L12 - isolating DNA Flashcards
Why would we want to isolate DNA?
Genetic manipulations & DNA analysis
What are the steps of DNA isolation?
Cell lysis, DNA purification from the cell extract, concentrate DNA, measurements of DNA purity and concentration
Why do we need cell lysis?
Release the DNA from the cell by breaking down the cell membrane
What are the three types of cell lysis
biological, physical and mechanical methods
What are things we don’t want in our sample of DNA?
Protein, ribosomes, mtDNA (mitochondrial) , lipid and plasmids
Cell lysis via biological methods - how can that be achieved?
Use enzymes to disrupt cell membranes. Different enzymes for different cells
What enzymes do we use for plant cells?
Cellulase
What enzymes do we use for bacteria
lysozyme
What enzymes do we use for animal cells?
Sappanin
What are the physical methods of cell lysis
Osmotic pressure, freeze-thaw,
How can we lyse cells using osmotic pressure
Excess water moves into the cell When cells are
placed in a hypotonic solution
How can we lyse cells using freeze-thaw
Repeated cycles of freezing and thawing ruptures cell membranes through ice crystal formation
What are the mechanical methods of cell lysis
- pestle and mortar 2. bead mill 3. vortex
How do we purify DNA?
DNA purification by Phenol-Chloroform
extraction, commercial kits
How do we perform DNA purification by phenol-chloroform extraction
Lysed cells or tissue are mixed
with equal volumes of a Phenol:
Chloroform mixture.
Centrifugation = two distinct
phases as the Phenol:Chloroform
mixture does not mix with water.
DNA concentration - 0.3M
Sodium Acetate and 2.5 volumes
Ethanol can be used to
precipitate DNA from salt and
sugar to concentrate it.
What concentration of sodium acetate is used in DNA purification by phenol-chloroform extraction
0.3 M
How do we perform DNA purification using commercial kits
Column contains a silica membrane that binds DNA in the presence of a high concentration of salt.
Impurities such as salts are washed away and then a low salt buffer such as water or 10 mM Tris·Cl, pH 8.5 is used to release the DNA from the membrane and collect it.
Using these kits is not as hazardous, is less time-consuming and results in purer DNA than phenol–chloroform extraction.
How do we measure quantity and quality of DNA
- UV absorbance
- fluorescence dyes
- agarose gel electrophoresis
- capillary electrophoresis
- diphenylamine method
Why is isolating DNA important?
Efficient extraction = efficient science
Without a good starting point you will never have good output!
Genomic testing would be impossible
PCR/cloning wouldn’t work
Forensic science would be unreliable
What are restriction endonucleases and what
do they do?
Enzymes produced by bacteria to protect
against viral DNA infection, cut the foreign DNA
Why are they used in the laboratory?
i. To make recombinant DNA molecules
(cloning)
ii. To cut DNA into defined fragments (DNA
fingerprinting and mutation analysis)
How many different restriction endonucleases are there?
over 300
How do restriction enzymes cut DNA?
Make one cut in each of the sugar phosphate backbones of thedouble helix (breaks bond between 3’O and P) at their recognition site in the presence of Mg2+
Hydrolyses the phosphate group
Cut ends have a 5’ phosphate
Different types of ends achieved from different restriction endonucleases
blunt and sticky
Recognition sites for restriction enzymes are often…..
Palindromes
Give examples of a palindrome recognised by restriction enzymes
5’ GAATTC 3’
3’ CTTAAG 5’
True or false: Restriction endonucleases can be used to make recombinant DNA molecules
True
What is star activity?
Relaxation or alteration of the specificity – chemical/drug intervention (from outside the host)
What causes star activity?
When reaction conditions differ significantly from the optimum for the enzyme
Why do we have star activity?
low ionic strength, high pH, high (> 5% v/v) glycerol
concentrations, can happen because of the presence of Mg2+ (HindIII)
What does star activity result in?
Enzyme cuts where it shouldn’t
What does electrophoresis do?
separates DNA fragments
Is DNA positively or negatively charged?
Negatively charged
What properties of agarose allows for the movement of DNA?
Polymerised agarose is porous,
allowing for the movement of
DNA (acts as a “molecular sieve”)
Where does agarose come from?
What does the % of an agarose gel mean?
What impacts the migration of samples in electrophoresis?
charge, size and shape
How are results visualised?
Intercalating dyes
How do we determine the size of the DNA fragments
Graph of log DNA
