L13 - preparation of DNA Flashcards
(15 cards)
what are the 5 general ‘rules’/concerns when purifying DNA
Lyse the cells
remove protein contamination
precipitate to concentrate nucleic acids
remove salt concentration
remove RNA concentration
describe step 1 of general rules when purifying DNA - lysing the cell
break open cells to release the DNA
Cell membranes (and nuclear membranes if eukaryotic) are disrupted
= Detergents like SDS or enyzmes like proteinase K
= You can’t purify DNA unless it’s out of the cell!
describe step 2 of general rules when purifying DNA - removing protein contamination
Get rid of protein contaminants such as enzymes like DNases that can degrade DNA.
Proteins are broken down or separated from DNA.
Use proteinase K (digests proteins), salt (helps precipitate them), or organic solvents like phenol-chloroform (phase separation/centrifugation)
= Protein contamination can interfere with downstream reactions like PCR or sequencing.
describe step 3 of DNA purification - precipitating to concentrate the nculeic acids
Make the DNA easier to isolate and purify by concentrating
Add cold ethanol or isopropanol + a salt (like sodium acetate or NaCl).
you need to do this as you can’t spin down and collect DNA unless it’s precipitated into a visible pellet.
= DNA becomes insoluble and clumps together (precipitates).
describe step 4 of DNA purification - removing salt concentration
Clean up the DNA pellet so it’s free of salts and contaminants.
Residual salts and alcohols are removed from the DNA pellet.
Wash the pellet with 70% ethanol, then air- or vacuum-dry.
= Leftover salt or alcohol can inhibit PCR, ligation, or other enzymatic reactions.
describe step 5 of puyrifying DNA - remving RNA contamination
make up the solution in water and add RNAse
= cuts RNA leavining only purified DNA
what is SDS and which step might it be used in for DNA purification
a strong detergent/denaturant
= used in steps 1 and 2 –> cell lysis and removal of protein contamination
disrupts cellular membranes helping lysis and unfolds proteins making them more soluable and easy to precipitate out with CTAB
what is CTAB and how does it help in DNA purification
precipitates proteins by forming insoluble complex with them in prescence of high salt conc
= proteions can then be removed by centrifugation
give an example method for DNA purification:
- Make cells up in water
- add SDS and proteinase K
- precipitate proteins with CTAB and NaCl
- clean DNA with chloroform
- spin down precipitate and remove the supernatatnt with DNA
- precipiate DNA with isopropanol
- remove DNA and rinse in ethanol
- makle upin water and treat with RNase
what is a commonly used and fast way to seperate and purify DNA in an automatted wy
magnetic bead separation
= coated in silica with a high affinity for DNA and washed in multiple buffers
what is what is solid phase phosphoramidite chemistrty
method used to synthesise short RNA and DNA sequences from scratch
= simpluy type/copy and paste your sequnece
uses a solid surface and adds nucleotides 1 at a time to the 5’ OH sugar of the previous base
describe the differences of synthetic and natural DNA synthesis
Natural:
Growing end: 3′-OH (sugar)
Incoming nucleotide: dNTP with 5′-triphosphate
Link formed: 3′ → 5′ phosphodiester
Direction: 5′ → 3′
Synthetic:
growing end: 5′-OH (sugar)
Incoming nucleotide: Phosphoramidite with 3′-P group
Link formed: 3′ → 5′ phosphodiester
Direction: 3′ → 5′
= so we are still forming a 5’-3’ phosphodiester bond between sugar and phosphate
= difference is the 5’ is now the OH and the 3’ is the phosphate
why in syhthetic chemistry do we synthesise in the 3’-5’ way unlike normal DNA synthesis
phosphoramidites (the reactive 3′ group on the incoming nucleotide) are very stable until activated
= ideal for precise, stepwise synthesis.
5′-triphosphates (like in natural synthesis) are much less stable and more reactive = harder to handle in an organic chemistry setting
DMT can be used to proterct the 5’ OH of the growing end = allows it to be protected and unprotected for controlled addition of each base
once weve created our oglionucletides by phosphoramidite chemistry how do we create synthetic full genes
Assembly PCR
= assembles multiple short overlapping oligos into one long double-stranded DNA
each oglio overlaps the next by 20 bases = overlaps allow annealing of fragments in correct order and then polymerase and added dNTPs fill in gaps
give the basic steps of creating a synthetic GENE from scratch
- create the oglionucletide fragments by phosphoarmaidite chemistry
- Assembly PCR - No primers
= create the full gene by overlapping oglios
- Amplification PCR - with Fwd and Rvs primers that match the full length of gene
= amplify the desired gene
- purify and put in plasmid for cloning
