Laboratory Isolation of Nucleic Acids Flashcards
What is the process of DNA isolation?
- Release cells from samples
- Disrupt cell integrity and prepare cell free extract.
- Remove debris from cell free extract
- Purify DNA from treated extract
- Measure quantity and quality of purified DNA
What are the steps of nucleic acid isolation dependent on?
The type of starting material
Nucleic acid isolation from nucleated cells?
Specimen have to be pretreated to make nucleated cells available e.g WBCs from blood
-differential density gradient centrifugation- FICOLL GRADIENT - where WBCs settle and separate from other plasma componnents and cells
-layers can be isolated and washed
-differential lysis - uses differences in osmotic fragility of cells
-incubation of whole blood in a hypotonic buffer - lyse RBCs before WBCs
-centrifuge to get pellet of WBCs
Pre-centrifugation VS Post-centrifugation in differential density gradient centrifugation - Layers
-Pre - blood sample (diluted), ficoll-plqque media
-Post - Plasma, mononuclear cells, ficoll-paque media, granulocytes, erythrocytes
Nucleic acid isolation from tissue samples
-Fresh or frozen tissues must be dissociated before DNA isolation by: grinding frozen tissue in liquid nitrogen OR homogenising the tissue OR mincing using scalpel
-Embedded tissue has to be deparaffinated
-tissue must be rehydrated
-From released cells must prepare cell fee extract - lyse cells
What can be used to deparaffinate fixed embeddedtissue?
-Using xylene
-Less toxic xylene alternatives
What does making cells into cell free extract depend upon?
-Cell type
-Whether cell is walled
-Composition of wall
How can cells be made into cell free extract?
-can occur naturally in gentle buffer
-enzymes
-grinding or vigorously mixing w beads
-treatment with detergents and bases (NaOH)
What does purification of DNA require?
Removal of contaminationg proteins, lipids, carbohydrates and cell debris
What can be used to dissolve hydrophobic contaminants like lipids and lipoproteins?
A combination of high salt, low pH and an organic mixture of phenol and chloroform can be used
What can be used to degrade RNA?
RNAse
What will the phenol and chloroform lead to the formation fo?
What will centrifugation do?
-Forms a biphasic emulsion
-Centrifugation will settle zhe hydrophobic layer at the bottom and the hydrophilic layer on top with a white interface containing amphipathic components.
What are the nexts steps of organic isolation after the separate layers are obtained from centrifugation?
-The upper phase containing DNA is collected
-DNA is precipitated using ethanol or isopropanol in a high conc of salt
-DNA percipitate is collected and rinsed to remove excess salt
-DNA is usually then dissolved in a buffer
Why are inorganic isolation methods used and what is the process of inorganic isolation?
-Used as safety issues with phenol lead - called salting out
-Uses low pH and high salt conditions to selectively precipitate proteins, leaving DNA in solution
-DNA is precipitated in alcohol and pelleted and resuspended in buffer or water
Why is solid phase isolation used and what is the process of this?
-DNA extraction using solid matrices can be used to rapidly and efficiently isolate DNA
-Commercial kits come with solid matrices in columns of various sizes
-Preparation of samples for isolation of DNA -cell lysis and release of nucleic acids
-Cell lysate in high salt buffer is applied to column and DNA absorbs onto solid matrix
-After washing the immobilised DNA is washed w buffer and DNA is eluted in specific vol of water or buffer
