Genetic Manipulation Flashcards
Why do we clone DNA
To obtain DNA in sufficient quantities and in a useful form for study (e.g. in a plasmid vector)
What is the purpose of plasmid vectors in gene cloning
They allow easy replication and manipulation of cloned DNA in host cells
What are the steps in the alkaline lysis protocol for plasmid DNA isolation
- Lyse bacterial cells with SDS and NaOH; chromosomal DNA becomes single-stranded, plasmid stays intact.
- Neutralise to pH 7, single-stranded linear DNA aggregates.
- Centrifuge: plasmid DNA remains in supernatant as supercoils.
How does Sanger sequencing work
Uses chain-terminating ddNTPs which lack a 3’ OH group, fragments are size-separated and read by fluorescence
How is gene structure determined from sequences
By comparing cDNA (exons only) to genomic DNA (introns + exons)
Why can’t gel electrophoresis estimate gene copy number
It only shows presence/absence, not copy number due to smearing or single bands
What is Southern Blotting
A method to estimate gene copy number by hybridising a labelled DNA probe to restriction fragments on a blot
How do you isolate a cloned DNA fragment from a vector
Cut vector with restriction enzyme, run gel, extract band, and purify
How do you label a DNA probe using Klenow fragment
Use random hexamers to prime synthesis on denatured DNA with labelled nucleotides; Klenow incorporates labels
What is autoradiobiography used for
To detect hybridised, radiolabelled probes on blot membranes
How is chromosomal localisation of a gene determined
Using FISH (fluorescent in situ hybridisation) on metaphase chromosome spreads
What does Northern blotting measure
RNA levels and mRNA size in tissue samples
What are the steps of Northern Blotting
RNA gel electrophoresis → blotting → hybridisation with labelled probe → autoradiography
What is RT-PCR and its advantage over Northern Blotting
It’s faster and easier, involving cDNA synthesis from RNA and PCR amplification
What does in situ hybridisation detect
The specific location of mRNA accumulation in tissues
Why are RNA probes used instead of DNA
RNA probes (single-stranded) have higher sensitivity and specificity
What is a digoxigenin-labelled RNA probe used for
It hybridises to mRNA and is detected via enzyme-linked antibodies (e.g. alkaline phosphatase
Why might mRNA expression not reflect protein levels
Post-transcriptional regulation may alter protein accumulation
What is Western blotting used for
To detect presence, size, and quantity of specific proteins
What are the key steps of Western blotting
SDS-PAGE → transfer → blocking → antibody probing → detection
What is the role of the lac operator in bacterial expression systems
Controls transcription via IPTG-inducible promoter system
How is His-tagged protein purified
Using nickel-affinity chromatography; imidazole elutes the protein
Whats the difference between transient and stable transformation
Transient: non-integrated DNA, short-term Stable: integrated into genome, heritable
How does calcium phosphate transformation work
DNA co-precipitates with CaPO₄ and enters cells via endocytosis
What is electroporation
DNA enters cells via pores created by a short, intense electric pulse
What is microinjection used for
Direct DNA injection into embryos or cells (e.g. to create transgenic animals)
What is microprojectile bombardment
Gold/tungsten particles coated with DNA are fired into plant tissues
What are key features of adenoviral vectors
dsDNA, high expression, no genome integration, strong immune response, high capacity
How does Agrobacterium-mediated transformation work
T-DNA from Ti plasmid integrates into plant genome after infection of wounded tissue
What limits Agrobacterium’s use
Limited host range (mostly dicots, not monocots or gymnosperms)
What is RNAi
A cellular mechanism that uses siRNA to degrade complementary mRNA, silencing gene expression
What is needed for RNAi gene silencing
A construct producing dsRNA (usually via a hairpin structure), processed by Dicer and RISC
What promoters are used for constitutive expression
Viral promoters (e.g. CMV) or ubiquitously expressed native gene promoters
What are examples of genome editing nucleases
Zinc-Finger Nucleases, TALENs, CRISPR/Cas9
What is CRISPR/Cas9 used for
Targeted gene editing via RNA-guided Cas9 creating a double-stranded break in DNA
Why use RNAi for polygalacturonase suppression in tomatoes
To delay fruit softening and allow on-vine ripening, improving flavour
What makes crops glyphosate resistant
Expression of a bacterial version of the enzyme not inhibited by glyphosate
Why are some therapeutic proteins made in mammalian cells
They require post-translational modifications not possible in bacteria (e.g., Factor VIII, tPA)
What is gene therapy
Introduction of a functional gene into patient cells to treat genetic disease
