CMB2000/L12 Isolation of DNA & Genes Flashcards
Give 2 reasons why DNA is isolated.
For genetic manipulations
For DNA analysis
Give the 4 steps of DNA isolation.
Cell lysis
DNA purification from cell extract
Concentrate DNA
Measurement of DNA purity and concentration
Describe how cell lysis/extraction of DNA can be achieved. (3)
Biological methods
Physical methods
Mechanical methods
Give 3 detergents used for biological extraction of DNA.
Cellulase (plants)
Lysozyme (bacteria)
Sappanin (eukaryotic cells)
What does lysostaphin target?
Cross-links of peptidoglycan layer of cell wall in Staphylococcus
Describe a physical method of cell lysis.
Osmotic pressure
Excess water moving into cell when cells placed in hypotonic solution
Freeze-thaw
Repeated cycles of freezing and thawing ruptures cell membranes through ice crystal formation
Give 2 mechanical methods of cell lysis and their apparatus.
Grinding - pestle & mortar, bead mill, vortex
Shearing - homogeniser, rotor-stator, syringe-needle
Describe DNA purification by phenol-chloroform extraction. (3)
Lysed cells or tissue mixed with equal vols phenol:chloroform mixture
Centrifugion forms 2 phases
0.3M Sodium acetate and 2.5 vols ethanol precipitates DNA from salt and sugar
Describe DNA purification using commercial kits. (4)
Lyse cells
Add high salt buffer
Wash with ethanol buffer
Elute with very low salt
Explain how commercial kits purify DNA.
Silica membrane of column binds DNA in high [salt]
Impurities washed away and low salt buffer (water or 10mM Tris.Cl pH 8.5) used to release DNA from membrane and collect
Compare phenol-chloroform extraction and commercial DNA purification kits.
Kits are not as hazardous
Less time consuming
Results in purer DNA
Give 2 ways to measure the quantity and quality of DNA.
UV absorbance
Fluorescence dyes
Agarose gel electrophoresis
Capillary electrophoresis
Diphenylamine method
What is the absorbance of pure DNA?
> 1.8 at 260/280
What are restriction endonucleases and what do they do?
Molecular scissors that cut DNA in precise locations
Why are restriction endonucleases used in the laboratory?
To make recombinant DNA molecules (cloning)
To cut DNA into defined fragments (DNA fingerprinting and mutation analysis)
How do restriction enzymes cut DNA?
Make one cut in each of the sugar phosphate backbones of the double helix (between 3’O and P) at recognition site
In presence of Mg2+
Hydrolyses phosphate group
Cut ends have 5’ phosphate
How are restriction enzymes named?
Bacterial genus, species, strain and type
Define star activity.
Relaxation or alteration of the specificity
Chemical/drug intervention from outside the host
Why can star activity occur?
Low ionic strength
High pH
High (>5% v/v) glycerol concentrations
Presence of Mg2+
How is Southern blotting different to Western blotting?
Southern blotting uses DNA not proteins
Uses hybridisation probes not Abs
Describe Armstrong’s experiments in chickens.
Looked for AvBD gene expression levels in GIT using qPCR
Analysed environmental effects
Extracted genomic DNA from 10 tissues in chickens and pooled blood samples
Looked for SNPs and AvBDs
Cloned AvBDs into plasmid
Hyper-expressed in E. coli and tested antimicrobial potency
What is the aim of electrophoresis?
Separate DNA fragments
How does polymerised agarose gel act as a molecular sieve?
It is porous and allows for movement of DNA
Which dye is used to visualise DNA in agarose gel electrophoresis and why?
Nancy Red
It is safe
