21: Recombinant DNA technology Flashcards
1
Q
recombinant dna technology; 3 components
A
- creating dna fragments (gene machine, reverse transcriptase, resitriction endonucleases)
- genetic fingerprinting (forensic, medial diagnosis, breeding, tests)
- genetic screening, counselling and locating genes (personalised medicine)
2
Q
Dna fragments
A
- combining of diff organisms dna to alter genes to improve industrial processes and medical treatment
- three methods to create fragments of dna
1. reverse transcription
2. restriction endonucleases
3. gene machine
3
Q
reverse transcription (dna fragments)
A
- enzyme makes DNA copies from mRNA
- naturally occurs in viruses
- cell that naturally produces the protein of interest is selected
- these cells should have large amounts of mRNA for protein
- reverse transcriptase enzyme joins dna nucleotides with complementary base to mrna sequence
- single stranded dna is made (cDNA)
- to make double stranded, enzyme DNA polymerase is used
- cDNA is intro free as template of mRNA
4
Q
restriction endonucleases
A
- enzymes that cut up dna
- naturally occur in viruses
- have active sites complementary in shape to dna base sequences - recognition sequences. so each enzyme cuts dna at specific location
- some enzymes cut up at same location in double stand and create a blunt end, others r sticky ends which create staggered ends and expose dna bases. able to join to dna with complementary base pair
- sticky ends are palindromic
5
Q
gene machine
A
- created in lab using computerised machine
- scientists examine protein of interest to try and identify the amino acid sequence and from that work out what the mRNA and DNA sequence would be
- dna sequence entered into computer, checks biosafety and biosecurity that the dna created is safe and ethical to produce
- computer creates small sections of overlapping single strands of nucleotides that make up gene called oligonucleotides
- oligonucleotides joined to create dna for entire gene
- PCR can be used to amplify quantity, make double strand
- quick, accurate, intron free
6
Q
once dna fragments are creating whats next
A
- in vivo or in vitro cloning
7
Q
in vivo cloning
A
1) - restriction endonuclease enzymes cuts gene at recognition site leaving sticky ends
- dna fragments need to be modified so transcription can occur
- promotor region is added at start of dna fragment, as bindiing site for rna polymerase so transcription can occur
- terminator region at end of gene so rna polymerase can detach stop transcription so only one gene at time is copied into mrna
2) - insert dna into vector
- plasmid cut open by same restriction endonuclease enzyme so same sticky ends are created for complementary bases
- dna fragment and cut plasmid stuck together by enzyme ligase
- condensation reaction, phosphodiester bonds
- to get plasmids into host cell, calcium ions and heat shock to make membrane more permeable so can enter cytoplasm
8
Q
in vivo cloning part b
A
- some host cells dont take up recombinant plsasmid as it doesnt make it inside cell, plasmid rejoins before dna enters or dna fragement sticks to itself
- marker genes on plasmid to identify which took up recombinant plasmid
1. antibiotic resistance genes
2. genes coding for fluorescent proteins
3. genes coding for enzymes
9
Q
antibiotic resistance marker gene
A
- bacteria plasmid inserted with gene for resistant to tetracycline antibiotic and resistance to ampicillin gene
- dna fragment is inserted into tetracycline gene, disrupts it and no longer creates functional protein
- grow bacterial on agar, transfer colony to plate with ampicillin antibiotic
- any colonies still grow must have plasmid in it
- transfer bacteril colonies to plate with tetracycline antibiotic, any colonies that grown on both must be original plasmids, any that dont have the recombinant plasmid
10
Q
fluorescent markers
A
- jellyfish contains gene codes for green fluroscent protein
- inserted into bacterial plasmid
- insert dna fragment into GFP gene, disrupts and prevents production
- any that dont glow must have recombinant plasmid
11
Q
enzyme lactase
A
- turns blue from colourless
- gene for this enzyme is inserted into plasmid
- dna fragment is inserted in middle of this gene to disrupt it
- bacteria grown on agar plate
- colonies that cant turn colourless substance blue contain recombinant plasmid
12
Q
where do you grow the host cell
A
- fermenter
- host cells multiplied which contains recombinant plasmid
- larged, cloned population of host cell then produce the protein coded for by the inserted dna fragment
13
Q
genetic fingerprinting
A
- dna made of intros which consist of variable number tandem repeats
- probability of vntrs in two individuals is very low
- closely related more similar vntrs
- vntrs are anaylsed in genetic fingerprinting
14
Q
process of genetic fingerprinting
A
- collection from blood, cells or hair
- PCR used to amplify if sample is small
- digestion; if its large it can be cut by restriction endonucleases, cuts dna ust before and after VNTRs
- dna samples loaded into small wells in agar gel with electrical voltage, dna is negatively charged so more towards positive end of gel
- gel has reistance, so smaller lengths of VNTRs can move along aster
- all lengths are seperated
- alkalie is added to seperate double strands of VNTRs
- dna probes (short, single stranded pieces of dna) added to gel. labelled with fluroscent label. add diff dna probes and complementary in base sequence, mix and wash away ones dont bind
- transfer to nylon sheet and xray or uv light
- positon of dna bands are compared
15
Q
in vitro cloning (not living)
A
- polymerase chain reaction:
- automated machine, thermocycler, happens over to amplify dna fragment
- temp increased to 95 to break h binds split dna strands
- drop temp to 55 so prmers can attach
- enzyme dna polymerase attaches complementary free nucleotides to make new strand, temp increased to 72 (optimum)
