Biology nucleic Isolation Flashcards
It refers to a set of laboratory techniques used to extract, refine, and measure nucleic acids ( such as DNA and RNA) from biological samples.
N u c l e i c A c i d I s o l a t i o n , P u r i f i c a t i o n , a n d Q u a n t i f i c a t i o n
N u c l e i c A c i d I s o l a t i o n ,
Extracting DNA or RNA from cells
or tissues by breaking them open
and separating nucleic acids from
other cellular components.
Nucleic Acid Isolation
Basic Steps in Isolating NA from Clinical Specimens
(Example is from blood)
- Separate WBCs from RBCs, if necessary
- Lyse WBCs or other nucleated cells
- Denature/digest proteins
- Separate contaminants (e.g., proteins) from DNA
- Precipitate DNA if necessary
- Resuspend DNA in final buffer
Isolation Methods: Liquid Phase Organic Extraction
o Phenol chloroform/isoamyl alcohol
o Cell membranes are lysed and proteins are denatured by detergent
(such as SDS)
Isolation Methods: Solid Phase Procedures
o Uses solid support columns and magnetic beads.
o Solid support columns: Fibrous or silica matrices bind DNA allowing
separation from other contaminants.
o Magnetic beads: DNA binds to beads; beads are separated from other
contaminants with a magnet.
o Uses solid support columns and magnetic beads.
o Solid support columns: Fibrous or silica matrices bind DNA allowing
separation from other contaminants.
o Magnetic beads: DNA binds to beads; beads are separated from other
contaminants with a magnet.
Isolation Methods: Solid Phase Procedures
o Uses solid support columns and magnetic beads.
o Solid support columns: Fibrous or silica matrices bind DNA allowing
separation from other contaminants.
o Magnetic beads: DNA binds to beads; beads are separated from other
contaminants with a magnet.
Isolation Methods: Solid Phase Procedures
Refining extracted nucleic acids
to remove contaminants like
proteins and salts, ensuring
sample purity for downstream
applications.
Nucleic Acid Purification
Purification Method:
Phenol- Chloroform Extraction
Mixing: Your sample, likely containing DNA along with cellular debris
and proteins, is mixed with a solution of phenol and chloroform.
Separation: Phenol is a solvent that disrupts protein structures, while
chloroform helps separate the mixture into layers.
Purification Method:
Phenol- Chloroform Extraction
Mixing: Your sample, likely containing DNA along with cellular debris
and proteins, is mixed with a solution of phenol and chloroform.
Separation: Phenol is a solvent that disrupts protein structures, while
chloroform helps separate the mixture into layers. Upon vigorous
shaking and centrifugation,
two distinct phases appear:
1.) Lower Phase
2.) Upper phase
contains lipids, denatured proteins, and other cellular components.
Lower Phase
aqueous phase that contains only nucleic acid.
Upper phase
Step-by-step Process of nucleic Purification
- Disruption of the cellular structure to
create a lysate - Separation of the soluble DNA from
cell debris and other insoluble material, - Binding the DNA of interest to a
purification matrix - Washing proteins and other
contaminants away from the matrix and - Elution of the DNA.
Determining the concentration of
nucleic acids in the purified sample using methods such as UV-visible spectrophotometry, or fluorometry.
Nucleic Acid Quantification
One of the most commonly used practices to quantitate DNA or
RNA is the use of
spectrophotometric analysis
- One of the most commonly used practices to quantitate DNA or
RNA is the use of spectrophotometric analysis using a
spectrophotometer.
A____________ is able to determine the average concentrations of the nucleic acids DNA or RNA present in a mixture,
as well as their purity.
spectrophotometer
It depends on the production of a
fluorescent compound as a result of
enzyme activity between a
substrate and enzyme. The rate of
production of the fluorescent
compound is related to both the
enzyme concentration and
substrate concentration.
Flourometric Analysis
After a brief period, the electrons
in the fluorophore return to their
ground state, emitting light at a
longer wavelength than the excitation light. This emitted light is the fluorescence that is measured.
EMISSON
The fluorometer separates the emitted
light from the excitation light using a
monochromator or filter, allowing
only the fluorescence emission to
reach the detector. The intensity of the
emitted light is then measured.
DETECTION
The sample is exposed to light, usually
from a laser or a lamp, which excites
the fluorescent molecules, causing
them to absorb energy and move to a higher energy state.
EXCITATION
In a molecular biology laboratory, scientist may perform nucleic acid isolation, purification, and quantification to extract DNA from a blood sample. They would first isolate the DNA molecules from other cellular components using techniques like ____ and _____.
Cell lysis and Centrifugation
