Dr R Alili

1. Lecture Title Principles of DNA Isolation and PCR
Lecturer Rubigilda Paraguison-Alili
Lecture Objectives  The participants will be able to
understand the principles of DNA and
RNA Isolation
 The participants understand the basic
theoretical background of the PCR and
LAMP techniques
 The participants are able to understand
these techniques and the usage of
reagents and chemicals incorporated in
the kits
TOPICS
1. Title: Principles of DNA Isolation/Extraction and PCR
2. DNA Location: DNA is located mainly in the nucleus, but can also be found in other cell
structures called mitochondria. Since the nucleus is so small, the DNA needs to be
tightly packaged into bundles known as chromosomes.
3. Principles of DNA isolation and purification
DNA/RNA can be isolated from animal cells, plant cells, bacteria, protozoa or viruses
DNA isolation is a process of purification of DNA from sample using a combination of
physical and chemical methods. Currently it is a routine procedure in molecular biology.
Good quality DNA is a prerequisite for all experiments of DNA manipulation. All DNA
extraction protocols comprise of the basic steps of disruption of cell membrane and
nuclear membrane to release the DNA into solution followed by precipitation of DNA
while ensuring removal of the contaminating biomolecules such as the proteins,
polysaccharides, lipids, phenols and other secondary metabolites.
4. Breaking the cell, layer by layer releases the cellular constituents.
Accomplished by lysis of the tissue or any sample and homogenizing in the extraction
buffer.
5. Components of Extraction/Lysis Buffer
Tris (Tris-hydroxymethyl aminomethane) buffers the pH of the cells at 8.0.
EDTA (Ethylene diamine tetraacetic acid) chelates the metal ions,cofactors for DNases;
weakens the cell membrane stability.
Sodium chloride helps in maintaining the osmoticum of the cells. High concentration
weakens the membrane integrity.
6. Components of Extraction/Lysis Buffer
Mercaptoethanol cleaves the disulphide bridges of proteins and helps in denaturation
proteins.

2. Proteinase K – degrade proteins at 56oC
SDS is a detergent, which also denatures the membrane proteins and disrupts the cell
membrane. SDS also helps in inhibition of nucleases.
7. PCI - The use of Phenol Chloroform-Isoamyl alcohol) is to remove the proteins, most
lipids, and cellular debris that can cause an impure DNA result.
8. DNA Precipitation:
DNA in the nucleoplasm is neutralized by Potassium acetate. K+
ions bind with negative
phosphate backbone of the DNA and shield them. Also, high concentration of NaCl. This
favors DNA precipitation from ethanol in cold condition.
DNA precipitate is settled down as a pellet by centrifugation, purified by 70 % ethanol
wash, hydrated in TE buffer.
9. RNA Isolation/Extraction
Take place in the presence of RNase inhibitory agents (typically strong denaturants like
guanidine salts-guanidine isothiocyanate (GITC), sodium dodecylsulfate (SDS), or
phenol-based compounds (e.g.Trizol)
It is typically prior to and after the isolation when RNA integrity is at highest risk.
10. You may have the option in choosing the procedure in extracting the DNA or RNA: Using
the commercial kit or the conventional method.
11. Things to remember in RNA Extraction
This can be problematic when tissues or cells are hard (e.g., bone, roots), when
workflows prevent processing immediately after collection (e.g., transport from a site of
collection to another location for processing), or when samples are numerous (making
rapid processing difficult).
12. Things to remember in RNA Extraction
RNA are easily degraded.
RNAses are mostly ubiquitous, there should be a designated area for RNA work.
RNA area usually uses DNAses and DNA area uses RNAses..
DEPC – RNAse inhibitor
13. Questions
Principles of Polymerase Chain Reaction
Introduction
Polymerase chain reaction (PCR) is a technique which is used to amplify the number of copies
of a specific region of DNA, in order to produce enough DNA to be adequately tested. This
technique can be used to identify with a very high-probability, disease-causing viruses and/or
bacteria, a deceased person, or a criminal suspect.
In order to use PCR, one must already know the exact sequences which flank (lie on either side
of) both ends of a given region of interest in DNA (may be a gene or any sequence). One
need not know the DNA sequence in-between. The building-block sequences (nucleotide
sequences) of many of the genes and flanking regions of genes of many different organisms are
known. We also know that the DNA of different organisms is different (while some genes may
be the same, or very similar among organisms, there will always be genes whose DNA
sequences differ among different organisms - otherwise, would be the same organism (e.g.,
same virus, same bacterium, an identical twin; therefore, by identifying the genes which are
different, and therefore unique, one can use this information to identify an organism.

3. Objectives: At the end of the lecture, the participants should be able to understand the
principles of PCR.
14. Title: Polymerase Chain Reaction
15. Definition of PCR
Developed by Kary Mullis in 1984, leading to the invention of the Thermocycler/Thermal
cycler
Thermal cycler is complex heatblock wherein different temperatures are set for in vitro
amplification or reproduction of target DNA
Polymerase Chain Reaction (PCR):
A molecular technique which can amplify a specific DNA region
Uses:
 Yields millions of copies of the target region
 Makes enough DNA for further molecular work
 Or diagnosing or detecting the presence of certain pathogens
PCR performs the chemistry of DNA duplication in vitro
Numerous PCR applications make this process a staple in most biology laboratories
16. PCR Targets
The targets in PCR are the sequences of DNA on each end of the region of interest,
which can be a complete gene or small sequence. Crucial in primer or DNA marker
designing
17. Steps in PCR Amplification
18. Denaturation
Denaturing is the first step in PCR, in which
the DNA strands are separated by heating to
95°C to 97C.
19. Heating separates the double stranded DNA -Denaturation
Slow cooling anneals the two strands- Renaturation
20. Annealing
Annealing is the process of allowing two sequences of DNA to form hydrogen bonds.
The annealing of the target sequences and primers is done by cooling the DNA to about
28 to 65°C.
Two primers are supplied to bind to the complementary region
As the DNA cools, they wedge between two template strands
Optimal temperature varies based on primer length and melting temperature
Typical temperature from 28 to 65 C
21. Elongation/Extension
DNA polymerase duplicates DNA
Optimal temperature 72C
22. Summary of the Polymerase Chain Reaction

4. Separating the Target DNA - Denaturation
During the first step of PCR, called denaturation, the tube containing the sample DNA is
heated to more than 90 degrees Celsius (194 degrees Fahrenheit), which separates the
double-stranded DNA into two separate strands. The high temperature breaks the
relatively weak bonds between the nucleotides that form the DNA code.
Binding Primers to the DNA Sequence - Annealing
PCR does not copy the all of the DNA in the sample. It copies only a very specific
sequence of genetic code, targeted by the PCR primers. For example, PED has a
unique pattern of nucleotides specific to the bacteria. The PCR will copy only the specific
DNA sequences that are present in PED and absent from other bacterial species. To do
this, PCR uses primers, man-made oligonucleotides (short pieces of synthetic DNA) that
bind, or anneal, only to sequences on either side of the target DNA region.
Two primers are used in step two - one for each of the newly separated single DNA
strands. The primers bind to the beginning of the sequence that will be copied, marking
off the sequence for step three. During step two, the tube is cooled and primer binding
occurs between 40 and 60 degrees Celsius (104 – 140 degrees Fahrenheit).
Step two yields two separate strands of DNA, with sequences marked off by primers.
The two strands are ready to be copied.
3. Making a Copy - Extension
In the third phase of the reaction, called extension, the temperature is increased to
approximately 72 degrees Celsius. Beginning at the regions marked by the primers,
nucleotides in the solution are added to the annealed primers by the DNA polymerase to
create a new strand of DNA complementary to each of the single template strands.
After completing the extension, two identical copies of the original DNA have been
made.
After making two copies of the DNA through PCR, the cycle begins again, this time using
the new duplicated DNA. Each duplicate creates two new copies and after approximately
30 or 40 PCR cycles, more than one billion copies of the original DNA segment have
been made. Because the PCR process is automated, it can be completed in just a few
hours.In a healthcare setting, PCR makes enough copies of target DNA from the clinical
sample to allow analysis; the results of these diagnostic and monitoring tests
provide clinicians and other healthcare providers with information to guide treatment
http://molecular.roche.com/pcr/Pages/Process.aspx
23. DNA Sequences of any organism, microorganism, viruses can be searched at the
NCBI web pageThe Primers/DNAMarkers
24. Roles of PCR Reagents
PCR Mix
 Taq polymerase

5.  Enzyme that extends growing DNA strand of the PCR target
 MgCl2
 Provides ions needed for enzyme reaction. Mg is a co-factor of the
enzyme
 dNTP’s ( DeoxynecleoTriphosphate)
 Nucleotides (Adenine, Cytosine, Guanine, Thymine) building blocks for
new DNA strands
 Buffer
Maintains optimal pH for enzyme (tris, KCl)
Basic Components
20mM Tris-HCL pH 8.4
50mM KCl
Magnesium –optimal concentration of MgCl2 has to be selected for each
experiment. Too few Mg2+ ions result in a low yield of PCR product, and
too many increase the yield of non-specific products and promote
misincorporation.
 Primers
Anneal to single-stranded DNA template
Provide initiation site for extension of new DNA
Forward primer
Reverse primer
 DNA template
In this case, the product of our DNA extraction
25. Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the
complementary building blocks of the target sequence.
A primer for each target sequence on the end of your DNA is needed. This allows both
strands to be copied simultaneously in both directions.
DNA template - In this case, the product of our DNA extraction which is not included in
the master mix.  1 µg or 10 to 100ng is enough

6. 26. Sample PCR Profile
27. Considerations in PCR
Contamination can easily lead to erroneous results
 Avoid contaminating with DNA or PCR product…
 DNA stocks, PCR reagents
 Gloves, tips, pipettors, benches
Carefully measure reagent quantities
Use appropriate cycling conditions
Run with positive and negative controls
28. How are PCR products analyzed?
Gel electrophoresis is a widely used technique for the analysis of nucleic acids
(Agarose gel electrophoresis)
Gel electrophoresis is a procedure that separates molecules on based on size through a
gel under the influence of an electrical field.

7. 29. DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA
will migrate towards the positively charged electrode.
Shorter strands of DNA move more quickly through the gel than longer strands resulting
in the fragments being arranged in order of size.
30. Example of the DNA bands ran on an agarose gel.
Principles of Loop-Mediated Isothermal Amplification (LAMP)
Introduction
This is a one-step alternative DNA technique other than PCR in detecting various diseases that
caused by viruses, bacteria, protozoa and many parasites. LAMP uses a single
temperature/isothermal incubation thereby it does not utilize expensive PCR machines and can
be performed simply with a heating block or water bath. Although the inception of LAMP refers
back to 1998, it became popular only after 2003 following the emergence of West Nile and
SARS viruses. Since then LAMP assay has increasingly been adapted by researchers mostly
from Japan for the clinical diagnosis of emerging diseases (Parida et al., 2008). Detection of the
culprit can be by photometry for turbidity caused by increasing quantity of Magnesium
pyrophosphate in solution or with addition of a DNA dye wherein a color change can be seen
using naked eye (Mori et al, 2001). In LAMP, the target DNA is amplified at a constant
temperature of 60 - 65 °C using either two or three sets of primers or DNA markers specific for
the target organism, microorganism or viruses which makes the detection more specific and a
polymerase, an enzyme for amplifying the target DNA.
Objectives:At the end of the lecture, the participants should be able to learn the principles of
Loop-Mediated Isothermal Amplification or LAMP and its applications.
Subtopics:
1. Definition of LAMP
2. Advantages and Disadvantages of LAMP
3. Components of LAMP reaction
4. LAMP mechanism
36. What is Loop-Mediated Isothermal Amplification (LAMP)?
 Developed by Eiken Chemical Co., Ltd. (Japan)
 Uses 4-6 different primers designed to recognize distinct regions on the target gene. it
is expected to amplify the target sequence with high selectivity.
 Reaction process proceeds at constant temperature (about 60°C- 65°C)
 May be combined with a Reverse Transcription step to allow the detection of RNA
37. Advantages
1. Amplification of DNA takes place at an isothermal condition (63 to 65°C) with greater
efficiency.
2. Thermal denaturation of double stranded DNA is not required.
3. LAMP helps in specific amplification as it designs 4 to 6 primers to recognize distinct regions
on the target gene.
4. LAMP is cost effective as it does not require sophisticated equipment.
5. This technology can be used for the amplification of RNA templates in presence of reverse
transcriptase.

8. 6. LAMP assay takes less time for amplification and detection.
38. Disadvantages
1. Complicated primer design
2. Too sensitive and prone to contamination and small changes in conditions
39. Comparison with PCR and ELISA
40. Nucleic Acid extraction
RNA extraction includes 50 µL or about 20mg of biological sample is crushed with 100µL of
0.5N NaOH using mini grinder. A 10µL is diluted 150µL of 100mM Tris-Cl, pH 8.0 and directly
used for the LAMP assay.
41. LAMP Components
LAMP
Components
Purpose Volume:
12.5uL
Nuclease-free
Water
5.45
10x LAMP buffer
---Tris-Cl,
MgSO4,
(NH4)2SO4, KCl
minimize the change in pH making nucleic acid stable 1.25

9. 5M Betaine Betaine was used in the LAMP reaction mixture to reduce
base stacking and to increase not only the overall rate of
reaction but also target selectivity by significantly reducing
amplification of irrelevant sequences
2.5
10uM dNTP mix (deoxy-nucleotide-tri phosphate) Nucleotides (Adenine,
Cytosine, Guanine, Thymine) building blocks for new DNA
strands 20-200µM
1.0
Primer Mix:
F3
B3
FIP
BIP
F-loop
B-loop
short nucleic acid sequences (generally about 10 base
pairs) that serves as a starting point for DNA synthesis
1
8u/uL Bst
Polymerase
DNA polymerase derived from Bacillus
stearothermophillus, able to unwind DNA strands. Its
optimum functional temperature is between 60 and 65 °C
and it does not require the high temperature (96 °C) step
required to denature DNA.
0.25
Reverse
Transcriptase
(RT) is an enzyme used to generate complementary
DNA (cDNA) from an RNA template, a process
termed reverse transcription.
0.05
DNA/RNA
template
Your DNA or RNA extract ~10 to
500ng
42. LAMP reaction can be incubated at 60 to 65oC for 30 to 1 hour. And then added with nucleic
acid dye to indicate the positive and negative reactions.