The document provides information about DNA and RNA extraction techniques from animal and plant cells. It discusses extracting nucleic acids using kits with varying costs and protocols for extracting DNA from animal tissue and plants. It also summarizes analyzing extracted nucleic acids through electrophoresis on agarose and polyacrylamide gels and using polymerase chain reaction (PCR) for applications such as DNA sequencing, forensics, and population genetics.

2. PRESENTATION OUTLINE
This presentation is going to cover the following
areas
DNA and RNA extraction from animals and plant cells
RNA and DNA analysis on Agarose and polyacrylamide
gels
Polymerase Chain Reaction (PCR) and its application

3. DNAAND RNA EXTRACTION
INTRODUCTION
Nucleic acid extraction has a long history, it was first done by Friedrich
Miescher in 1869 when he was struggling to isolate DNA from the lymph
node (Dahm, 2008).
Following the great need for DNA and RNA analysis, various protocols
have been developed to easy the extraction process (Tan & Yiap, 2009).
Molecular biology through nucleic acid extraction has helped in the
determination of fingerprinting, paternity tests, diagnosis of disease,
genetic engineering, and population genetic studies the attributes that
increased human security, human and animal health as well as invention
of number of drugs.

4. With tremendous improvements in science and technology various
laboratory extraction kits have been developed with varying costs.
Depending with the needs and cost effectiveness each laboratory has its
own extraction kit

5. DNA EXTRACTION FROM ANIMAL
Materials
DNA wash buffer, Column, DNA elusion buffer, Solid
tissue buffer, Proteinase k, Eppendorf tube, and
Micropipette.
Procedures
To a tissue sample (sample 1-11) in a deep well block, solution of
45µl water, 45 µl solid tissue buffer, and 10 µl proteinase K were
added. Mixed thoroughly, sealed, with film, and then incubated at
550C for 1-3 hours or until tissues clarified. Then mixed
thoroughly.
2 volumes of genomic binding buffer were added to each well and
mixed thoroughly. The debris were removed by centrifugation at ≥
1000 x g for 5 minutes. The acquas supernatants were transferred
to the 96 well plate.

6. The lysates were transferred to the wells of the Zymo-spin 1-96-XL Plate on
a collection plate. Centrifuged at ≥3,500 x g for 5minutess. The flow through
was discarded.
200 µl of DNA pre-wash buffer were added to each well and centrifuged for
5 minutes. The flow through was discarded
500 µl of g-DNA wash buffer were added to each well and centrifuged for
5 minutes. The flow-through was discarded
200 µl of g-DNA wash buffer were added and centrifuged for 5 minutes. The
flow through was discarded
Samples containing the DNA were transferred to an Elution plate. 15 µ DNA
Elution buffer were added, and incubated for 3 minutes, and centrifuged for
5 minutes

7. DNAAND RNA EXTRACTION FROM PLANTS
Materials
Eppendorf tube, water bath set at 65oC, pestle, sand or glass
beads, CTAB buffer freshly added 1% beta –mercaptoethanol,
Chloroform, Ice cold isopropanol, Ethano 70% and TE buffer.
Procedure
For plant extraction, the initial step that needs to be done is to grind the
sample after freezing it with liquid nitrogen. The purpose of doing this
step is to break down cell wall material of sample and allow access to
nucleic acid while harmful cellular enzymes and chemicals remain
inactivated.

8. After grinding the sample, it can be resuspended in a suitable buffer such as CTAB.
Cetyltrimethylammonium bromide (CTAB) is a nonionic detergent that can precipitate
nucleic acids and acidic polysaccharides from low ionic strength solutions.
Meanwhile, proteins and neutral polysaccharides remain in solution under these conditions.
In solutions of high ionic strength, CTAB will not precipitate nucleic acids and forms
complexes with proteins. CTAB is therefore useful for purification of nucleic acid from
organisms which produce large quantities of polysaccharides
This method also uses organic solvents and alcohol precipitation in later steps.
 Insoluble particles are removed through centrifugation to purify nucleic acid.
Soluble proteins and other material are separated through mixing with chloroform and
centrifugation.
Nucleic acid must be precipitated after this from the supernatant and washed thoroughly to
remove contaminating salts.
The purified nucleic acid is then resuspended and stored in TE buffer or sterile
distilled water.

9. RNA AND DNA ANALYSIS ON AGAROSE
AND POLYACRYLAMIDE GELS
INTRODUCTION
Electrophoresis is the motion of colloidal particles relative to a fluid medium
under the influence of an electric field that is uniformly spaced (Tunjin, 2017).
Electrophoresis is the process of moving charged molecules in a solution by
applying an electric field (Ayoub, 2011). During electrophoresis mobility is
dependent on charge, shape and size of the molecules.
Electrophoresis is influenced by the type, concentration and the PH of the
buffer.
Two prominent gels widely used for analysis of DNA, RNA and proteins are
agarose and polyacrylamide gels which are describe in details as follows.

10. Nucleic acid analysis on Agarose gel
Agarose is a polysaccharide purified from seaweed. An agarose gel is
created by suspending dry agarose in a buffer solution, boiling until the
solution become clear, and then pouring it into a casting tray and allowing
it to cool.
Agarose gel electrophoresis is one of several physical methods for
separating DNA fragments according to size.
The lower the concentration of agarose the faster the DNA fragments migrates.
If the aim is to separate the large DNA fragments, a lower agarose
concentration should be used and if the aim is to separate the small DNA
molecules the higher concentration of agarose should be used.
 In this method, DNA is forced to migrate through highly cross-linked
agarose matrix in response to an electric current.
In solution, the phosphate on the DNA are negatively charged, and the
molecule will therefore migrate to the positive (red) pole

11. In most applications only single agarose component
is needed and no polymerization is required.
Agarose gels are simple and rapid to prepare, most
popular medium for separation of moderate and large
sized nucleic.
Agarose gels use low resolving power since the
bands formed in the gels tend to be fuzzy and spread
apart, this is the results of pore size and cannot be
largely controlled

12. There are several factors that affect migration rate through gel: the
size and conformation of the DNA, the size and conformation of the
DNA, the concentration of agarose, and the ionic strength of the
running buffer.
It is wise to use TBE as a running buffer so ionic strength was
constant throughout at all our experiment.
The following procedures are to be followed when running agarose
gel
1% agarose gel was prepared by suspending 2 agaroses in 200ml of
TBE (45mM Trisborate, 1mM EDTA) in a 150 Erlenmeyer flask.
The mixture was homogenized by boiling in a microwave at 1000C
for 2minutes or until the agarose powder completely melts

13. The agarose mixture was cooled in air for 10 minutes after which 10µl of blue light DNA
stain and thoroughly mixed by swirling the flask until the agarose stain mixture was
homogenized.
The agarose blue light DNA mixture was poure into a well prepared casting tray with well-
placed combs. The agarose gel was left to solidify by cooling at room temperature for 20
minutes
The cover tapes on a casting tray were removed and the solidified agarose gel was placed
into a gel box filled with TBE buffer. The combs were removed careful to make the well
visible
5µl of 1kb ladder was loaded into the first lane of the gel. 8µl of each DNA samples was
mixed with 1µl of loading dye on a parafilm and the dye DNA solution was loaded into
each well. The sample type loaded in each well was recorded on a note book for further
reference.
The gel box was covered with its lid and its terminals were connected to the electrophoretic
machine and set at 80V for 1 hour. The gel box was disconnected from power and the
agarose gel was removed on its casting tray
The agarose gel with resolved band was placed on UV light source stage and the DNA
bands were observed. A photo image of the gel was taken with ist bands for futher reference

14. OBSERVATION

15. From the above results, the ladder at the beginning and that at the
end were the reference indicating the genomic DNA base pair/Kb.
Samples 1 and 10 show that DNA was present in a small amount
that’s why the bands were not well seen. For samples 2,3,4,5, 8
and 11 were containing large amount of DNA because the bands
were clearly seen.
In case of samples 6, 7, and 9 the bands were completely not
detected meaning that the sample contained no DNA.
The presence or absence of DNA detection depend is affected
much on the quantity of the sample collected and also depends on
the correctness of following the procedures..

16. NUCLEIC ACID ANALYSIS ON POLYACRYLAMIDE GEL
Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in
biochemistry, molecular biology and biotechnology to separate and purify
biological molecules such as nucleic acid and and proteins according to their
electrophoretic mobility and molecular size.
 Polyacrylamide gels are chemically cross linked gels formed by
polymerization of acrylamide with a cross linking agent usually N,N-
Methylenebisacrylamide.
The reaction is free radical polymerization, usually carried out with
ammonium persulfate as initiator and N,N,N,N-Tetramethylendiamine
(TEMED) as a catalyst
PAGE is a powerful tool for purifying RNA samples, it is commonly used to
separate and purify RNA species after in vitro transcription.
The purified RNA can be preserved and usually visualized by UV shadowing
or stained with ethidium bromide or SYBR green dyes.

17. These gels are so difficulty to prepare and handle
as they are classified as neurotoxin causing harm
when poorly handled.
Polyacrylamide gels are chemically stable, having
high resolving power making them able to
accommodate large quantity of DNA without
significant loss in resolution and the DNA obtained
from these gels are extremely pure.
These gels are good in separation of molecules
with low molecular weight.

18. Procedures
Mix Tris borate EDTA (TBE), acrylamide, water and Ammonium
persulphate (APS). Then Tetramethylendiamine (TEMED) and mix by
swirling to prepare the PAG.
Pour the gel apparatus with TBE and remove the comb. Use the syringe
to wash out the wells in order to remove un-polymerized acrylamide
which may disrupt the running of the sample.
Load the samples on the gel and run at 200-300V (approx. 10 V/cm) until
the bromophenol blue band is two – third of the way down the gel, this
takes about 2.5 hours.
Place the gel to stain in 1mg/mL of ethidium bromide for 3 min. view the
stain gel on transilluminator.

19. In other ways,
During electrophoresis the mixture of electrophoresis is firstly dissolved in a
solution of SDS (Sodium dodecyl sulfate) which is an anionic detergent that
disrupts nearly all non-covalent interaction in native proteins.
Mercaptoethanol also added to reduce disulfide bonds. SDS nucleic acid
complex is then subjected to electrophoresis.
Microliter pipette is used to place solution of nucleic acid in the wells of the
slab, a cover is then placed over the gel chamber and the voltage is applied.
The negatively charge SDS protein complexes migrate in the direction of the
anode at the bottom of the gel. The sieving action of porous PAG separate
nucleic acid according to size with the smallest moving most rapidly.
When electrophoresis is complete, the nucleic acid in gel can be visualized
by staining them with Ethidium bromide, when the adequate migration occurs
this fluorescent intercalates between bases of DNA and RNA.

20. POLYMERASE CHAIN REACTION (PCR)
PCR is a method widely used to rapidly make million to billions of copies of a
specific DNA sample allowing scientist to take a very small sample of DNA and
amplify it to a large enough amount to study in detail (Gupta, 2017).
 PCR is also called Molecular photocopying and it is inexpensive technique used to
amplify small segment of the DNA (NHGRI, 2018).
DNA replication occurs in cell where polymerization of nucleotide using a template
DNA strand with the help of enzyme DNA polymerase.
This reaction require a polymer strand to which further nucleotide can be added by
using DNA polymerase enzyme (NHGRI, 2018).
According to Gupta (2018) in PCR similar process takes place in Eppendorf tube in
which the primer fragment is added from the outside in the form of
deoxyligonucleotide and DNA polymerase enzyme is added to help polymerization.

21. STEPS OF POLYMERASE CHAIN REACTION (PCR)
The denatured DNA (at 90o-98oc) and two primers, one recognizing
each strand bordering the sequence to be amplified were mixed.
The mixture was then cooled to 40o-60oC this allowed primers to
find their complementary bases through renaturation.
To this mixture was then added
DNA polymerase enzyme (taq polymerase) and
All four essential nucleoside triphosphate
The mixture was incubated and this allowed synthesis of
complimentary strand using primers with the help of DNA
polymerase in the usual manner.
The process should be repeated 20-30 times so that in a single
afternoon billion copies of DNA be produced.

22. The above explanations, can be
summarized into three steps
1.Denaturation of the template into single
strand
2.Annealing of primers to each original
strand for new strand synthsis
3.Extension of the new DNA strands

24. APPLICATION OF CHAIN REACTION (PCR)
The PCR can be applied in range of areas such as in
1. DNA sequencing
PCR in the presence of the dideoxynucleoside triphosphate (ddNTPs), chain
terminating inhibitors of DNA synthesis used for DNA sequencing, allows
DNA sequencing reaction to be run successfully with very small amounts of
template. The process requires the special a specially developed enzyme that
combines the properties needed for the sequencing with thermostability.
2. Diagnostic
PCR is useful as a diagnostic tool. Example in the identification of specific
genetic traits or for the detection of pathogens of food contaminants. One of
the earliest diagnostic application of PCR was for sickle cell anemia allowing
disease analysis to be completed for a day rather than the weeks as used to be
done by conventional approach of hybridization of DNA from cells.

25. APPLICATION CONT…..
3. Forensic
The ability to amplify DNA from region of genome that are highly
polymorphic starting with the sample that contains a very small portion of
DNA such as single hair or traces of body fluid leads to application in
forensic work
4. Present day population genetics
The ability to amplify materials rapidly from a large number of DNA
preparation leads to applications in the population genetics allowing for
example the determination of frequencies of particular alleles in a large
collection of individuals. PCR also used in the population genetics studies
of microsatellites. Advantages of using PCR in population genetics is that
with appropriately designed specific primers it may be possible to amplify
DNA from one organism that cannot be separated from others such as
particular bacterial strains in a mixed population

26. APPLICATION CONT…..
5. Archeology and evolution
PCR can be used with old material as well as more recent
samples and it is often to amplify ancient DNA from museum
specimens and archaeological remains. Multiple copy sequences
such as mitochondrial DNA or chloroplast DNA are particularly
useful target. Comparison from the ancient DNA with the today
observed DNA allow inferences to be made about the origin of
particular population and or species

27. THANK YOU
THE END