction enzymes in 1st place)
inserting a gene into a vector
- the same restriction endonuclease as used to produce sticky ends cuts a plasmid (ensures ends are complementary)
- DNA ligase forms phosphodiester bonds
- forming recombinant DNA
recombinant DNA
DNA from more than 1 source eg plasmid + target gene
plasmid
- double stranded loop of DNA
- used to transfer genes between bacteria
vector
used to move DNA from one place to another
inserting a vector into bacteria
- forms a transgenic organism (contains recombinant DNA)
- put recombinant plasmid and bacteria into ice cold calcium chloride then heat shock it
- -> this increases the permeability of the bacterial cell wall
marker gene
genes which are paired with target genes to allow you to identify if the vector has been inserted properly
why are marker genes useful?
vectors are often not taken up properly by bacteria so they allow you to test which have become transgenic
what to marker genes do?
- they fluoresce under UV light
2. they are resistant to antibiotics
only bacteria that have taken up the vector…
will fluoresce/be resistant to antibiotics if put into them so these transgenic bacteria can be selected and cultured
what is PCR used for?
to amplify DNA samples
describe the method of PCR
- heat sample to 95’c - to break H-bonds and make DNA single stranded
- cool sample to 50’c - allows primers to anneal by complimentary base pairing and form a double strand so DNA polymerase can bind
- heat to 70’c - DNA polymerase forms phosphodiester bonds between compimentary nucleotides
- repeat
VNTR’s
Variable Number Tandem Repeats
~unique for each person!
what type of DNA is used for genetic fingerprinting and why?
- non-coding DNA
- it mutates more regularly and is more different
gel electrophoresis
- chromatography for DNA using a current
- where the shortest sequences move the furthest
- DNA is negatively charged so will move towards the positive end
genetic fingerprinting: extraction
extract of sample of DNA taken and amplified using PCR
genetic fingerprinting: digestion
- DNA sample cut into fragments using restriction endonucleases
- fragments are different lengths for different people
genetic fingerprinting: separation 1
- samples placed in wells with agarose gel
- electrophoresis - current run through, shortest move furthest
genetic fingerprinting: separation 2
- nylon membrane placed over gel so DNA attaches to membrane
genetic fingerprinting: hybridisation
radioactive DNA probes added which bind to VNTR’s
