gene technology Flashcards
what is recombinant DNA technology?
transfer of fragments of DNA from one organism to another
DNA that contains nucleotides from other organisms
3 ways of producing DNA fragments
- reverse transcriptase
- restriction endonucleases
- gene machine
how can reverse transcriptase make DNA fragments?
- remove mRNA from a cell
- add reverse transcriptase and DNA nucleotides
- converts the mRNA into DNA (double stranded)
uses mRNA as template
= cDNA (complementary)
why is mRNA used for reverse transcriptase?
easier to isolate from cells than DNA
introns already removed in splicing
cDNA produced is complementary to mRNA, therefore is a copy of original DNA
how can restriction endonucleases make DNA fragments?
- obtain sample of DNA from a cell
- incubated with specific restriction endonuclease
- cuts DNA fragment using hydrolysis
how do restriction endonucleases work?
- different endonucleases cut at different base sequences
- shape of sequence is complementary to enzymes active site
- used to obtain gene between the sequences to cut it out, hydrolysis
(gene cant contain specific sequence or it will be cut)
leaves sticky or blunt ends
what are sticky and blunt ends?
when a gene is cut using a restriction endonuclease, leaves:
sticky ends - one strand of fragment longer than the other
- easier to insert gene as hydrogen bonds can form between bases
blunt ends - when both strands are same length
- nucleotides can be added to create sticky ends
how does a gene machine produce DNA fragments?
- determine amino acid sequence of the protein
- first nucleotide fixed to support
- nucleotides added step by step in order
- creates oligonucleotides (short sections of DNA)
- oligonucleotides joined to make longer fragments
ways of amplifying DNA fragments
PCR (in vitro)
in vivo
describe PCR as a method to amplify DNA fragments
- reaction mixture made
- heated to 95c
- hydrogen bonds break, strands separate - cooled to 55c
- allows primers to bind - heated to 72c
- allows complementary nucleotides to attach (optimum) - heat stable (taq) DNA polymerase joins nucleotides together
- with phosphodiester bonds
repeats
- each PCR cycle doubles amount of DNA
what does the reaction mixture for PCR contain?
- DNA sample
- free DNA nucleotides
- primers
- DNA polymerase (taq)
in vivo method of amplifying DNA fragments
- use restriction endonuclease to obtain desired gene from DNA
- use same RE to cut open a plasmid
- leaves sticky ends complementary to sticky ends of fragment - join desired gene to plasmid using DNA ligase
- joins sticky ends
creates recombinant DNA (vector and fragment)
- vector (plasmid) inserted into a bacteria cell
- plasmid replicated during binary fission to create more recombinant DNA (and therefore gene)
what are transformed cells and how can you identify them?
cells that have taken up the vectors, contain plasmids with recombinant DNA
- marker genes
how do marker genes identify transformed cells?
used to identify which cells contain the plasmid with the desired gene
- another gene (for an identifiable characteristic) is inserted with the desired gene
- means transformed cells also contain that gene
- eg antibiotic resistance, so only transformed and resistant cells survive
or gene inserted within a gene for antibiotic resistance
tranformed are killed
benefits of recombinant DNA tech in agriculture
crops can be transformed to be:
- more nutritious
- higher yields
reduce risk of famine
- pest resistant (fewer pesticides needed, cheaper)
benefits of recombinant DNA tech in industry
industrial process often use enzymes
- can be made from transformed organisms
- made in large quantities and cheaper
benefits of recombinant DNA tech in medicine
transformed organisms used to make drugs and vaccines
- can be made cheaper and quicker
eg insulin
issues of recombinant DNA tech in agriculture
monoculture of transformed crops - genetically identical
- makes whole crop vulnerable to same disease - famine risk
- reduces biodiversity
long term effects of GM food on health unknown
issues of recombinant DNA tech in industry
some consumer markets won’t import genetically modified food - producers suffer economically
no labelling - no choice over eating GM food or not, unethical
issues of recombinant DNA tech in medicine
could lead to unethical uses
eg designer babies - modifying and choosing characteristics
can be used to save lives but companies may limit use for economic reasons