MOLECULAR AND CELLULAR BIOLOGY

LAB REPORT SESSION 1- 4

BM2102

DNA BARCODING, AMPLIFICATION & SEQUENCING

Pamela Wijaya
Bio Medicine
19010203

9th February 2021

i3L - Indonesia International Institute of Life Sciences
Jakarta
 Introduction
Various unidentified organisms among the biodiversity are still commonly found. DNA barcoding
can be suggested as one of the methods to identify the unknown eukaryotic species based on the DNA
sequences (Shneer, 2009). In this process, the standard short DNA fragment of the samples would be
compared to the references for the recognition and identification. According to Kress & Erickson (2008),
the DNA fragment that is used as the DNA barcode should have a short sequence length, able to be
developed into universal PCR primer, and conserved within species while diverse between species.
DNA barcoding involves several steps, including DNA isolation, DNA amplification, DNA
sequencing, and end up by matching the samples with the references (Kress & Erickson, 2008). The cell
membranes will be degraded in the DNA isolation step which will only leave the purified DNA (Dewey et
al., 2012). This purified DNA then needs to be copied million times in the DNA amplification step by
using polymerase chain reaction or PCR (Desjardins & Conklin, 2011). In order to know the details of the
PCR products, the DNA fragment should undergo a sequencing process. This process will characterize
the DNA fragment by identifying the exact nucleotides sequences which will also enable the detection of
any variation or mutation on that fragment (Dewey et al., 2012). The results will then be inputted into
the databases to be matched with the references list and the unknown organisms can now be identified.
Quality control after each step is also crucial to avoid any unwanted results at the end of the
processes. In this case, nanodrop and gel electrophoresis can be used. Nanodrop is used to check the
concentration and the purity of the DNA so that only the proper concentration of pure DNA could
undergo the next processes (Desjardins & Conklin, 2011). Meanwhile, gel electrophoresis is used to
check the integrity or quality of the DNA by separating the DNA fragments based on their size (Dewey et
al., 2012). Regarding the theories above, these studies aim to learn about the basic principles and
functions of DNA barcoding steps. These studies are done in several cases for different purposes: (1)
Blood DNA isolation, (2) Identify a murderer based on the PCR result, (3) Identify a peanut butter thief,
and (4) Identify the physical appearance of ancient Greenlandic man. Primer design for DNA
amplification, as well as making the agarose gel for gel electrophoresis are also discussed in these
studies

Materials & Methods

I. DNA Isolation & DNA Barcoding
Safety Protocols
 Personal protective equipment was used during any experiments done in the laboratory. Prior to
all of the experiments, the area was kept sterile and has been decontaminated through the sterilization
procedure. A 10% bleach solution and 70% ethanol were used respectively to clean the bench and all the
equipment used. Any residual bleach was wiped away after 15 to 30 minutes then the 70% ethanol was
sprayed to all the equipment and gloves.
DNA Isolation/Extraction
All the materials and equipment needed were prepared and kept in place. Two microtubes were
labelled with sample IDs (1a and 1b) on the lid and the side of the tube in case the ethanol came in
contact. The blood sample was inverted 10 times in order to obtain a homogenous mixture. Additionally,
A 20 µL of proteinase K was added into 200µL blood in order to form interpeptide cross-links which
enable digestion of any contaminating protein, degradation of nucleases, and protects the DNA samples
(Tan & Yiap, 2009).
Buffers and other solutions were added to the samples, including AL buffer, ethanol, AW1 buffer
AW2 buffer, and AE buffer. A 200 µL of AL buffer was added to each sample in order to promote cell
lysis or degrade the cell membrane of the blood (Ashram, Nasr, Suo, 2016). These samples were then
incubated with the heating block at 56​°C to stimulate the enzyme to work optimally​. Addition of 200 µL
absolute ethanol to the samples was also used to precipitate and isolate the DNA from the organic
phase (Tan & Yiap, 2009). Both AW1 and AW2 buffers (500 µL each) were then added to the samples as
the wash buffer that would bind and wash all the remaining contaminants, leaving only the purified DNA
(Zheng et al., 2013).. The last buffer added was the 200 µL of AE buffer that contained Tris-EDTA which
was used to protect DNA degradation by metal nucleases during storage as well as to elute the DNA and,
hence, DNA became soluble (Ashram, Nasr, Suo, 2016).
In the present study, samples were frequently put into a vortex and centrifugation machine.
Vortex was used to mix the solution via the mechanical energy, while centrifugation was to obtain the
supernatant of the solution mixed (Zheng et al., 2013). Spin columns with the collection tubes were also
used for more sensitive purification. These spin columns would filtrate the samples so only the insoluble
part (DNA) would stay in the spin columns, while the lysate went to the collection tubes (Tan & Yiap,
2009). At the end of the DNA extraction process, dilution steps or samples incubation would need to be
repeated to optimize separation of the lysate, resulting in the purified DNA.
Quality Control
The quantity and quality of DNA yield were checked through nanodrop and gel electrophoresis
technique. Approximately 5 µL of each sample were transferred into two new labelled tubes for the
 yield checking using nanodrop. About 1.5 µL of DNA samples along with AE buffer were used as the
blank instrument in the spectrophotometer as it has been used to dilute the DNA before. The
absorbance ratios used were 260/230 as well as 260/280. Moreover, the samples were also run on an
1% agarose gel for the gel electrophoresis process. Loading buffer or loading dye, as well as the DNA
ladder were added alongside the samples followed by addition of 1X TBE to the gel tank. After the buffer
system has been set up, it was heated up at high voltages about 100 volts for 20 to 30 minutes. This led
to the breakdown of protein bands and loss of resolution. The gel was removed from the gel tank at the
end of run time and was put into a gel doc system or transilluminator in order to visualize the DNA.
II. Polymerase Chain Reaction
Noted that personal protective equipment and sterilization process were done before doing any
experiments in the lab.
Blood samples from the victim and the three suspects, as well as the mysterious blood pool on
the floor of the crime scene were collected and isolated. Each of the samples was transferred into the
PCR tube and was denatured. This process would use helicase with a high temperature to break the
double stranded into single stranded DNA (Garibyan & Avashia, 2014). The exact section of the single
DNA fragment could be identified by adding primers that would attach to the specific site of the
targeted DNA, in which was called as the annealing process. Primers designed were followed these
regulations: the length was around 18-24 base pairs that was started and ended with guanine-cytosine
clamp, the melting temperature was 50-60​°C within 5°C of each other, and did not form a primer dimer
(Lorenz, 2012).
Nucleotides (dNTPs) that were the building blocks of DNA were used to continue adding bases
to the growing DNA strand with the help of cold DNA polymerase that was added to the samples
thereafter (Ghannam & Varacallo, 2020). This process was called the elongation process in which these
strands were joined to the DNA template strand through ligase. DNA polymerase was used in a cold
condition due to room temperature could cause DNA degradation. When all the components needed for
PCR were completed, the PCR tubes were placed into the PCR machine then were run into the gel
electrophoresis. The PCR machine aimed to modify the DNA so the DNA amplification process could
occur, where the cycles were repeated for around 30 cycles (Ghannam & Varacallo, 2020). A DNA ladder
was also put into the first column of the electrophoresis gel wells and at the end of the electrophoresis
step, the result was being analyzed.
III. Gel Electrophoresis
 Noted that personal protective equipment and sterilization process were done before doing any
experiments in the lab.
Agarose gel was prepared for the gel electrophoresis by mixing agarose with 1x sodium borate
buffer that would create alkaline pH to prevent hydrolysis and bind to magnesium ion to prevent DNA
degradation (Sanderson et al., 2015). The mixture was heated then was mixed with ethidium bromide
that would stain and illuminate the DNA under the UV light (Sigmon & Larcom, 1996). These solutions
were poured into the cast carefully with no presence of bubbles. After it was set, the agarose gel was
put into the gel electrophoresis box, facing toward the negative electrode. Running buffer (1x sodium
borate) was then added over the gel electrophoresis box.
DNA samples were collected from the crime scene and two other suspects of the peanut butter
jar thief. These samples have been amplified by PCR before and were centrifuged for 30 seconds. A DNA
ladder was loaded to the first electrophoresis gel well followed by a crime scene DNA sample at the
second then the two other suspects at the third and fourth gel wells. The solution was pipetted and
dispensed only to the first stop as pushing the plunger down to the second stop might displace the
sample from the well. Power supply leads were then connected to the power supply and the lid of the
gel electrophoresis box. Red lead indicated a positive charge, while black lead indicated a negative
charge. Electrophoresis was started with 130 V for 10 minutes and as the time had elapsed, result was
obtained and being analyzed.
IV. DNA Sequencing (NGS)
Noted that personal protective equipment and sterilization process were done before doing any
experiments in the lab.
Hair samples were collected for carbon dating and the result stated that the samples were
obtained from 2,500 to 4,750 years ago of ancient greenlandic man. Bone sample was then isolated for
DNA sequencing. This was to predict the physical appearance of the ancient Greenlandic man. The DNA
fragmentation into smaller pieces was skipped as the sample was ancient DNA in which the DNA had
already partially degraded.
Several steps must be done in the thermoshaker before the DNA sequencing process took place.
It involved sticky ends repairment, generation of A-overhang and T-overhang adapter, as well as ligation
of the adapters. The extracted DNA was mixed with the end-repair mix in order to repair the sticky ends
of the DNA which resulted in the blunt ended DNA (Zhang et al., 2018). The sticky ends repair process
began by attaching the DNA polymerase to complete the missing DNA in 5’ end and used the
exonuclease to digest the 3’ overhangs (Harkins et al., 2020). Generation of A-overhang was done by
 mixing A-overhang mix with the supernatant of the samples. This process would allow the binding of
adenine into the 3’ end of DNA strand with the help of Taq polymerase (Heather & Chain, 2016). The
T-overhang adapter was then obtained by the addition of adapter mix. This would enable the ligase to
join the adapter to the DNA strand (Harkins et al., 2020). After each process was done, magnetic beads
were added to the solution that would use magnetism to separate the particles of purified DNA and the
solution added (Heather & Chain, 2016).
Briefly, PCR master mix contained primers, nucleotides, and Taq polymerase was added to the
purified DNA obtained to undergo DNA amplification in the PCR machine. The DNA amplification process
began with denaturation, followed by annealing, elongation, and the cycle would be repeated for 10-12
times. DNA sequencing by using the NGS machine began after this process. This would create plenty of
DNA clusters in the flow cell, allowing the samples to undergo sequencing. These samples were then run
into the sequencing program and processed several cycles. The short DNA would act as the primer which
DNA polymerase would bind onto it (Zhang et al., 2018). Each nucleotide was then labeled with a
specific fluorophore and photograph was taken to record the fluorescence of the base, followed by
cleavage of the fluorophore (Harkins et al., 2020). Four pictures were taken every single cycle and sent
to the computer to be analyzed

Results & Discussion

I. DNA Barcoding & DNA Isolation
There were four DNA isolation methods, including organic, non-organic, adsorption, and
magnetic separation. Organic method involved phenol-chloroform, while the non-organic method
involved salt-proteinase K. The most common used was the adsorption method which involved silica-gel
membrane, whereas the magnetic separation involved magnetic beads (Gimberg et al., 1989). This study
used a non-organic method to extract the DNA from the blood samples.
The DNA concentration was checked with nanodrop and the result showed that only sample 1a
could be used for the next DNA barcoding step. Pure DNA should yield around 1.8 dsDNA with a 260/280
absorbance ratio and around 1.5-3 dsDNA with a 260/230 absorbance ratio (Desjardins & Conklin, 2010).
All the samples within the 260/280 ratio were in range, but sample 1b showed low concentration within
260/230. Low yield in 260/280 ratios indicated protein contamination, while low yield in 260/230
indicated a high salt contamination (Desjardins & Conklin, 2010). In this case, sample 1b might be
contaminated with the buffer solution
 Ethanol precipitation steps could be repeated to improve DNA yield as it would precipitate the
DNA from the salt solution, such as ammonium acetate (Qamar et al., 2017). Another pitfalls possibility
might also occur, including DNA degraded due to poor storage conditions and inappropriate lysis time
due to the sample being mixed too vigorously when the lysis buffer was added. The samples could be
stored at -20​°C prior to DNA extraction and mixed gently with the lysis buffer to prevent the pitfalls (​Tan
& Yiap, 2009).
The DNA integrity and molecular weight were checked with gel electrophoresis and the result
shown as in the ​picture 1​. All the samples showed bold stains at the top of the wells and did not degrade
into the bottom which indicated that all the samples were at a high molecular weight. Error might occur
if there were too many samples loaded into the well which would form an overloaded separation
(Desjardins & Conklin, 2010). A lot of practices might be needed to load the samples properly.

A 260/280 A 260/230

Control DNA 1.83 2.4

Sample 1a 1.72 1.67

Sample 1b 1.76 1.44

Table 1. Result Obtained from Nanodrop

Picture 1. Result Obtained from Gel Electrophoresis

II. Polymerase Chain Reaction
A DNA amplification process with polymerase chain reaction was used in order to determine the
unknown organisms and in this case were the murderer. This process resulted in multiple copies of a
specific DNA fragment which could be used for visualization in gel electrophoresis, DNA sequencing, and
cloning (Garibyan & Avashia, 2014). Gel electrophoresis result was as shown in the ​picture 2. The fifth
gel stain was similar to the crime scene sample so that the murderer could be identified which was the
rival scientist.
Temperatures during the denaturation, annealing, and elongation process varied as stronger
bonds required higher energy or temperature. A higher temperature was needed for the denaturation
process if the sample used was a genomic DNA with a high guanine-cytosine content and high salt
concentration (Hite et al., 1996). Two types of primers, including forward primer which binded to
antisense strand of DNA and reverse primer that binded to sense strand of DNA were used to optimize
 the annealing process. The annealing process would involve melting and annealing temperature in which
annealing temperature was set to be 3-5​°C lower than the melting temperature ​(Hite et al., 1996)​. On
the other hand, around 70-75°C was known to be the optimum temperature for the elongation process
(Lorenz, 2012).
The PCR result might fail and result in non-specific or even absence of products due to the
setting of temperature also primers. Too low annealing temperature could lead to nonspecific PCR
product, while too high annealing temperature could lead to low or absence of amplification (Lorenz,
2012). Primers designed more than 24 bases with too high guanine-cytosine content needed high
temperature as they were harder to bind. By contrast, primers less than 18 bases with annealing
temperature near the melting temperature led to non-specific binding (Hilte et al., 1996). Nucleotides
sequences should also not be repeated frequently as it could cause a mispriming. Most of the PCR
problems could be solved by using the optimum temperature and primers, as well as using titration of
magnesium ions (Lorenz, 2012).

Picture 2. Gel Electrophoresis Result Obtained to Determine The Murderer

III. Gel Electrophoresis
Another criminal case was investigated in this gel electrophoresis method to identify the peanut
butter thief. The result obtained was visualized by the gel doc system and it was seen that the second
and the fourth gel stains were similar so that the thief of the peanut butter could be identified which
was the suspect 2. The DNA ladder was used to gauge the size of the bands in the sample, while the
crime scene sample helped to identify the thief. Each solution showed different numbers of bands which
indicated that each of them had different molecular weight. Sodium borate buffer was used as it had
low conductivity to resist heat and high voltage (Sanderson et al., 2015). In addition, gel was put facing
the negative pole as DNA was negatively charged and would migrate toward the positive pole ​(Sigmon &
Larcom, 1996).
 Several errors in the result might occur due to several factors. Addition of EtBr to the gel should
lead to final concentration of 0.2-0.5 µg/mL. If the concentration exceeded those ranges, the result
would show unclear bands with a fuzzy background (Sigmon & Larcom, 1996). Extended running time
would also cause the samples to go out of the gel, while insufficient running time could lead to unclear
separation of DNA fragments (Sanderson et al., 2015). In addition, voltage used could not be too low as
it led to fuzzy bands and could not also be too high as it led to U-shaped band results. Appropriate
materials and equipment used, as well as technique to load the samples into the well were needed to
prevent all the pitfalls.

Picture 3. Gel Electrophoresis Result Obtained to Determine The Thief

IV. DNA sequencing
Bone samples obtained from the archaic site were interpreted using the NGS machine that
showed the single nucleotide polymorphisms (SNPs) of the sample. The obtained SNPs were related to a
specific feature (Alderborn et al., 2000) and this study had predicted that ancient Greenlandic man
would have a risk of developing baldness, brown eye color, dry earwax, shovel-shaped teeth, and
non-light skin. Potential pitfalls might be correlated with the implementation, standardization, and
analysis of the NGS result. This might be overcomed by using synthetic microbial community (SMC)
mixes which contain multiple fully characterized microbial species (Boers et al., 2019).
Three types of DNA sequencing methods were identified, including sanger method,
Maxam-Gilbert method, and next generation sequencing. Sanger sequencing method involved ddNTP to
terminate elongation at specific bases irreversibly and the result could be seen from electropherogram
(Dewey et al., 2012). By contrast, the Maxam-Gilbert method involved radioactive chemicals and the
result was taken from the X ray photo (Heather & Chain, 2016). The most commonly used was the NGS,
as used in this study, which involved illumina sequencing. Illumina sequencing included isothermal
bridge amplification in which each spaced end of adapter-ligated DNA consisting of oligonucleotides
were subjected as the substrates for repeated amplification, forming a clonal cluster generation (Slatko
et al., 2018).

Body Part SNPs Result

Hairs Allele of G in SNP Rs6152 Developing baldness

Eyes Allele of C/C on Rs1129038 Brown eye color

Ears Allele of T/T on Rs17822931 Dry earwax

Mouth Allele of C/C on Rs3827760 Shovel-shaped teeth

Skin Allele of G in SNP Rs1426654 Non-light skin

Table 2. Result Obtained from Next Generation Sequencing Machine

Picture 4. Prediction of The Physical Appearance of The Ancient Greenlandic Man

Conclusion
Unidentified organisms can be done by analyzing the blueprint of life which is the DNA. This
process is done through DNA barcoding that involves isolation, amplification, and sequencing of the
DNA. By isolating the purified DNA, it will then be multiplied in vitro, as well as being characterized. The
result will be checked through the nanodrop and gel electrophoresis technique which will show the DNA
concentration and bands of the samples that will be compared to the references and, hence, organisms
can be identified. This study is important for various aspects of life as it can be used for forensic study,
archeological study, and many more. However, some errors might still occur. Therefore, appropriate
materials, equipment, and technique are needed to obtain the expected result.
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