4 - Pharmaceutical Biotechnology (PD523-CCS518) - Lecture Four.

Pharmaceutical
Biotechnology
(PD523, CCS518)
Lecture Four
Dr. Asmaa Ramadan
Lecturer, Microbiology & Biotechnology Department – College of Pharmacy
Arab Academy for Science, Technology and Maritime Transport (AASTMT)
Different Molecular
Techniques
• Nucleic acid Extraction
• Conventional PCR
• Gel Electrophoresis
Nucleic Acid Isolation
PCR
Conventional PCR
Real-Time (Quantitative) PCR
Reverse transcriptase PCR
Nested PCR
Multiplex PCR
Etc…..
1- Nucleic Acid Isolation
Nucleic Acid Isolation
- DNA is found within the cell nucleus while RNA is found either in the
nucleus or cytoplasm.
- To study DNA, we first have to get it out of the cell.
- AIM: The DNA extraction process frees DNA from the cell and then
separates it from cellular fluid and proteins, leaving behind pure DNA.
- Basic Steps of DNA Extraction:
1.Cell Lysis 2.Precipitation 3.Purification

1. Cell Lysis
• In this step, the cell and the nucleus are “lysed” to release the DNA inside.
• Cell lysis results in the release of all organic components within the cell into solution,
such as nucleic acids, proteins, carbohydrates & lipids (proteins are most abundant).
There are two ways to do this:
a. Mechanical disruption: breaks open the cells. This can be done with a tissue
homogenizer or with Sonication. Mechanical disruption is particularly important when
using plant cells because they have a tough cell wall.
b. Lysis using Detergents (such as Sodium Dodecyl Sulphate - SDS) & Enzymes
(such as Proteinase K) to free the DNA and dissolve cellular proteins.
• Phospholipids from the cell membrane and the nucleus are disrupted
with detergents and surfactants.
• Breaking down proteins by adding a protease
• In addition, RNase enzyme may be added to break down RNA.
2. Precipitation
Now DNA is mixed with mashed up cell parts and debris. Precipitation aims to
separate the DNA from this cellular debris.
1) First, Na+ ions (e.g. sodium acetate) may be added to neutralize the negative
charges on the DNA molecules, which makes them more stable and less water
soluble.
2) Next, alcohol (such as 70% ethanol or isopropanol) is added, which facilitates

Na+ to interact with the PO3- (in DNA). Alcohol also removes the hydration shell of
H2O molecules around the phosphate. The total effect is that the DNA molecules
can come closer together due to neutralization of charge and removal of water that
eventually leads to the precipitation from the solution.
3) Upon centrifugation, DNA will aggregate together forming a pellet.

3. Purification
• Now that DNA has been separated from the aqueous
phase, it can be rinsed with alcohol to purify it &
remove any remaining unwanted material and
cellular debris.
• At this point the purified DNA in pellet is then

re-dissolved in water for easy handling and storage.
N.B: Mini-column purification is another method that

relies on the fact that the nucleic acids may bind
(adsorption) to the solid phase (e.g. silica)
Organic Phenol–Chloroform extraction method
1. Cell Lysis: sodium dodecylsulfate (SDS) + proteinase K (for the enzymatic
digestion of proteins and non-nucleic acid cellular components)
2. A mixture of phenol: chloroform: isoamyl alcohol (25:24:1) is then added to

promote the partitioning of lipids and cellular debris into the organic phase,
leaving isolated DNA in the aqueous phase. Chloroform mixed with phenol is
more efficient at denaturing proteins than either reagent is alone.
3. When the mixture is vortexed and centrifuged,
the denser organic layer containing lipids settles
at the bottom of the tube while aqueous phase
containing nucleic acids remains at the top.
Proteins may be found at the interface of the
aqueous and organic phase.
4. The aqueous phase containing the purified DNA

can be transferred to a clean tube for analysis.
2- Polymerase Chain Reaction (PCR)
PCR: Where does the name come from?
Polymerase: DNA polymerase enzyme

DNA polymerase duplicates DNA
Chain Reaction: The product of a reaction is used to amplify

the next reaction
Results in rapid increase in the amount of product
Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is a molecular biology technique
used in the lab to amplify (make millions of copies) a particular section of
DNA. It is a revolutionary method developed by Kary Mullis in the 1980s.
Kary Mullis
1. The developer of PCR Technique – 1987
2. Published paper - 1990.
3. Nobel Prize- 1993.
Polymerase Chain Reaction Technique
- It is a fast technique for amplifying a specific segment of DNA into many
fragments with identical size and sequence by enzymatic method and special
cycling conditions.
- This requires the use of a special device called a PCR thermal cycler.
-Repetitive cycling at specific temperatures

allows the amplification of a unique gene to
millions of copies
- The newly generated DNA strands

serve as template DNA for the next
cycle, leading to an exponential increase in
the number of amplicons.
Polymerase Chain Reaction (PCR)
17
Components Required forthe(PCR)Reaction
a. DNA template (sample) to be amplified.
b. DNA polymerase:
• TaqDNA polymerase : a special enzyme with high optimal temperature
• Uses single-stranded DNA as a template.
• Requires primers to initiate DNA synthesis.
c. Nucleotides: Called Deoxynucleoside triphosphates (dNTPs)- dATP, dTTP, dGTP, dCTP

d- Pair of primers:
• Generally, 20-30 nucleotides long.
• Synthetically produced.
• Designed/Chosen to flank the targeted DNA region.
• One primer binds to the 5’ end of one of the DNA strands.
• The other primer binds to the 3’ end of the anti-parallel DNA strand.
• Delineate the region of DNA you want to be amplified.
e- Water. f- Buffer/ Mg+2 12

Steps of PCR
15
Steps of PCR
1. Denaturation Step:
DNA sample is heated to 95oC to separate the two DNA template strands.
2. Annealing:
Cooling to 55oC to allow short single-stranded primers to anneal to
complementary sequences on template DNA.
3. Extension (Synthesis of a new dsDNA):
Heating to 72oC; DNA polymerase (Taq) synthesizes new DNA strands
creating 2 new double-stranded DNA.
Then: Repeating the cycle:
Each time the cycle repeats the amount of DNA doubles;
After 20 cycles, i.e. 220 =1 million dsDNA copies are formed, and after 30 cycles,
i.e. 230 = 1 billion dsDNA copies are formed
14
PCR Cycler
(Temperature vs Time (Hr))
1
6
Post-PCR processing
Detection of the target is accomplished
by different methods:
• gel electrophoresis,
• by use of labeled probes (in real time PCR)
• by end-product analysis like melt curve

analysis of the amplified product
3. Gel Electrophoresis
ELECTROPHORESIS
- Electrophoresis is a technique used in laboratories in order to separate
macromolecules (protein, DNA and RNA) based on size.
- The technique applies an electric charge; so the negatively charged
proteins and DNA move towards the positively charged electrode.
Agarose gel equipment/chemicals
An electrophoresis chamber and power supply
Gel casting trays
Sample combs
Agarose
Electrophoresis buffer
Loading buffer
Staining
UV Transilluminator
1- Agarose
2- Electrophoresis Buffer Solutions
- Effective separationof nucleic acids by agarose gel electrophoresis depends upon the
effective maintenance of pH within the matrix.
- Therefore, buffers are an integral part of any electrophoresis technique.

E.g.
Tris/Acetate/EDTA (TAE)
Tris/Borate/EDTA (TBE)
at a concentration of approximately 50 mM (pH 7.5-7.8).

3- Loading buffer
• This is the buffer to be added to the DNA fragment that will be electrophoresed.
• This buffer contains glycerol or sucrose to increase the density of the DNA
solutions; otherwise, the samples would dissolve in running buffer tank and not
sink into the gel pocket.
• Loading buffer also contains dyes that facilitate observation of the sample during
gel loading and electrophoresis, such as bromophenol blue or xylene cyanol.
These molecules are small, hence migrate quickly
through the gel during electrophoresis, indicating the
progress of electrophoresis.
4- Voltage/current applied
- The higher the voltage/current, the faster the DNA migrates.
- It is usually recommended to set the voltage at 5-15 Volts per cm of Gel.
- If the voltage is too high:

- band streaking may result.
- increase in buffer temperature in very short time leads to the melting
of the gel, DNA bands smiling, decrease of DNA bands resolution and
fuse blowout.
- If the voltage is too low, the mobility of small (≤1kb) DNA is reduced
and band broadening will occur due to dispersion and diffusion.
Visualizing the DNA
- The separated DNA species in the gel is visualized and

localized by using Ethidium bromide stain
(interchalating fluorescent dye) in very low concentrations
under UV light.
- The dye can be included in both the running buffer tank

and the gel, the gel alone, or the gel can be stained after
DNA separation.
- Instant photos are taken from the gels under UV light in a

dark room.
- It is important to note that ethidium bromide is a

potent mutagen and should be handled with great care.
Applications
1- Analysis of PCR products,
e.g. in molecular genetic diagnosis, genetic fingerprinting, etc…
2- Separation of DNA fragments for extraction and purification.
3- Estimation of the size of DNA molecules using a DNA marker/ladder which

contains DNA fragments of various known sizes.
4- Separation of restriction enzyme digested genomic DNA.
5- Allows for a rough estimation of DNA quantity and quality.

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Pharmaceutical

- Basic Steps of DNA Extraction:

1.Cell Lysis 2.Precipitation 3.Purification

2) Next, alcohol (such as 70% ethanol or isopropanol) is added, which facilitates

3) Upon centrifugation, DNA will aggregate together forming a pellet.

• At this point the purified DNA in pellet is then

N.B: Mini-column purification is another method that

2. A mixture of phenol: chloroform: isoamyl alcohol (25:24:1) is then added to

4. The aqueous phase containing the purified DNA

Polymerase: DNA polymerase enzyme

Chain Reaction: The product of a reaction is used to amplify

-Repetitive cycling at specific temperatures

- The newly generated DNA strands

c. Nucleotides: Called Deoxynucleoside triphosphates (dNTPs)- dATP, dTTP, dGTP, dCTP

e- Water. f- Buffer/ Mg+2 12

• by use of labeled probes (in real time PCR)

• by end-product analysis like melt curve

- Therefore, buffers are an integral part of any electrophoresis technique.

at a concentration of approximately 50 mM (pH 7.5-7.8).

- It is usually recommended to set the voltage at 5-15 Volts per cm of Gel.

- If the voltage is too high:

- The separated DNA species in the gel is visualized and

- The dye can be included in both the running buffer tank

- Instant photos are taken from the gels under UV light in a

- It is important to note that ethidium bromide is a

2- Separation of DNA fragments for extraction and purification.

3- Estimation of the size of DNA molecules using a DNA marker/ladder which

5- Allows for a rough estimation of DNA quantity and quality.

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