Lecture 26 Flashcards
(17 cards)
Recombinant DNA
Using enzymes and lab techniques to manipulate and isolate target DNA. Can combine DNA from different species, create new genes with new functions.
Expression and purification of target proteins: research purposes
Enzymes like polymerases and ligases.
Expression and purification of target proteins: monitoring devices
Glucose monitors use glucose oxidase
Expression and purification of target proteins: industrial purposes
High fructose corn syrup uses main enzymes
Expression and purification of target proteins: therapeutic agents
Insulin, clotting factors
Expression and tagging of proteins for visualisation: Research processes
Immunofluorescence for visualisation of tissues and cells in vivo
Expression and tagging of proteins for visualisation: Commercial uses
Glofish o.g to detect toxins in H2O but sold to public
Improving traits of crops and animals
Bt cotton makes crops resilient to pests (decreases pesticide use).
Golden rice (great for vit A deficiencies)
Sterile mosquitoes (males mate with females but offspring are sterile)
Cloning prokaryote DNA
PCR
Cloning eukaryote DNA
Introns and exons means that target gene cannot be copied straight away. You also cannot do PCR using mRNA. Instead, you can use cDNA which is formed by reverse transcribing mRNA.
Technical challenges of cDNA
Does tissue/cell type matter? Where protein is expressed = increase [mRNA]
Genomic DNA
Total DNA in cell (incl. exons and introns) used for GWAS
Complementary DNA
Only expressed genes therefore, undergoes splicing. Used for seeing protein expression
Converting mRNA to cDNA
Need RNA-dependent DNA polymerase (RdDp) which originates from retroviruses.
Technical challenges of making cDNA
Total RNA includes tRNA, microRNA, snRNA, and mRNA, but only mRNA is needed for cDNA synthesis. To isolate mRNA, the poly(A) tail can be targeted using oligo(dT) primers, which are short sequences of thymidine that bind specifically to the poly(A) tail. This can be done by: adding free oligo(dT) primers to bind mRNA directly, or using magnetic beads coated with oligo(dT) primers, which selectively bind mRNA when incubated with total RNA, allowing separation with a magnet.
Making cDNA
1) Synthesise 1st strand of cDNA from purified mRNA. Oligo(dt) primers at 3’ end, reverse transcriptase can make cDNA.
2) cDNA used in PCR by adding random or specific primers. Creates cDNA library which has all amplified genes or specific gene PCR product.
After
To express target gene, need promoter and coding sequence. Promoters can be site or time specific. Consider: appropriate promoter, gene of interest, incentive for bacteria to keep target gene, successful propagation of gene leads to later generations.
