L11: Isolation of DNA Flashcards
How is DNA isolatied
Through cell lysis and DNA purification (add high salt buffer, wash with ethanol buffer and elute with low salt)
What is the purpose of cell lysis?
To release DNA from the cell by breaking down the cell membrane
What are the 3 types of cell lysis?
Biological, physical and mehchanical lysis
Explain biological lysis
Uses enzymes to disrupt cell membrane. Different enzymes used for different cells
- for plants: cellulose
- for bactria: lysozyme (mainly gram+ as targets are peptidoglycans)
- for euk. cell: sappanin
Explain the different mechanisms of physical lysis
- osmotic pressure: excess water moves into cell when cells placed in hypotonnic solution and cell bursts
- free thaw: repeated cycles of freezing and thawing ruptutres cell membranes through ice crystal formation
Explain mechanisms of mechanical lysis
- Grinding: pestle+motar usde to disrupt plant cells and hard tissue (e.g. mice or human organ tissue)
- Bead mill: used to beat and grind tough samples
- Vortex: used with beads or alone for small cell number
- Shearing:
- -Homogeniser: tissue suspensions are forced through narrow space
- -Rotor- stator: uses a combo of turbulence and mechanical shearing
- -Syringe-needle: purification of longer DNA fragments
Name 2 methods used for DNA purification
Phenol-Chloroform extraction and commercial kits
How is DNA purified by Phenol-Chloroform extractin
- lysed cells are mixed with equal volumes of a phenol:chloroform mixture
- centrifugation occurs, giving 2 distinct phases as the the phenol:chloroform mixture doesn’t mix with water
- 0.3M sodium acetate & 2.5 volumes ethanol can be usd to precipitate DNA from salt and sugar to concentrate it
Name advantages of using phenol chloroform extraction
- very cost effective
- fast
- big sample in one go
- DNA still has salts& impurities so purifies with alcohol
Name cons of using phenol chloroform extraction
- dangerous to us and environement
- chloroform is an easy anaesthetic
- phenol can cause v serious chemical burns
How can DNA be purified 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
- Low salt buffer, such as water or 10mM Tris, used to release DNA from membrane and collect it
Why may commercial kits be prefered to purify DNA?
- They aren’t as hazardos
- Less time consuming
- Results in purer DNA
What are the functions of restriction endonucleases (REs)?
- Used to make recombinant DNA molecules (cloning)
- Used to cut DNA into defined fragments (DNA fingerprinting & mutation analysis)
What enzyme is dubbed as ‘molecular scissors’?
Restriction endonucleases
How do REs cut DNA in precise locations?
By making one cut in each of the sugar-phosphate backbones of the double helix (breaks bond between 3’ O and P) at their recognition site in the presence of Mg2+
- hydrolyses phosphate group
- cuts ends have a 5’ phosphate
State the naming convention
Bacterial genus → species → strain → type
What are the 2 main types of REs and how do they work?
Blunt end: cuts in the middle of sequene
Sticky ends: cuts from one end to the opposite
The recongition sites for REs are often what?
Palindromes (read the same backwards and forwards)
What is the function of MCSs (multiple cloning sites)?
They indicate all restriction sites and have to be in the correct reading frame
What is ‘star activity’?
The relaxation/ alteration of the specificity (chemical or drug intervention from outside the host)
When does star activity occur?
When reaction conditions differ significantly from the optimum for the enzyme e.g. low ionic strength, high pH
Explain the use of gel electrophoresis
Gel agorase acts as a molecular sieve with DNA travelling downwards from large to small.
-DNA is -vely charged so migrates to positive electrode
How is DNA fragment size determined?
- The size of the product of interest is compared with MW DNA ladder.
- Can also graph log DNA size of the markers by distance migrated creating a straight line
