DNA AND RNA ISOLATION TECHNIQUES AND DETECTION Flashcards
(23 cards)
DNA isolation is first done by
Friedrich Miescher in 1869
DNA isolation is first done thru
Density Gradient Centrifugation
STEPS IN DNA ISOLATION:
Lysis if cell in which the nucleic acids will be extracted
CELL LYSIS
STEPS IN DNA ISOLATION:
Removing proteins, lipids and other contaminants from the nucleic acids
REMOVAL OF DEBRIS
STEPS IN DNA ISOLATION:
Transferring the nucleic acids to water, or a buffer solution that will preserve them without interfering with subsequent work
DNA PURIFICATION
Types of samples for DNA Isolation
Nucleated cells in suspensions (WBC)
Tissue samples
Microorganisms
DENSITY GRADIENT CENTRIFUGATION:
A highly branched sucrose polymer that does not penetrate biological membranes.
Ficoll
NUCLEATED CELLS IN SUSPENSIONS:
whole blood or bone marrow mixed with isotonic saline is overlaid with Ficoll
DENSITY GRADIENT CENTRIFUGATION
NUCLEATED CELLS IN SUSPENSIONS:
differences in the osmotic fragility of RBCs and WBCs wherein incubation of whole blood or bone marrow in hypotonic buffer or water that results in the lysis of the RBCs before the WBCs happen. The WBCs are then pelleted by centrifugation, leaving the empty RBC membranes (ghost) and hemoglobin, respectively, in suspension and solution.
DIFFERENTIAL LYSIS
TISSUE SAMPLES:
Less toxic xylene substitutes such as
Histosolve, Anatech Pro-Par, or ParaClear
Means of dissociation of tissue samples
Grinding (the frozen tissue sample in liquid nitrogen), Homogenizing or Mincing (using a scalpel)
TISSUE SAMPLES:
Fixed embedded tissue has to be deparaffinized by soaking in
xylene (dimethylbenzene)
TISSUE SAMPLES:
After xylene treatment, the tissue is usually rehydrated by soaking it in
decreasing concentrations of ethanol
MICROORGANISMS:
Means to break down cell walls
- Enzyme (Lyzozyme or Zymolyase)
- Grinding or by vigorous mixing with glass beads
- Gentler enzymatic method (preferred for methods involving larger chromosomal targets as opposed to plasmid DNA)
- Detergent (1% sodium dodecyl sulfate) and strong base (0.2 M NaOH) in the presence of Trisbase, EDTA, and Glucose
- Boiling in 8% sucrose, 8% Triton X-100 detergent, Tris buffer, and EDTA
SAMPLES FOR RNA ISOLATION:
lysed by osmosis or separated from WBCs by centrifugation
Reticulocytes in blood and bone marrow
SAMPLES FOR RNA ISOLATION:
kept frozen in liquid nitrogen or immersed in buffer that will inactivate intracellular RNAses (true for tissues such as pancreas that contain large amounts of innate RNAses)
Tissue samples
SAMPLES FOR RNA ISOLATION:
chemical lysis or by grinding in liquid nitrogen
Bacterial and fungal RNA
SAMPLES FOR RNA ISOLATION:
can be isolated directly from serum or other cell-free fluids by means of specially formulated spin columns or beads
Viral RNA
METHODS FOR DNA/RNA EXTRACTION:
DNA and RNA can be analyzed for quality by resolving an aliquot of the isolated sample on an agarose gel (DNA/RNA)
ELECTROPHORESIS
METHODS FOR DNA/RNA EXTRACTION:
nucleic acids absorb light at 260 nm through the adenine residues
SPECTROPHOTOMETRY
METHODS FOR DNA/RNA EXTRACTION:
measures fluorescence related to DNA concentration in association with DNA-specific fluorescent dyes
FLUOROMETRY
METHODS FOR DNA/RNA EXTRACTION:
a type of spectrophotometer with a smaller sample size and a novel technology which allow us to measure nano-liter volumes (pico concentration) of the nucleic acid (DNA or RNA) sample.
NANODROP
METHODS FOR DNA/RNA EXTRACTION:
has higher resolution and utilizes “probe sequence” for DNA or RNA, and when it finds its intended target, binds to it by hybridization process.
HYBRIDIZATION TECHNIQUES
