TABLE OF CONTENTs:

DESIRED LEARNING OUTCOMES:

At the end of the session, the students must have:
1.Discussed DNA isolation techniques, processes of
conducting PCR, DNA Sequencing
2.Described the CRISPR technology
3.Discussed developments in the conduct of cell culture
4.Explained the types of cell culture: primary and
secondary cell cultures
5.Explained the processes involved in cell culture
6.Relate the contributions of cell culture in the quality of
human life
7.Present ethical concerns related to cell culture
ISOLATION OF
INTRODUCTION
DNA ISOLATION: PURPOSE
SOURCES
EXTRACTION
OF
PROCEDURES:
1. PREPARATION OF A CELL EXTRACT
1. PREPARATION OF A CELL EXTRACT
1. PREPARATION OF A CELL EXTRACT
1. PREPARATION OF A CELL EXTRACT
1. PREPARATION OF A CELL EXTRACT
2. PURIFICATION OF DNA FROM CELL EXTRACT
PROCEDURE 1: ETHANOL PRECIPITATION
Concentration of
70 % ethanol is
added to your
sample.

It uses ice-cold
ethanol or
isopropanol.
PROCEDURE 2: PHENOL-CHLOROFORM
Phenol denatures proteins
in the sample.
Denatured proteins stays in
the organic phase.
Aqueous phase containing
nucleic acid is mixed with
chloroform that removes
phenol residues from the
solution.
PROCEDURE 3: MINICOLUMN PURIFICATION
Relies on the fact that
the nucleic acids may
bind (adsorption) to the
solid phase (silica or
other) depending on the
pH and the salt
concentration of the
buffer.
3. CONCENTRATION OF DNA SAMPLES
4. MEASUREMENT OF PURITY OF DNA CONCENTRATION

DNA concentrations can be accurately measured by UV

absorbance spectrometry.
Absorbance is measured by at 260 nm. An absorbance
of 1.0 corresponds to 50 µg of double stranded DNA per
ml.
The following table indicates a general acceptable
range of these ratios and does not assure sample purity.

Sample Type Ideal High Low

DNA ~1.8 >2.0 <1.7
RNA ~2.0 >2.2 <1.9
Possible – Basic Acidic Phenol
contamination or Protein

With a pure sample DNA the ratio of the absorbancies at

260 nm and 280 nm (𝐴260 /𝐴280 ) is 1.8.
POLYMERASE CHAIN REACTION (PCR)
POLYMERASE CHAIN REACTION (PCR)
A powerful and versatile method for
amplifying DNA or to make copies of
DNA.
Invented in the 1983, conceived by
Kary Mullis of Cetus Corporation
The technique depends on the use of
a special DNA polymerase.
PCR INGREDIENTS
 PCR INGREDIENTS

Strand/s of the The building “Tell” the For the DNA Necessary to
target DNA. enzyme. polymerase polymerase create
which part of to build with. optimal
Uses Taq the genome conditions for
polymerase. to copy. activity of Taq
DNA
polymerase
PCR SEQUENCE
 STEP 1: DENATURATION

Addition of heat in order to separate the

strand of a DNA.
 STEP 2: ANNEALING

Two separated strands that are separated by

the heat are going to be cooled and be
joined by primers.
STEP 3: DNA SYNTHESIS

Make more copies of DNA

 DNA SEQUENCING

DNA sequencing refers to the general laboratory

technique for determining the exact sequence of
nucleotides, or bases, in a DNA molecule.
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4 5

1. Take a sample.
2. Use PCR to amplify sample to generate DNA fragments.
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4 5

3. The Taq polymerase adds dNTPs (deoxyribonucleotide triphosphate) to the end of

the primer that are complementary to the template molecule, synthesizing a new
complementary strand of DNA. Every now and then, the polymerase inserts a ddNTP
(dideoxyribonucleotides triphosphates) instead of a dNTP.
4. Dideoxynucleotides lack a hydroxyl group at both their 2 and 3
positions. When one of these nucleotides has been incorporated
onto the end of a growing chain, the lack of the 3 OH makes it
impossible for the polymerase to add another nucleotide, thus
causing chain termination.
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4 5

5. The cycle of hybridization, synthesis, and denaturation is repeated many times, generating
a large population of daughter DNA strands that by now includes molecules in which a
ddNTP has been incorporated at every position. For every A on the template strand, for
example, there will be daughter molecules that terminate in a ddNTP at that position.
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4 5

6. When all cycles are complete, the reaction products are separated by electrophoresis on
very thin capillary gels. High‐resolution gel electrophoresis can separate fragments that differ
by only one nucleotide in length. Each ddNTP was labeled with a unique fluorescent dye, the
color of the band. For example: G, C, T, A
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4 5

7. Reveals the identity of the terminal nucleotide on each daughter

molecule. The order of the colors in the gel therefore corresponds to the base
sequence of the template molecule.
SANGER SEQUENCING METHOD
Fred Sanger won his first Nobel Prize for
Chemistry in 1958 for his work on the
structure of protein.

Sanger sequencing is also known as

chain termination sequencing.

Based on dideoxynucleotides.

If 3’ OH group is absent, there won’t

be addition of new nucleotides in the
polynucleotide chain! The reaction will
stop
 SANGER SEQUENCING METHOD
CLASSICAL SANGER MODERN SANGER
SEQUENCING METHOD SEQUENCING METHOD
 CRISPR-Cas9 System

CRISPR means Clustered regularly Interspaced

Short Palindromic Repeats (CRISPR).
 CRISPR-Cas9 System

Represents genome editing technology that has revolutionized

molecular biology due to ties precise and site-specific gene
editing capability.
 CRISPR-Cas9 System

CRISPR system is a part of prokaryotic adaptive

immunity against viral DNA, bacteriophage and
plasmids.
Composed of two
components:
Cas9 protein that
can cut DNA and
Guide RNA that
can recognize the
sequence of DNA
to be edited.
To use CRISPR Cas9:
▪ Scientist identify the sequence of
the human genome that’s
causing a health problem.
▪ Create a specific guide RNA the
nucleotides in the DNA.
▪ The guide RNA is attached to the
DNA cutting enzyme cas9 and
introduce to the target cells.
▪ Locate the target sequence and
cuts the DNA.
▪ Scientist can now edit the existing
genome by either modifying,
deleting or inserting new
sequences.
▪ This makes the CRISPR Cas9 a cut
and paste tool for DNA editing.
THANK YOU FOR LISTENING!