DNA and RNA extraction Flashcards
starting point in any molecular diagnostic kit
nucleic acid extraction
these methods rely on biochemical properties of the cellular components to elicit the desired molecular separation and might exhibit preference or exclusivity in extracting DNA or RNA depending on its intrinsic characteristics
NAE chemical method
enumerate the chemical techniques
- osmotic shock
- enzymatic digestion
- detergents
- alkali treatment
osmotic rupture of membrane
osmotic shock
principle of enzymatic digestion
digestion of cell wall
principle of detergent
solubilization of membranes
principle of alkali treatment
solubilization of membrane
mode of lysis (chemical)
- osmotic shock - gentle
- enzymatic digestion - gentle
- detergents - gentle
- alkali treatment - harsh
enzymatic digestion cost
cheap at small scale
expensive at large scale
most usual application of osmotic shock
spheroplasts and protoplasts
most usual application of enzymatic digestion
gram-positive and gram-negative bacteria
most usual application of detergents
general use
most usual application of alkali treatment
plasmid DNA
Cesium Chloride Gradient Centrifugation with Ethidium Bromide is baded on the phenomenon ____
used to ____
phenomenon of buoyant and specific density
used to extract DNA from bacteria
separation of RNA from DNA and proteins after extraction with an acidic solution
Guanidinium Thiocyanate-Phenol-Chloroform Extraction
Guanidinium Thiocyanate-Phenol-Chloroform Extraction consist mainly of?
- GuSCN
- sodium acetate
- phenol
- chloroform
followed by centrifugation
in Guanidinium Thiocyanate-Phenol-Chloroform Extraction, total RNA remains in the ____ while most of DNA and proteins part remain either in the ____
total RNA = upper aqueous phase
DNA and proteins = interphase or in the lower organic phase under acidic condition
Guanidinium Thiocyanate-Phenol-Chloroform Extraction: total RNA Is then recovered through?
precipitation by isopropanol
Chelex extraction is used in the field of?
forensics for DNA extraction from various sources such as:
- hair
- blood stain cards
- buccal swabs
inhibiting DNA degradation by chelating metal ions which cause DNA breakdown at high temperature and lower ionic conditions
Chelex extraction
alkaline extraction is dedicated to?
plasmid DNA isolation
principle of alkaline extraction
selective alkaline denaturation of high molecular weight chromosomal DNA, while covalently bond circular plasmid DNA remains intact. After neutralization, chromosomal DNA renatures and makes an insoluble precipitate, while plasmid DNA remains in the supernatant
involves harvesting the bacteria of interest from culture media and exposing them to alkaline solution (consisting basically of SDS and NaOH)
alkaline extraction
poly(A) RNA hybridizes with an oligo(dT)-cellulose matrix, under high-salt conditions
purification of Poly(A) + RNA by Olido(dT)-Cellulose chromatography
based on the liquid and stationary phases, which selectively separate the target analyte from the solution based on specific hydrophobic, polar, and/or ionic properties of both solute and sorbent
Solid-Phase nucleic acid Extraction
- normal/regular SPE
- reverse SPE
- ion exchange SPE
solid phases use (6)
- silica matrices
- glass particles
- diatomaceous earth
- magnetic beads-based nucleic acid purification
- anion exchange material
- cellulose matrix
enumerate the mechanical techniques
- homogenization (blade or pestle)
- ultrasonication or cavitation
- pressure cell “french press”
- ball mill
method of choice for large scale
homogenization
most usual application of homogenization
animal tissues
most usual application of ultrasonication or cavitation
good for spheroplasts but not primary cells
most usual application of pressure cell
gram negative and some gram positive bacteria
most usual application of ball mill
bacteria, yeast, microalgae, unicellular animal cells
principle of homogenization
shredding of cells
principle of ultrasonication or cavitation
disruption of cells by pressure
principle of pressure cell
disruption of cells by shear force
principle of ball mill
cells crushed between glass/steel balls/beads
most common technique to determine DNA yield and purity?
measurement of absorbance
needed for absorbance method
spectrophotometer with UV lamp, UV-transparent cuvettes, and a solution of purified DNA
absorbance to evaluate DNA purity
260 - 280 nm (based ni maam abi)
260 - most common
good quality DNA will have the ratio of?
1.7 - 2.0
ratio that indicates more contaminants present
<1.6
ratio indicates too much protein and is not acceptable
3
ratio indicates contaminants and still acceptable
2-2.5
strong absorbances around 230 nm can indicate that?
organic compounds or chaotropic salts are present
the __ the ratio, the ___ the amount of thiocyanate salt is present
the LOWER the ratio, the GREATER the amount of thiocyanate salt is present
absorbance best for greater than 1.5
260/320
a reading at 320nm indicate?
turbidity in the solution and possible contamination
DNA concentration absotbance
260 nm
do not use ___ if no correction
A320
