V L7: DNA isolation and quantification Flashcards
what are the Principle stages of DNA extraction from cells?
- Cell lysis to disrupt cell and nuclear membranes including denaturing and degrading proteins which are abundant in cells and bound to DNA in chromosomes
- Separating DNA from other cell components including cell debris, proteins, lipids, CHO and RNA
- Isolation of the DNA into a aqueous phase.
- Storing DNA for future analyses.
what does cell lysis involve?
heating the sample
why do we use ethanol precipitation?
because if the DNA is in water it forms a pallet of DNA
a detergent does what?
denatures
in what ways can we separate and isolate DNA form other cell components?
– Organic or phenol-chloroform method
– Ethanol precipitation
– Silica based extraction (affinity binding) and magnetic separation
– Filtration and centrifugation
what does SDS stand for?
sodium dodecyl sulphate
what is SDS?
anionic detergent that
denatures proteins.
what is proteinase K?
protease (enzyme) that degrades proteins – cleaves peptide bonds.
what does EDTA stand for?
Ethylenediamine tetra acetic acid
what does EDTA do?
chelating agent, DNase inhibition.
what does DTT stand for?
dithiotreitol
what is DTT?
reducing agent for disulphide bonds.
what does DNase do?
chew up DNA
for DNA storage what temperature is needed?
-20 degrees and lower
what tube material is best to store DNA in?
PTFE
To store DNA we need maintain ____ ____.
pH levels
whats an FTA card?
a card containing different chemicals so they can be stored at room temperature
what the organic method for storing DNA?
phenol-chloroform method
phenol-chloroform method is not used ______.
frequently
phenol-chloroform method is used more often on a ___ sample.
dirty
phenol-chloroform method involves how many steps ?
2
phenol-chloroform method is like a ____ _____ extraction.
liquid - liquid
phenol-chloroform method requires the constant removal of a _____ layer.
aquaous
phenol-chloroform method involves what 3 steps?
lysis, solvent extraction and DNA seperation
the Chelex method involves how many tubes?
1
How does the Chelex method work?
- add chelex resin which inhibits DNAase
- add proteinase K +boil to denture and degrade
- centrifuge, pellet at bottom to throw away.
what exactly does DNase break?
phosphodiester bonds found in DNA
How do chelating agents inactive DNase?
Chelating agents bind with metal ions that are cofactors for DNase, inactivating them.
DNA binding method
- cell lysis + denature proteins, detergent, proteinase K and chaotropic salt.
- DNA binds to magnetic beads everything else passess through
- beads collected by magnetic force
what are FTA cards sued for?
to store blood and saliva samples.
what do FTA cards contain?
- Contains chemicals that causes cell lysis
- Protects DNA from nuclease degradation
- Prohibits microbial growth
vaginal swabs, how to pull out male DNA?
differential lysis
How is differential lysis carried out?
SDS EDTA proteinase K, incubate, centrifuge, remove supernatant, spermatozoa are still in tact, add SDS EDTA proteinase K + DTT, centrifuge, disulphide bonds broken, opens membranes of spermatozoa, remove supernatant (male DNA).
whats an advantage of bones and hard tissue?
- DNA is protected from degradation
- bone surface can be cleaned with abrasives and bleach or cross-linked with UV
what are problems with bones?
- need to be grinded up, hard to obtain homogenous sample
- high PCR inhibitor, high calcium (EDTA can remove it)
what can you use to quantify DNA?
Ultraviolet spectroscopy
fluorescent dye
Ultraviolet spectroscopy gives you the concentration of what?
DNA
protein
CHO
whats an issue with Ultraviolet spectroscopy ?
it can’t differentiate between different types of DNA.
Fluorescent dye
_____ to DNA.
binds
what two dyes are mainly used?
– Sybergreen
– Ethidium Bromide
whats more sensitive Interchelating Fluorescent Dyes or Ultraviolet spectroscopy?
Interchelating Fluorescent Dyes
Fluorescence is recorded using what?
spectrofluorometer, or gels
Interchelating Fluorescent Dyes requires a…
calibration graph
what is the hybridisation method?
- Sample is denatured and a small spot of sample is immobilised onto a nylon membrane
- Addition of a 40-nucleotide labelled probe which hybridises to a primate-specific highly repetitive α- satellite D17Z1 locus in the genome – located on chromosome 17
whats the best method for DNA quantification?
real-time PCR
