gene technologies Flashcards
genome project, fingerprinting
proteome =
the full range of proteins a cell can prod
genome =
all of the DNA in a cell/organism
sequencing a genome requires…
working out the DNA base seq for all the DNA in a cell
the human genome project
took 13 years –> completed in 2003
why is it easier to use the genome of simpler organisms to identify potential antigens for vaccines?
- they dont contain introns but eukaryotes do contain introns and regulatory genes (alternative splicing occurs)
- the genome can be directly used to obtain the proteome of the organism –> this can be used to identify potential antigens
examples of recombinant DNA technologies include…
- creating DNA fragments for in vitro/in vivo cloning
- genetic fingerprinting
- genetic screening, counselling & location of specific genes
the 3 ways of prod. DNA fragments
- restriction endonucleases
- reverse transcriptase
- gene machine
recombinant DNA technology =
the combining of diff organisms’ DNA –> enables scientists to manipulate & alter genes to improve industrial prcesses/medical treatment
restriction endonucleases
(1/3 method of prod. DNA)
naturally occur in bacteria as defense mechanism
1. active sit of endonucleases comple. to specific DNA base seqs –> recognition seqs
2. therefore, each enzyme cuts the DNA at a specific location
3. some endonucleases cut at same location of double strand to create blunt end, some endonucleases cut to create staggered ends & exposed bases
4. staggered ends = ‘sticky ends’ –> palindromic –> have ability to join to DNA with comple. base pairs
reverse transcription
(2/3 method of prod. DNA)
- isolate the mRNA transcribed for the desired gene
- the reverse transcriptase joins the comple nucleotides to the mRNA seq.
- single-stranded ‘cDNA’ is prod.
- the DNA polymerase joins nucleotides to the cDNA to prod double-stranded DNA
why is the cDNA intron-free?
it’s based on the mRNA template
gene machine
(3/3 method of prod. DNA)
- uses computerised machine
- a.a seq of protein used to determine DNA seq
- PCR amplifies amount of oligonucleotides (which make up gene)
- DNA fragments prod = intron-free –> can be transcribed in prokaryotic cells
why is use of a gene machine faster than reverse transcription to prod DNA. fragments?
reverse transcription has slow enzyme-controlled reactions
name the 2 methods of amplifying DNA fragments to obtain a large amount
- in vivo cloning
- in vitro cloning
in vivo cloning
- restriction endonucleases cut at recognition sites, leaving ‘sticky ends’
- promotor and terminator regions added –> RNA polymerase can attach and detach –> transcription starts & stops
- insert DNA into vector –> plasmid cut using same res. endo. –> comple ‘sticky ends’ -> ligase enzyme anneals DNA & vector –> DNA ligase forms phoshodiester bond between adj. nucleotides on sticky ends
- vector inserted into host cell –> cell membrane of host cell needs increased permeability –> mixed w Ca^2+ & heat shocked
- addition of marker genes
issues w recombinant plasmids
(which makes identification important)
- recombinant plasmid not inside cell
- plasmid re-joins before DNA frag enters
- DNA frag sticks to itself (instead of plasmid)
marker genes used to identify which genes successfully took up the recombinant plasmid
in vitro cloning
uses ‘polymerase chain reaction’ in a thermocycler
describe and explain how PCR is used to amplify a DNA fragment (4)
- thermocycler contains DNA fragment, DNA polymerase, nucleotides & primers
- heat to 95 degrees to break H-bonds –> splits DNA into single strands
- reduce temp to 55 degrees so primers anneal/bind to DNA
- increase temp to 72 degrees so DNA polymerase joins free comple nucleotides to exposed DNA strand
in every PCR cycle, how does the amount of DNA increase?
exponential increase –> doubles each cycle
advantages of PCR
- automated –> more efficient
- rapid –> billions of copies within hrs
- doesn’t req living cells
what is a DNA probe? (2)
- short single-strand of DNA
- with bases comple to the DNA/gene
why are DNA probes used?
- fluorescent/radioactive label on probe allows identification of specific base seqs of DNA
DNA hybridisation (used for medical diagnosis)
(as a result of use of DNA probes and identifying a particular allele)
- DNA heated –> double strand split into single strands
- split DNA mixed w comple single-stranded DNA
- strands will anneal
genetic fingerprinting =
analysis of VNTR DNA fragments
general process of genetic fingerprinting
- extraction of DNA –> from blood, saliva etc (PCR used if sample too small)
- digestion –> restriction endonucleases cut DNA into fragments –> then VNTRs added
- separation –> DNA samples loaded into small wells in agar gel placed in buffer liq w voltage applied –> -vely charged DNA moves towards +ve end of gel –> gel electrophoresis
- hybridisation –> DNA probes comple to VNTRs –> bind
- development –> VNTRs & probes transferred to nylon sheet –> x-ray film/uv light used to show probe
- analysis
genetic fingerprinting mark scheme ans
- extract DNA & add restriction endonucleases
- seperate fragments using gel electrophoresis
- treat DNA to expose bases
- probe will bind/hybridise to gene
- use autoradiography/x-ray film to show the bound probe
gel electrophoresis
analysis of genetic fingerprinting
- position of DNA bands (VNTRs) used to compare genetic relationships
