recombinant DNA tech

what applications does genetic manipulation have in medicine?

-determine molecular basis of disease

-develop diagnostic tests

-develop new therapies

-whole genome sequencing- to see change in cancer cell, pre-natal test, identify drug resistant infections

what are the biotech applications of genetic manipulation?

-pharmaceutical

-forensic sciences

-genetic manipulation of plants and animals

how could you insert a fragment of DNA into a plasmid?

digest w restriction endonuclease EcoRI, add in fragment, DNA ligase

what are isoschizomers?

recognize same site and cut in same way

what are neoschizomers?

recognise same site but cut differently

what are the stages of E.coli transformation?

incubate at 4degrees, heat shock (42degree for ~30s)/ electroporation (15kV/cm, 5microsecond pulse), recover w/o selection, grow on selective agar

what do you need for PCR?

target DNA, Primers (one forward and one reverse), Taq DNA polymerase, dNTPs, buffer containing Mg2+, thermal cycler

what are the 3 stages of PCR cycle, what are the temperatures?

denaturation 95degrees (30-60s, denature DNA into ssDNA)

annealing 40-65degrees (30s allow primers to anneal ssDNA)

extension 72degrees (1-3min DNA synthesis)

what are some issues with taq polymerase?

error prone, can fix via proofreading polymerases (e.g. Pfu)

what are some examples of intercalated dyes used to visualise DNA w UV?

Ethidium bromide, SYBR safe

what are 3 proteins e.coli used to make

insulin, Human growth hormone, chymyosin (in cheese making)

what can transgenic goats be used to make?

antithrombin III

what was yeast used to make?

hepatitis b vaccine

what were culture mammalian cells used for?

making herceptin

explain how to make mRNA e.g. for vaccines n vitro?

plasmid vector- pGEM3Z, add T7 or SP6 RNA polymerase

how to make RNA vaccine against SARS-Cov2?

insert gene encoding virus spike into plasmid vector

transcribe in vitro (pseudouridine not uridine)

add 5’ cap and poly A tail to RNA

remove vector DNA & purify spike protein mRNA

package pure spike protein mRNA into lipid nanoparticles

how do DNA probes work?

probe hybridises once DNA or mRNA recognised

what is southern blotting?

DNA molecules separated on gel, probe is labelled DNA or RNA

what is northern blotting?

RNA molecules separated on gel, labelled DNA or RNA is probe (tell you extent of gene expression)

what is western blotting?

proteins are separated on a gel, antibodies are the probe

what is the difference between qPCR and qRT-PCR?

quantitative PCR: detect presence DNA, quantify no. of copies of target DNA in sample

quantitative reverse transcription PCR: detect presence of cDNA, e.g. copies of a virus, measure gene expression relative to control gene

both use probes or fluorescent dye- measured after each cycle

how is conc. measured through these RT-PCR?

ct= cycle threshold, the sooner it is crossed the higher the concentration, can compare to housekeeping gene

what were the 3 genes the COVID-19 PCR tests?

S, N and ORF1ab

what would a test positive for omicron variants look like?

+ve for N & ORF1ab genes, negative for S

how to DIY transgenic animals?

can do pronuclear microinjection on a newly fertilised egg (100-200 copies)→ implant into pseudopregnant mother

who are the AquaAdvangtage salmons?

salmon promotor + chinook growth hormone cDNA→ microinject into fertilised salmon eggs

-imporves utilisation of diet

-increases growth rate

what are concerns regarding Aquaadvantage salmon?

alteration to human health-hormones/allergens

environment- escaped transgenic fish breed w wild pop

who are transgenic goats?

goat promoter + human antithrombin gene→ microinject goat fertilised egg

milk contain antithrombin

what can transgenic plants be used for?

-manufacture commercial important proteins

-resistance to insects & herbicides

-improve nutrition

what is the T region and vir region in the Ti plasmid?

T= promotes tumour formation, integrates into a host cell chromosome

Vir= req to transfer T region into plant chromosme

in the Ti plasmid, what region do u insert the exogenous DNA into?

T region

what are 2 examples transgenic plants?

-Bt Corn & cotton (express insecticidal protein)

-Herbicide resistant soya (makes EPSPS that cannot be inhibited by glyphosate)

what are some concerns about transgenic plants?

could transfer genes to weeds→superweeds

could encourage use of single herbicide→resistance

could be unforeseen harm to consumer

what is the Cas9/ tracrRNA/ crRNA complex?

Case 9= DNAse enzyme

tracrRNA= trans-acting RNA

crRNA= CRISPR RNA (transcription of fragments makes this)

this whole complex recognises bacteriophage DNA and cleaves it

how can CRISPR/Cas9 be used in targeting a gene?

Cas9 & fused crRNA-trancrRNA fused= a single guide sgRNA, engineered to recognise a target gene

what are the 2 ways double stranded breaks are repaired in eukaryotes?

homology directed repair- accurate, has crossover, one chromatid donates sequence to replace gap, only in S-phase & early G2 (utilised in precise alteration)

non-homologous end joining-error prone, happens in all phases cell cycle (utilised by CRISPR Cas9 for inactivation of gene)

what does deletion of ADE2 in yeast cause?

red colonies due to accumulation of substrate

what deos CAR T-cell therapy do?

get t cell from patient

make CAR t cells in lab (counteract cancer cells)

grow more

infuse CAR T cells into patient