Characterisation of Cloned Genes Flashcards
What the two main reasons for cloning DNA
- To obtain DNA in sufficient quantity
- To keep DNA in a usable form, such as in a plasmid vector
What is the first step in plasmid preparation
Grow bacterial culture containing the plasmid and lyse the cells using SDS + NaOH
What happens to chromosomal vs plasmid DNA during lysis
Chromosomal DNA becomes single-stranded and aggregates; plasmid DNA remains in supercoiled form
How is plasmid DNA separated
After pH neutralisation and centrifugation, plasmid DNA is in the supernatant
What makes ddNTPs different from regular dNTPs
They lack a 3’ OH group, so they terminate DNA chain elongation
How is sequencing done in modern methods
With fluorescently labelled ddNTPs in a single reaction tube, separated by capillary electrophoresis
What is the sequence read limit of Sanger sequencing
About 700 nucleotides, resolution decreases with longer sequences
How can comparing genomic and cDNA sequences help
It identifies exons, introns, UTRs, and promoter regions
Why cant gel electrophoresis estimate gene copy number
It shows either a single band or a smear, which isn’t informative
What steps are involved in a Southern blot
- Cut DNA with restriction enzyme
- Gel electrophoresis
- Denature and transfer DNA to nitrocellulose
- Fix DNA with heat or UV
- Hybridise with a labelled probe
- Detect with autoradiography
What indicates multiple gene copies
Multiple bands on X-ray film after hybridisation
How is a cloned DNA fragment isolated from a plasmid
Digest with restriction enzymes
Separate fragments via agarose gel electrophoresis
Excise the desired band
Purify the DNA
What is used as a template for probe synthesis
Purified DNA similar to or identical to the gene of interest
How is DNA probe labelled
Random hexamer primers
Klenow fragment of DNA polymerase I
Incorporation of a labelled nucleotide
What does Northern blotting measure
mRNA levels and transcript sizes in different tissues
What’s different from a Southern blot
Uses RNA, not DNA, and doesn’t require fragmentation
Steps in Northern blotting
- Isolate RNA
- Gel electrophoresis
- Transfer to filter
- Hybridise with probe
- Detect by autoradiography
What is RT-PCR used for
Detecting gene expression by amplifying cDNA from mRNA
What are the key steps
RNA isolation
Reverse transcription
PCR
Gel or real-time analysis
What is in situ hybridisation used for
Determines which cells/tissues express the gene
What type of probe is used in in situ hybridisation
A single-stranded, antisense RNA probe, usually labelled
How is detection performed
With an antibody-enzyme conjugate that produces a colour precipitate (e.g. purple)
What vector orientation is needed to make an antisense RNA probe
Inserted in reverse relative to the promoter in the plasmid
What labelling system is commonly used
DIG (dioxigenin) with detection by alkaline phosphatase-conjugated antibody
