COLLEGE OF NATURAL SCIENCES

DEPARTMENT OF BIOCHEMISTRY AND

SPORTS SCIENCES

Experiment on DNA Extraction from Whole Blood Sample and

its Quantitative determination using the Diphenylamine Assay

NAME: ANZO SIMON

REG NO: 22/U/5784
YEAR: 2
SEMESTER: 2

DATE OF PRACTICAL: 12TH FEBRUARY, 2023

DATE OF SUBMISSION: 22nd MARCH, 2023

PARTNERS.
NAMES REG. NO.
1. Ampire Arnold 22/U/5765
2. Balyawo Hope 22/U/5870

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ABSTRACT
DNA extraction and Quantification from biological samples are two critical first steps in many
molecular biology techniques and thus proves their importance in various fields. Basing on the
experiment, the main aim was to extract DNA from a blood sample followed by its quantitative
determination.

The DNA extraction technique involved the disruption of the cells (cell lysis) to release the
highly intact DNA into solution followed by precipitation of DNA and removal of the
contaminating biomolecules such as the proteins, polysaccharides, lipids, phenols, and other
secondary metabolites which effectively achieved by subjecting the blood sample to a set of
reagents including a RBC lysis buffer for lysing red blood cells, an extraction buffer which
contained EDTA for removal of metal ions present, SDS was to give a net negative charge to
DNA as a function of its size and also denatures proteins whereas Isoamyl alcohol and
chloroform were to partition lipids and debris into an organic phase leaving DNA in the
aqueous phase. The samples were centrifuged at different intervals to separate DNA from other
cellular components hence pure DNA was obtained.

To quantify DNA, the pure samples obtained from isolation were subjected to Diphenylamine
reaction based on the principle that, when DNA is treated with diphenylamine under the acidic
condition, a blue-green colored complex is formed where in acidic solutions deoxyribose are
converted into a highly reactive β-hydroxy laevulin aldehyde which reacts with diphenylamine
giving a blue-green colored complex which intensity is then measured at 595 nm. A standard
curve was constructed using standard DNA samples and acted as a reference for the
determination of the concentration of DNA in the sample provided.

Calculations based on the standard curve yielded 15 μg DNA, corresponding for 5 µg/ml DNA
concentration in Unknown 3 and 13.89 μg DNA corresponding for 4.63µg/ml DNA
concentration in Unknown 4 giving an average of 4.815µg/ml DNA concentration in the
sample.

This results indicate that the DNA extraction process was not highly effective thus resulting in
a lower yield of DNA from the sample consequently when DNA concentration is low, it can
affect downstream applications such as PCR, sequencing, or other molecular biology
techniques, potentially leading to unreliable or inconclusive results.

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INTRODUCTION
Genome is the entirety of an organism's hereditary information. It is encoded either in DNA
or, for many types of virus (HIV, Coronavirus), in RNA. The genome includes both the genes
and the non-coding sequences of the DNA.
Deoxyribonucleic acid (DNA) on the other hand is the hereditary material that carries the
genetic instructions for building and maintaining an organism, also known as "molecule of
life," existing in nearly every cell and contains the blueprint for all known living things right
from the simplest bacteria to the most complex humans.

DNA consists of a double helix, formed by two strands of nucleotides that coil around each
other. Each nucleotide consists of three key components, the deoxyribose sugar, a phosphate
group and a nitrogenous base.

The Phosphate group plays a vital role in provision of energy for cellular processes. When a
phosphate group is attached to a nucleotide, it stores potential energy. When the phosphate
group is broken off, it releases energy that can be used by the cell to power various functions,
the 5-carbon sugar, in DNA, the sugar is deoxyribose, while RNA uses ribose and the sugar
acts as a backbone, linking the nucleotides together to form the long chains of DNA or RNA
and the Nitrogenous bases are the key to the informational role of nucleotides. There are two
types of nitrogenous bases: purines (adenine and guanine) and pyrimidines (cytosine, thymine
in DNA, and uracil in RNA). The specific pairing of these bases (A with T and C with G)
determines the genetic code in DNA and allows for complementary base pairing during
processes like DNA replication and protein synthesis.

The two strands of the DNA molecule are held together by specific base pairing with Adenine
pairing with Thymine and Cytosine pairing with Guanine which is referred to as the
complementary base pairing rule and is absolutely crucial for DNA replication and information
transfer within the organisms.(Travers & Muskhelishvili, 2015)

Structure of DNA

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DNA is very important in cells and genetic information storage is one of the most critical role
of DNA, it stores the instructions for building and maintaining an organism, including its
physical characteristics. This genetic information is passed from parents to offspring and is
responsible for inherited traits. DNA also replicates itself to ensure that each new cell receives
a complete copy of the genetic information. It also contains the instructions for synthesizing
proteins, which are essential for the structure, function, and regulation of the body's tissues and
organs.

The information encoded in DNA is transcribed into messenger RNA (mRNA), which carries
the instructions to ribosomes for protein synthesis and protein synthesis, the building blocks of
life that is proteins are synthesized based on the instructions carried by mRNA, which
ultimately dictate cellular functions and organisms traits.

Additionally, DNA also plays a crucial role in regulating the functioning of cells as it controls
the expression of genes, which are segments of DNA that code for specific proteins or RNA
molecules. Gene expression is tightly regulated and can be influenced by various factors, such
as environmental signals and internal cellular processes.

In cell division, it is replicated and distributed to daughter cells, ensuring that each new cell
receives a complete set of genetic information. This process is essential for growth,
development, and repair of tissues in multicellular organisms.

For the purpose of evolution, DNA contains a record of an organism's evolutionary history.
Researchers and scientists can infer evolutionary relationships and study the mechanisms of
evolution by comparing the DNA sequences of different species.

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DNA extraction is a process that is used to purify DNA by using chemical or physical methods
from a biological sample separating it from protein, cell membranes, plus other cellular
components and purifying it for further analysis. DNA extraction is a fundamental step in many
biological and medical research applications, including molecular biology, genetics, forensics,
and biotechnology.(Green & Sambrook, 2018)

Various fields like genetics make use of DNA fundamentals for studies of how traits are passed
down through generations and how variations in DNA can lead to genetic diseases, in the field
of Medicine uses DNA analysis for diagnosis of genetic disorders, development of personalized
medical approaches as well as guide in forensic investigations during crime solving. In the field
of biotechnology, Recombinant DNA technology allows scientists to manipulate DNA for
various applications such as producing therapeutic proteins or genetically modified organisms.

The method used for the DNA extraction process literally depends upon the sample type, that
is the DNA extraction method for plant DNA is different from that of the blood. Likewise, the
bacterial DNA isolation method is different from other types. So, varieties of DNA extraction
methods are needed for different samples.

DNA extraction methods are broadly categorized into: Chemical-based DNA extraction
methods and Solid-phase DNA extraction methods (Physical method) of which the choice of

DNA extraction method depends on factors such as the sample type, the amount of DNA
needed, the purity required, and the downstream applications. It is important to select a method
that is suitable for the specific requirements of the experiment.

To effectively extract DNA from a sample, first, methods such as mechanical disruption,
enzymatic digestion or chemical lysis are required to break down the formidable cell wall and
nuclear envelope and once the DNA has been liberated, it has to be separated from other
cellular constituents like lipids and proteins and this can be achieved through a multitude of
techniques including differential centrifugation, organic extraction, salting out,
chromatography, or silica-based methods with each method exploiting specific characteristics
of DNA and other biomolecules to achieve separation.

Some common DNA extraction methods include: Phenol-Chloroform Extraction which

involves lysing cells with a detergent to release the DNA, followed by extraction with a mixture
of phenol and chloroform to separate DNA from other cellular components. The DNA is then
precipitated with ethanol and purified. Another method is Spin Column Extraction where Spin

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column kits use silica membrane technology to bind DNA in the presence of a chaotropic salt,
which disrupts protein interactions. The DNA is washed to remove contaminants and eluted in
a buffer suitable for downstream applications. Another method is Chelex Extraction in which
Chelex resin can selectively bind metal ions, including magnesium ions that are required for
many enzymes that degrade DNA. Chelex extraction involves incubating the sample with
Chelex resin, followed by heat treatment to release the DNA.

The CTAB Extraction: CTAB (cetyltrimethylammonium bromide) is a cationic detergent that

can be used to extract DNA from plant tissues. It involves a series of steps, including cell lysis,
protein precipitation, and DNA precipitation.(Barnett & Larson, 2012)

DNA precipitation is achieved by adding high concentrations of salt to DNA containing

solutions, as cations from salts such as ammonium acetate counteract repulsion caused by the
negative charge of the phosphate backbone. A mixture of DNA and salts in the presence of
solvents like ethanol (final concentration of 70%-80%) or isopropanol (final concentrations of
40%-50%) causes nucleic acids to precipitate. Some protocols include washing steps with 70%
ethanol to remove excess salt from DNA. Finally, nucleic acids are re-suspended in water or
TE buffer.(Baechtel & Quantico, 1989)

On the other hand DNA quantification is a scientific procedure used to determine the exact
amount, or concentration, of DNA present in a sample. This measurement is typically reported
in units of Nano grams per microliter (ng/uL).

DNA quantification plays a vital role in various molecular biology experiments because many
downstream applications require a specific amount of DNA to function properly. Knowing the
concentration allows researchers to determine if there's enough DNA in the sample to proceed
with techniques like PCR (polymerase chain reaction) or gene sequencing. These techniques
require a minimum amount of DNA to amplify or analyze the genetic material effectively. It
also provides quality check since certain quantification methods, like UV spectrophotometry,
can also offer insights into the purity of the extracted DNA. Contaminants like proteins or RNA
can absorb light at different wavelengths, and these measurements can indicate potential issues
with the sample.

In the quantitative determination of DNA, various techniques can be employed including

spectrophotometric analysis, fluorescence-based assays, or the colorimetric diphenylamine
(DPA) method.

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In this experiment the DPA method, also known as the Dische's test or diphenylamine (DPA)
method was employed due to its simplicity though it’s an old technique and is based on the
reaction between the sugar component of DNA (deoxyribose) and diphenylamine reagent.

Treatment of DNA with strong, hot acid, leads to the depurination of the molecule and the
sugar-phosphate backbone is cleaved thus the 2-deoxy-D-ribose monosaccharides are
dehydrated to T-hydroxylevulinyl aldehydes which condense with diphenylamine, the final
product is a blue-colored complex. The intensity of this blue color is proportional to the amount of
DNA present in the sample and absorbs at 595nm (blue-green in color); the range of this assay is
10-200 µg/ml.(Zhao et al., 2013)

A standard curve is drawn from standard DNA which is the used as a reference used in the
quantitative determination of DNA in the given sample

OBJECTIVES
The objectives of the experiment include the following;
To separate the genetic material from the rest of the cell components.

To purify DNA from a human blood sample provided.

To draw a calibration curve of Absorbance versus DNA concentration using diphenylamine

assay.

To determine the concentration of DNA present in unknown samples 3 and 4.

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PROCEDURES

Requirements

Reagents;

RBC lysis Buffer (RLB) Extraction Buffer TE buffer (10 mM Tris, 1

mM EDTA)
0.155 mol/L NH4CI 1.5 mol/L Tris (pH 7.6),
10 mmol/L KHCO3 0.4 mol/L Na2 EDTA, 2.5 Equipment
0.1 mol/L EDTA (Na2) mol/L NaCl, 2% (CTAB),
Distilled water Cetyltrimethylammonium Spectrophotometer,
bromide, Distilled H2O, Eppendorf tubes,
70% and 90% ethanol, volumetric flasks,
10% SDS Sodium dodecyl Beakers, Pipettes,
Sulfate, Chloroform; Centrifuge, Ice,
Isoamyl alcohol (24:1) Incubator, AB (analytical
solution balance)

Procedures for the preparation of Reagents:

1. Procedure for the preparation of RBC lysis Buffer (RLB)

NH4Cl (8.77 g), KHCO3 (100 mg) and Na2EDTA (37.22 g) were weighed using an analytical
balance and each of them was dissolved separately in a small amount of distilled water. The
solutions were all poured in to a 250ml volumetric flask topped up to the mark using distilled
water. The pH of the prepared solution was measured using a pH meter and a dilute solution
of HCl was added in few drops that adjusted the pH of the solution to 7.6.

2. Procedure for the preparation Extraction Buffer

Tris (180.15g), Na2EDTA (116.16g), NaCl (146.10g) and CTAB (20g) were separately
weighed using an analytical balance and each was separately dissolved in a small amount of
distilled water and poured/mixed in a 250ml volumetric flask and topped up to the mark using
distilled water. Using a pH meter and a dilute solution of NaOH, the pH of the solution was
adjusted to 8.0.

8
3. Procedure for the preparation of 10% SDS (Sodium dodecyl sulfate)

10g of SDS was weighed using an analytical balance and then dissolved in small amount of
water and poured in a 100 mL volumetric flask and topped up to the 100mL mark using distilled
water.

4. Chloroform: Isoamyl alcohol (24:1) solution

96mL of chloroform and 4mL of Isoamyl alcohol were separately measured using a measuring
cylinder, poured in a 100mL beaker and mixed gently.

5. Procedure for the preparation of TE buffer (10mM Tris, 1mM EDTA)

Tris (1.21g) and EDTA (0.37g) were separately weighed and dissolved in a small amount of
distilled water, poured in a beaker and distilled water was then added to reach a final volume
of 150 Ml of pH of approximately 8.15.

6. Procedure for preparation of Diphenylamine

1.0g of purified Diphenylamine was dissolved in 100 ml of glacial acetic acid and then 2.75 ml
of concentrated Sulfuric acid was after added to the solution.

Procedure for extraction of DNA from Blood

500 µL of blood sample was transferred from the vacutainer to an eppendorf tube. The plasma
was carefully aspirated by centrifuging at 2664 RCF for 7 minutes at 4°C. 1mL of RLB was
added to the precipitate, mixed gently and allowed to stand at room temperature for 1-2
minutes. The mixture was then centrifuge at 2664 RCF for 6 minutes at room temperature.

Later, the supernatant was discarded. This step was repeated 1-2 times until a white colored
pellet was obtained. 500µL prewarmed DNA extraction buffer was added to the pellet followed
by 30 µL of 10% SDS and mixed gently.

The mixture was then incubated at 56-60°C for 1 hour. 500 µL of Chloroform: Isoamyl alcohol
at a ratio of 24:1 was added to the mixture after incubation and shaken well and then centrifuged
at 10 656 RCF for 12 minutes at 4°C.

The supernatant was pipetted out into another fresh sterilized centrifuge tube containing chilled
ethanol. The tube was well shaken for a while until fine white threads appeared in the solution.
The sample tube was kept at -20°C for 20 minutes without shaking. The sample as then
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centrifuged at 10 656 RCF for 12 minutes at 4°C. The supernatant was then discarded without
disturbing the pellet and added 500 µl of 90% alcohol. The sample was then centrifuged at
10,656 RCF for 12 minutes at 4°C. The above procedure was repeated with 500 µL of 70%
alcohol and centrifuge at the same RCF and time.

The supernatant was discarded and the pellet allowed to dry at 37°C. Later the pellet was
dissolved in 100 µL of TE buffer and store at -20°C for quantification the next day.

Procedure for the Quantitative determination of DNA by Diphenylamine method.

Seven test tubes were thoroughly cleaned and rinsed with distilled water and dried, the test
tubes were then set as shown in the table below and the various reagents added to it.

Tubes 1 2 3 4 5 6 7

DNA std (200 µg/ml) -- 0.2 0.4 0.6 0.8 -- --

Unknown 3 -- -- -- -- -- 0.5 --

Unknown 4 -- -- -- -- -- -- 0.5

5 % TCA (ml) 1.0 0.8 0.6 0.4 0.2 0.5 0.5

Diphenylamine (ml) 2.0 2.0 2.0 2.0 2.0 2.0 2.0

The tubes were then shaken vigorously using the vortex mixer and were put in heat in a boiling
water bath for 10 minutes. The tubes were allowed to cool for some time and after cooling the
tubes were taken for analysis using a spectrophotometer set at a light wavelength of 590nm to
measure the absorbances of the solution in the tubes using Tube 1 as the Blank.

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RESULTS

The table below shows the absorbances of the content of each test tube obtained at 590nm
wavelength using a spectrophotometer.

Test tube number 1 2 3 4 5 6 7

Absorbance 0.000 0.115 0.162 0.217 0.263 0.027 0.025

(590nm)

Calculation of µg DNA in each test tube content.

DNA Std, 200 µg/ml

Tube 2 Tube 4

V2 = 0.2ml V = 0.6ml
0.2∗200 0.6∗200
µg DNA in T2 = µg DNA in T3 =
1 1
= 40 µg DNA = 120 µg DNA

Tube 3 Tube 5

V = 0.4ml V = 0.8ml
0.4∗200 0.8∗200
µg DNA in T3 = µg DNA in T3 =
1 1
= 80 µg DNA = 160 µg DNA

The table below shows the results used to construct the standard curve of absorbance
verses µg DNA using the absorbance data from tube 2 - 5.

Test Tube DNA Std (ml) µg DNA Absorbance (590nm)

2 0.2 40 0.115

3 0.4 80 0.162

4 0.6 120 0.217

5 0.8 160 0.263

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 A Standard Curve of absorbance versus µg DNA from Tube
2-5
y = 0.0018x
0.3 R² = 0.7763
0.25 T5
Absorbances

T4
0.2
0.15 T3
T2
0.1
0.05
0
0 20 40 60 80 100 120 140 160 180
µg DNA

Determination of the concentration of DNA in the sample

From the absorbance of the two unknown samples 3 & 4 of 0.027 and 0.025 respectively.

The regression line equation of the standard curve is y = 0.0018x

Where;

y = absorbance
x = µg DNA

Unknown 3

µg DNA in T5

Absorbance = 0.027

0.27 = 0.0018x
x = 0.027/0.0018µg
x = 15 µg

Concentration in 1ml

Total volume = 3ml

𝟏∗𝟏𝟓
= 𝟑

= 5 µg/ml

Therefore, the Unknown 3 in Tube 6 has 5 µg/ml DNA

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Unknown 4

Absorbance = 0.025

0.025 = 0.0018x
x = 0.025/0.0018 µg
x = 13.89 µg

Concentration of the DNA in 1ml

Total volume = 3ml

𝟏∗𝟏𝟑.𝟖𝟗
= 𝟑
= 4.63µg/ml

Therefore, the Unknown 3 in Tube 6 has 4.63µg/ml DNA

Average concentration of the DNA sample

𝟓+𝟒.𝟔𝟑
= 𝟐
= 4.815 µg/ml

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DISCUSSIONS
The experiment about DNA extraction effectively isolated DNA from the blood sample and
this success was typically based on the high effectiveness of the various components of the
lysis buffer, extraction buffer, 10% Sodium dodecyl Sulfate SDS, Isoamyl alcohol: chloroform
and the 70% and 90% ethanol.

The process involved breaking open the cells (cell lysis) to release the DNA, removing
proteins, lipids, and other contaminants, and then precipitating the DNA out of solution.

All these where achieved based on the following; The RBC Lysis Buffer solution offered the
lytic environment for the preferential lysis of red blood cells from whole blood which are the
most abundant of cells in whole blood, and thus permits the concentration of the nucleated
white blood cells ideal for the isolation of DNA and RNA from blood. This was achieved by
the presence of ammonium chloride which lyses the red blood cells by osmotically rupturing
the cell membrane thus hemoglobin which would otherwise interfere with the DNA extraction
was removed.

Extraction buffer used contained EDTA which removes metal ions potentially present on the
cell surface and thus reduced contamination in the final DNA extract. The CTAB played a
multi-faceted role in the DNA extraction process. It helped break open cells, inactivated
enzymes such as DNAses that protected DNA from degradation, as well as promoted
separation from unwanted components, and allows for the isolation of relatively pure DNA

A mixture of phenol, chloroform, and Isoamyl alcohol was added to tissue samples to promote
the partitioning of lipids and debris into an organic phase, leaving the DNA in the aqueous
phase. Isoamyl alcohol creates a biphasic solution. Denatured proteins partition into the organic
phase while DNA remains in the aqueous phase. This removes contaminating proteins.

Usually, about 70% ethanol solution is used during the DNA washing steps. This allows the
salts to dissolve while minimizing DNA solubility. The 90% ethanol wash which is mainly
employed helps to promote convenient ethanol evaporation from DNA pellet, thus preventing
any carryover.

Additionally, centrifuging the sample is vital since the molecular weight of DNA is lighter than
the other cell material, like proteins and cell walls. By spinning the sample with centrifuge, the
cell material are separated from the DNA, which give in a cleaner DNA sample.

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The TE buffer played a vital role in maintenance of the pH of the solution and solubilizing
DNA while protecting the nucleic acids from enzymatic lysis. TE (Tris-EDTA) buffer is made
up of Tris, a pH buffer and EDTA, a metal chelating ion. It is used in DNA extraction processes
to lyse, wash and dissolve DNA

In the quantitative determination of DNA, the DPA method, also known as the Dische's test or
diphenylamine (DPA) method was employed and was effective in the process since the
diphenylamine reagent effectively depurinated the molecule and the sugar-phosphate backbone
was cleaved thus the 2-deoxy-D-ribose monosaccharides were dehydrated to T-
hydroxylevulinyl aldehydes which condense with diphenylamine, in the presence of the strong,
hot acid, and a blue-colored complex was formed. Since the intensity of this blue color is proportional
to the amount of DNA present in the sample and it absorbs at 595nm (blue-green in color); the
range of this assay is 10-200 µg/ml the DNA in the sample was effectively determined.

A standard curve was constructed using known concentrations of DNA (standard DNA) and
their corresponding absorbance values. The equation of the standard curve was determined to
be y = 0.0018x, where y is absorbance and x is μg DNA.

Two unknown samples were used during the analysis where Unknown 3 gave of 15 μg DNA,
corresponding for 5 µg/ml DNA concentration whereas Unknown 4 with 13.89 μg DNA on the
standard curve corresponds for 4.63µg/ml DNA concentration and on average the sample
contains 4.815µg/ml DNA concentration.

The R2 value of 0.7763 of the standard cure which is approximately 1 indicates a high
correlation value of the absorbances with the concentration of DNA in the samples thus the
corresponding values of the DNA concentration of the unknowns are highly reliable though
improvements can be made to improve the assay in order to obtain an R2 value close to 1.

15
CONCLUSIONS
The DNA extraction method based on SDS cell lysis, precipitation, purification, concentration
and organic extraction using chilled ethanol under specific buffer conditions and several steps
of centrifugation was effective in isolating high quality genomic DNA from the human blood
sample.

The spectrophotometric quantification phenyl amine based determination of DNA using a

DNA standard curve allowed accurate determination of DNA concentrations in the samples.
The DNA concentrations of each unknown 3 and 4 was obtained to be 4.63 μg/ml and 5.0
μg/ml with average of 4.815 µg/ml.

RECOMMENDATIONS
Though the DNA extraction from the blood sample and its quantification was successful, there
exist few things that can be done to achieve high degree of efficiency including regular
calibration and maintenance of the spectrophotometer in order to obtain content and accurate
DNA quantification,

In a bid to obtain highly pure DNA samples, performing RNases digestion is a vital step that
could be important in the removal of most of the residual RNA that can occasionally
contaminate DNA in the process.

Engaging in further purification of DNA by ethanol precipitation, column chromatography or

magnetic beads could remove Polymerase Chain Reaction inhibitors like heme and urea among
others that will help to improve downstream application performance such as DNA
amplification.

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REFERENCES
1. Baechtel, F. S., & Quantico, V. (1989). The extraction, purification and quantification
of DNA. Proceedings of the International Symposium on the Forensic Aspects of DNA
Analysis, 25–28.
2. Barnett, R., & Larson, G. (2012). A phenol–chloroform protocol for extracting DNA
from ancient samples. Ancient DNA: Methods and Protocols, 13–19.
3. Green, M. R., & Sambrook, J. (2018). Isolation and quantification of DNA. Cold Spring
Harbor Protocols, 2018(6), pdb. top093336.
4. Travers, A., & Muskhelishvili, G. (2015). DNA structure and function. The FEBS
Journal, 282(12), 2279–2295.
5. Zhao, Y., Xiang, S., Dai, X., & Yang, K. (2013). A simplified diphenylamine
colorimetric method for growth quantification. Applied Microbiology and
Biotechnology, 97, 5069–5077.

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