(21) recombinant DNA technology Flashcards
define genome
entire set of DNA including all the genes in an organism
how is gene sequencing done
smaller fragments are sequenced first and then put back into order to give the whole genome
define proteome
all the proteins made by an organism
why is determining the proteome useful in medical research
identifying protein antigens on the surface of disease causing bacteria and viruses can help in the development of vaccines to prevent the disease
what are regulatory genes
they determine when the genes that code for particular proteins should be switched on and off
what is pyrosequencing
recently developed technique that can sequence around 400 million bases in a 10 hour period
what is recombinant DNA technology
transferring a fragment of DNA from one organism to another because genetic code is universal and transcription and translation mechanisms are pretty similar, the trasnferred dna can be used to produce a protein in the recipient cells
what are the 3 ways dna fragments can be produced
1) using reverse transcriptase
2) using restriction endonuclease enzyme
3) using a ‘gene machine’
how can dna fragments be made using reverse transcriptase
mRNA can be used as templates to make lots of dna. reverse transcriptase then makes dna from an rna template. the dna made is called complementary dna (cDNA)
how can dna fragments be made using restriction endonuclease enzyme
restriction endonuclease recognises palindromic sequences (recognitiion sites) and cuts the dna at these places. different enzyme used for different recognition sites as has to be complementary. dna sample is then incubated with specific restriction endonuclease which cuts dna fragment out by a hydrolysis reaction. this sometimes leaves sticky ends
what can sticky ends be used for
to bind the dna fragment to another piece of dna that has sticky ends with complementary sequences.
how can dna fragments be made using a ‘gene machine’
technology that synthesises dna fragments from scratch with no need for a pre existing dna template. database contains necessary info to produce the dna fragment. sequence is designed, first nucleotide is fixed to a bead and more nucleotides are added and protecting groups prevent unwanted branching. then broken off from bead and protecting groups are removed
why do dna fragments need to be amplified
so we have sufficient quantity to work with
outline the process of in vivo amplification
1) dna fragment inserted into vector (plasmid or bacteriophages). vector is cut open using same restriction endonuclease that was used to isolate fragment sticky ends of vector and dna are then complementary. vector dna and fragment dna are mixed together using dna ligase (new combination of DNA + DNA fragment is recombinant dna.)
2) vector transfers gene into cells (only about 5% of host cells will take up the vector)
3) marker genes can be used to identify the transformed genes
how can you produce proteins coded for by the dna fragment using transformed host cells
vector must contain specific promoter and terminator regions.
promoter regions- dna sequences that tell the enzyme rna polymerase when to start producing mRNA and terminator regions tell it when to stop. without the right promoter region the DNA fragment wont be transcribed and protein wont be made
what is in vitro cloning
when copies of a dna fragment are made outside of a living organism using the polymerase chain reaction.
what are the stages of the polymerase chain reaction (in vitro cloning to make dna fragments)
1) reaction mixture containing DNA sample, free nucleotides, primers and DNA polymerase is heated to 95 to break H bonds between 2 dna strands. mixture cooled to 50-65 so primers can bind to strands
2) heated to 72 so dna polymerase can work- lines up free dna nucleotides alongside each template strand and complementary strands are formed.
3) 2 copies of dna fragment are formed in one PCR cycle
what are transformed organisms and how can they be made
microorganisms, plants and animals can be transformed (genetic engineering). foreign dna can be inserted into:
1) microorganisms- to produce useful protein
2) plants
3) animals- desirable gene inserted into embryo or egg cell
how can recombinant dna benefit the agriculture industry
tranformed to give higher yields or more nutritious - reduces risk of famine and malnutrition
how can recombinant dna benefit the medicinal industry
many drugs and vaccines are produced from transformed organisms eg insulin for T1 diabetes used to come from animals but didn’t work that well for humans but transformed microorganisms can now use a cloned insulin gene to make insulin
what are the concerns associated with using recombinant dna in agriculture
1) using only one type of transformed crop (monoculture) could make the whole crop vulnerable to disease also reduced biodiversity
2) superweeds could occur if transformed crops interbreed with wild plants- uncontrolled spread of recombinant dna with unknown consequences
3) organic farmers could have their crops contaminated with wind blown seeds from nearby GM crops so would no longer be organic- less income
concerns of recombinant dna in medicine
1) companies who own genetic engineering technologies may limit the use of technologies that could be saving lives
2) could be used unethically e.g. designer babies (currently illegal)
what are the ownership issues arisen from recombinant dna
1) who owns human genetic material once it has been removed from the body- donor or researcher?
2) large corporations own patents to particular seeds and charge high prices and can require farmers to repurchase every year. if non GM is contaminated by GM farmers can be sued
what are the humanitarian benefits of recombinant dna technology
1) agricultural crops can be produced that reduce famine and malnutrition
2) transformed crops can be used to make useful pharmaceutical products so can be available for more people
3) medicine can be made more cheaply so more can afford it
4) potential to be used in gene therapy to treat human disease
