Biology nucleic Isolation

Question 1

It refers to a set of laboratory techniques used to extract, refine, and measure nucleic acids ( such as DNA and RNA) from biological samples.

Answer 1

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 ,

Question 2

Extracting DNA or RNA from cells
or tissues by breaking them open
and separating nucleic acids from
other cellular components.

Answer 2

Nucleic Acid Isolation

Question 3

Basic Steps in Isolating NA from Clinical Specimens

(Example is from blood)

Answer 3

1. Separate WBCs from RBCs, if necessary
2. Lyse WBCs or other nucleated cells
3. Denature/digest proteins
4. Separate contaminants (e.g., proteins) from DNA
5. Precipitate DNA if necessary
6. Resuspend DNA in final buffer

Question 4

Isolation Methods: Liquid Phase Organic Extraction

Answer 4

o Phenol chloroform/isoamyl alcohol
o Cell membranes are lysed and proteins are denatured by detergent
(such as SDS)

Question 5

Isolation Methods: Solid Phase Procedures

Answer 5

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.

Question 6

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.

Answer 6

Isolation Methods: Solid Phase Procedures

Question 7

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.

Answer 7

Isolation Methods: Solid Phase Procedures

Question 8

Refining extracted nucleic acids
to remove contaminants like
proteins and salts, ensuring
sample purity for downstream
applications.

Answer 8

Nucleic Acid Purification

Question 9

Purification Method:
Phenol- Chloroform Extraction

Answer 9

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.

Question 10

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:

Answer 10

1.) Lower Phase
2.) Upper phase

Question 11

contains lipids, denatured proteins, and other cellular components.

Answer 11

Lower Phase

Question 12

aqueous phase that contains only nucleic acid.

Answer 12

Upper phase

Question 13

Step-by-step Process of nucleic Purification

Answer 13

1. Disruption of the cellular structure to
   create a lysate
2. Separation of the soluble DNA from
   cell debris and other insoluble material,
3. Binding the DNA of interest to a
   purification matrix
4. Washing proteins and other
   contaminants away from the matrix and
5. Elution of the DNA.

Question 14

Determining the concentration of
nucleic acids in the purified sample using methods such as UV-visible spectrophotometry, or fluorometry.

Answer 14

Nucleic Acid Quantification

Question 15

One of the most commonly used practices to quantitate DNA or
RNA is the use of

Answer 15

spectrophotometric analysis

Question 16

• One of the most commonly used practices to quantitate DNA or
  RNA is the use of spectrophotometric analysis using a

Answer 16

spectrophotometer.

Question 17

A____________ is able to determine the average concentrations of the nucleic acids DNA or RNA present in a mixture,
as well as their purity.

Answer 17

spectrophotometer

Question 18

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.

Answer 18

Flourometric Analysis

Question 19

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.

Answer 19

EMISSON

Question 20

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.

Answer 20

DETECTION

Question 21

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.

Answer 21

EXCITATION

Question 22

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 _____.

Answer 22

Cell lysis and Centrifugation