week 2 Flashcards
(29 cards)
what is required to extract DNA
- it must effectively separate DNA from other cellular components
- necessary as other cellular components can :
- interfere with the DNA manipulation/analysis techniques to be used after DNA extraction
- cause the breakdown of DNA
- encourage the growth of microorganisms that can breakdown DNA
- the main cellular components that need to be removed are proteins, lipids (fats) and metal ions
reagents for DNA extraction
- phenol
- chloroform
- detergents
- proteinase
- guanidine thiocyanate
- EDTA
- pH buffers
- alcohols
- commercial resins
describe phenol
strongly denatures protein, dissolves lipids, does not mix with water or aqueous solutions, does not interact with DNA
describe chloroform
weakly denatures protein, dissolves lipids, does not mix with water or aqueous solutions, does not interact with DNA
describe detergents
strongly denatures protein, dissolves lipids, soluble in aqueous solutions, does not interact with DNA
describe proteinase
denatures protein, does not interact with DNA
describe guanidine thiocyanate
strongly denatures protein, chaotropic agent (cause DNA to bind reversibly to silica)
EDTA
binds to and effectively removes metal ions (which are used by DNAses)
describe pH buffers
stops the pH going too high or too low
describe alcohols
in combination with various salts will cause DNA to form a solid precipitate, which may be spun down
describe commercial resins
selectively bind to DNA or protein
describe DNA manipulation with enzymes
- most DNA manipulation is done with naturally occurring or genetically modified enzymes
- these enzymes
- cut DNA (nucleases)
- join DNA (ligases)
- copy DNA (polymerases)
- chemically modify DNA
describe exo and endonucleases
exonucleases - break off nucleotides from the end of the DNA or RNA molecules
endonucleases - cleave the DNA or RNA molecule internally
- some cleave indiscriminately
- some cleave at specific base sequences only (restriction endonucleases)
describe restriction endonucleases
- only cut DNA molecules at defined sequences
- in molecular biology, all are Type II
- they recognise a specific sequence then cut at a defined site within that sequence
- they are enzymes made by bacteria that serve as a defense against phage (viral) attack
- the bacterial DNA is protected from digestion by having its restriction endonuclease recognition sequences chemically modified by methylases
describe restriction endonucleases and methylases
in nature, type II REs form one half of a restriction/modification system
- this is composed of 2 enzymes
- the restriction enzyme recognises a specific DNA sequence and cuts within it
- the modification enzyme recognises the same sequence and methylates bases within it
- methylation prevents the sequence being cut by the restriction enzyme - essential to prevent the bacterium from self-destructing
target sequences of type II REs
- target sequences are usually palindromic
- the enzymes acts as homodimers, antiparallel pairs of identical subunits so the symmetry of the recognition site matches the symmetry of the enzyme: this ensures that both strands will be cut
different ways restriction enzymes can cut DNA
- blunt
- with an overhang at the 5’ end
- with an overhang at the 3’ end
describe overhangs
- overhangs may be 1, 2, 3, 4 or 5 bases long
- nearly all enzymes cut to leave a 5’ terminal phosphate
how often do REs cut?
- The number of times a RE would cut random DNA sequence is
determined by the target sequence length. - If an RE’s target sequence is 4 bp long, we would expect that
sequence to occur one in every:
4 x 4 x 4 x 4 bp (= 4^4 = 256bp). - If an RE’s target sequence is 6 bp long, we would expect that
sequence to occur one in every:
4 x 4 x 4 x 4 x 4 x 4 bp (= 4^6 = 4096bp)
how are DNA molecules joined
- enzymes called DNA ligases
- most REs leave ‘sticky ends’
- these are much easier to ligate than blunt ends
- don’t always have to use the same enzyme on both molecules: some have different recognition sequences but produce compatible sticky ends.
describe joining blunt ends
- sometimes it’s not convenient to join DNA molecules this way (maybe not suitable sites)
- in this case we can use blunt ends which are harder to join together (need to use special conditions and very concentrated DNA mixtures but can be done using T4 DNA ligase
-to cut blunt ends, use a blunt cutter or use an enzyme which digests the single-stranded overhang
mode of action of type II restriction enzymes
- they function as a homodimer, one subunit binding each strand of the DNA and cutting it. usually cut palindromic sequences
- the hydrolysis of the sugar-phosphate bonds provides the free energy required
- nearly all type II’s cut to leave a 5’ phosphate
- most restriction enzymes are highly specific for double-stranded DNA
- some enzymes show a preference for relaxed DNA rather than supercoiled
requirements for RE’s
- need Mg2+ to work
- usually pH 7-8 (to buffer the reaction mix)
- different enzymes have different requirements for NaCl (ionic strength) - could use low, medium or high salt buffers.
- usually work best around 37 degrees
- also some thermolabile
how are restriction endonucleases stored
- usually stored in a sterile buffered solution containing glycerol, at -20 degrees
- soon lose activity due to denaturation and oxidation if allowed to sit around at room temperature
