1

CHAPTER I

INTRODUCTION

1.1 INTRODUCTION
The development of bacterial resistance and the rapid spread of these pathogens has led to the
urgent need in utilization and thus development of a faster and efficient method in identification
of these microorganisms (for management of patients). Classical approach of microscopy-
based diagnosis that has been the gold standard in the bacterial species detection of clinical
specimen requires a minimum of 2 days for bacterial phenotypic identification, biochemical
identification and susceptibility testing after a discrete colony has been isolated. (Tang et al.,
1998) This has led to the excessive prescription thus usage of broad spectrum antibiotics by
apprehensive physicians before undertaking the complete diagnosis owing to the fear of
uncontrolled infection as they create a false sense of security. The rampant emergence of multi
drug resistant organisms thus will eventually lead to exhaustion of source of effective antibiotic
for susceptible population may lead to potential death by generally minor infections (Franco
B, Martínez M, Rodríguez M, 2018). Furthermore, in cases where low amounts of clinical
specimens are available, detection and species differentiation of pathogens requires highly
experienced technologist and even so at times due to inadequate sample, are impossible to
perform. In recent years, several advancements have entered clinical microbiology laboratories
enabling rapid results, amongst them are RFLP analysis. Restriction fragment length
polymorphism (RFLP) analysis is a rapid, sensitive, culture independent technique used to
study microbial communities based on variation in the 16S rRNA gene. 16s rRNA genes are
ubiquitous, with highly conserved and variable regions and present in all bacteria. RFLP
analysis consists of five major steps which are DNA isolation and purification, PCR
amplification and purification, restriction enzyme digestion and separation and detection of the
digested fragments via electrophoresis for differentiation and identification. Related molecules
of DNA, such as different versions of the same gene from two related organisms will have very
similar sequences. However, occasional differences in base sequence will result in
 2

corresponding differences in restriction sites. Therefore, two related but different DNA
molecules that are cut with a similar restriction enzyme may produce segments of different
lengths. Consequently, a difference between two DNA sequences that affects a restriction site
is known as a restriction fragment length polymorphism (RFLP) When these are separated on
a gel, bands of different sizes will be produced. RFLPs in useful in analysis of relationships or
discrimination of microorganisms even without knowledge of the true function of the altered
gene. (Clark, 2005)

2.0 OBJECTIVES
2.1 Protocol I (Day 1)
1. To extract bacterial DNA using extraction kit (spin column)
2. To quantify and determine purity of extracted DNA material
3. To perform DNA amplification using Polymerase chain reaction (PCR) techniques
2.2 Protocol II(Day 2)
1. To purify DNA using PCR purification combo kit
2. To perform restriction enzyme digestion.
2.2 Protocol III(Day 3)
1. To prepare agarose gel.
2. To visualize pieces of DNA by size, using a gel matrix and an electrical current.
3. To identify bacterial species based on the fragments produced by restriction enzymes
(BamHI, EcoRI, Hind III and Sma I)
 3

CHAPTER II

METHODOLOGY

3.1.1 PROTOCOL I: BACTERIAL DNA EXTRACTION, QUANTIFICATION OF DNA

AND DETERMINATION OF DNA PURITY
 4

1ml of bacterial suspension samples labelled After being centrifuged at 10,000rpm

S, T and U were collected in microcentrifuge for 1 minute, flow-through. was
tubes. (Maximum 2x10^9 cells). discarded.
 Centrifuge at 10,00rpm for 30 seconds

The supernatant was discarded and

remaining
The pelletwas
supernatant wasdiscarded
collected.and remaining
pellet was collected. The solution with flocculated precipitate were
 200µl buffer RB was added to the transferred into a Spin-column AC
 pellet.
 Spin-column AC was placed on collection
tube

After being vortexed for a few seconds, a

cloudy suspension is obtained.
 suspension was then centrifuged at
10,000rpm for 30 seconds.
The supernatant was discarded.
 200µl buffer RB was added to newly
formed pellet.and vortexed for a few
seconds to resuspend it.
50µl lysozyme (10mg/ml in 10mM Tris-
HCL,pH 8.0) were added
 Tube was overturn and mixed thoroughly
 Incubated at 37ºC water bath for 30
minutes.
After tube was cooled down to RT,
 100μl isopropyl alcohol was added
with vigorous inversions performed to
break up precipitates.
 flocculated precipitates are DNA
20μl Proteinase K (20mg/ml) were then
added and mixed thoroughly.
 After incubating at 70℃ water-bath
for 10 min, a clear lysate mixture was
obtained.
200 µl Buffer CB was then added
 Tube overturned vigorously and
mixed thoroughly

After being centrifuged at 10,000rpm for

2 minutes, supernatant was discarded. 20 µl RNase A (10mg/ml) solution was
 200µl buffer RB was added to newly added
formed pellet and gently pipetted to  Tube was vortexed to mix thoroughly.
resuspend it.  Incubated at 37ºC for 15minutes

Figure 3.1a: Bacterial DNA extraction, quantification and purity determination

 5

500μl buffer IR was then added

 Centrifuged at 12,00rpm for 1 minute
 Flow-through was discarded.
The supernatant was discarded and
remaining pellet
700μl buffer WBwas
werecollected.
then added (ethanol
was previously added to buffer WB)
 Centrifuged at 12,00rpm for 1 minute
 Flow-through was discarded.
Using a NanoDrop™ 2000/2000c
spectrophotometer, following values
were observed and recorded.
 Nucleic acid concentration
 The absorbance A260
 The ratio of A260/A280
The DNA was quantified and its purity
was analysed.
• The ratio of A260/A280

500μl buffer WB were then added

 Centrifuged at 12,00rpm for 1 minute
 Flow-through was discarded. 2 μl each of extracted DNA were subjected
to quantification and purity determination
 The rest of the samples were stored at 2-
8℃ for further use.
Spin-column AC was placed back to
Collection Tube
 Centrifuged at 13,00rpm for 1 minute
to remove ethanol residue in column

Flow through is elution solution containing

Spin-column AC was then placed into
eluted DNA
a clean new centrifuge tube
 50μl buffer EB was then loaded in  The tubes containing DNA obtained were
the middle of the column. labelled S(A), T(B) and U(C)).
 Centrifuged at 12,00rpm for 1
minute

Figure 3.1a: Bacterial DNA extraction, quantification and purity determination

 6

3.1.2 PROTOCOL I: AMPLIFICATION OF 16S RDNA USING PCR

2X PCR Master Mix was thawed at room

temperature and kept on ice after
thawing.
 Vortexed and spin down quickly.
Purified PCR product quantification
was determined using a NanoDrop™
2000/2000c spectrophotometer.

Reaction mix was prepared in nuclease-

 Purity of product was determined
free PCR tube on ice overlaid with 50µl
by the ratio of absorbance at 260
of mineral oil. Reaction volume of 50µl
was prepared as outlined in Table3.1.2. and 280 nm.

Table 3.1.2: Volumes required for reaction mix.

Sample S(A) T(B) U(C)

The PCR products were purified with
Components Volume(µl)
BioTeke Corporation PCR Purification
2X PCR 25 25 25 Kit.
Master Mix
Forward 2 2 2
Primer
(10µM)
Reverse 2 2 2
Primer
(10µM) Samples were placed in automated
thermal cycler and amplified by using the
DNA template 1.5 1.2 2.5 following protocol:
Nuclease-Free 19.5 19.8 18.5
 an initial denaturation step of 95°C
water
for 5 min
Total 50 50 50  34 cycles of denaturation at 95°C for
volume(µl) 1 min
 annealing at 55°C for 1 min
 extension at 72°C for 1.5 min.
 final extension at 72°C for 10 min
The tubes were capped and  Soak cycle is set at 4°C for several
contents were spun briefly. hours.

Figure 3.1.2: Amplification of 16s rDNA using PCR

 7

3.2.1 PROTOCOL II: PCR PURIFICATION.

500μl of buffer DB was added to 50μl of
sample
Spin-column AC was placed into
Collection Tube.
 The mixture was transferred to spin-
column AC.
Incubated for 5min at room
temperature.
 Centrifuged at 12,000rpm for 30-
60sec
 Flow- through was discarded.
50μl buffer EB was added in the middle of
column,
 Incubated for 2 min at room
temperature.
 Centrifuged at 12,000 rpm for 1 min.
To dry, the empty Spin-column AC was
centrifuged at 12,000 rpm for 2min.
 Spin-column AC was transferred to a
clean tube.
700μl buffer WB was added.
 Centrifuged at 12,000rpm for 1min.
 Flow-through was discarded.

 200µlPCR
Figure3.2.1: bufferpurification
RB was added to newly
formed pellet and gently pipetted to
resuspend it.

3.2.2 PROTOCOL II: RESTRICTION ENZYME DIGESTION

PCR product were labelled as S1, S2, T1, T2, U1 and U2.

The PCR product, RE and reagents (Table3.2.2) were pipetted

into individual microcentrifuge tubes and labelled accordingly.
 Briefly centrifuged up to 10,00rpm
 8

Table 3.2.2: Preparation sample mix for restriction enzyme action.

DNA sample Reagents Restriction enzymes - (1µl)
concentration (volume)
BamHI(A) EcoRI(B) HindIII (C) SmaI (D)
DNA 5 µl
S1(41.8ng) Buffer V2: 2.5 µl V3: 2.5 µl V2: 2.5 µl V5: 2.5 µl
H20 16.5 µl
DNA 6 µl
S2 (38.2ng) Buffer V2: 2.5 µl V3: 2.5 µl V2: 2.5 µl V5: 2.5 µl
H20 15.5 µl
DNA 5 µl
T1 (32.7ng) Buffer V2: 2.5 V3: 2.5 V2: 2.5 V5: 2.5
H20 16.5 µl
DNA 5 µl
T2 (35.4ng) Buffer V2: 2.5 µl V3: 2.5 µl V2: 2.5 µl V5: 2.5 µl
H20 16.5 µl
DNA 8 µl
U1 (11.8ng) Buffer V2: 2.5 µl V3: 2.5 µl V2: 2.5 µl V5: 2.5 µl
H20 13.5 µl
DNA 10 µl
U2 (6.9ng) Buffer V2: 2.5 µl V3: 2.5 µl V2: 2.5 µl V5: 2.5 µl
H20 12.5 µl
Total volume in each tube 25 µl

Each tubes were centrifuged briefly up to 10,00rpm

Incubated in thermocycler for 2 hours at 37°C

 followed by 80°C for 20 minutes to deactivate enzymes.

Figure 3.2.2: Restriction enzyme digestion

 9

3.3.1 PROTOCOL III: 1% Gel Preparation And Loading.

The ladder and DNA samples were then

0.5g agarose powder was added to 50ml of
gently expelled into the wells
0.5X TBE buffer in an Erlenmeyer flask.
accordingly.

10µl of samples were mixed with 2 µl DNA

Flask was placed into a microwave and
‘6X Runsafe’ DNA stain
heated.
 DNA ladder was prepared by adding 1
µl of dye and 2 µl deionized water to 3
µl marker

Melted agarose (clear solution) was cooled

down to approximately 40°C The gel was carefully transferred to the
electrophoresis tank.
 In the tank, the gel was then covered
with 0.5% TBE buffer solution.

Agarose solution is poured into gel casting

tray followed by comb insertion.
 Gel was allowed to cool and solidify The comb was carefully removed after gel
for about 45 min (solidified gel turns solidified
opaque)

Figure 3.3.1: 1% Agarose Gel preparation.and DNA loading

 10

3.3.2 PROTOCOL III: GEL ELECTROPHORESIS

Upon completion of DNA loading, the top is Gel is finally disposed in biohazard bag
placed back on electrophoresis chamber. and used TBE buffer in labelled beaker.

The black lead (-) was attached at the end

of the gel nearest the DNA.
The gel was visualized with Omega Fluor™
 The red lead (+) was attached at the Gel Documentation System and
other end. photographed.

Power supply was switched on.

 Voltage was set at 80 volts and gel was
Lid from electrophoresis chamber was
run for 45 min.
removed and gel was placed in container.

Once start button is pressed confirmation

of current flow is done by observation of After completion, power supply was
tiny bubbles rising from both the anode(+) turned off, the colours visible on gel was
and cathode(-). noted.

Figure 3.3.2: Gel electrophoresis.

 11

CHAPTER III

RESULTS AND OBSERVATION

4.1 PROTOCOL I: BACTERIAL DNA EXTRACTION, QUANTIFICATION OF DNA

AND DETERMINATION OF DNA PURITY

Table 4.1: Quantity of DNA obtained and purity of DNA extracted (samples S(A), T(B) and
U(C)).

Absorbance DNA
Samples Concentration(µg/ml)
A260 A280 A260/ A280

S(A) 1.148 0.665 1.73 57.4

T(B) 1.442 0.859 1.68 72.1

U(C) 0.682 0.466 1.46 34.1

 12

4.2 PROTOCOL I: AMPLIFICATION OF 16S RDNA USING PCR

Table 4.2.1: Results of PCR product (amplicon) quantification. Selected PCR product obtained
by other groups were included in this table. Sample T(B) of our group was later selected for
restriction enzyme action and re-labelled as T1.

Absorbance Nucleic acid

Samples concentration(µg/ml)
A260 A280 A260/ A280

S(A) 0.627 0.323 1.94 31.3

T(B) 0.654 0.341 1.92 32.7

U(C) 0.340 0.154 2.20 17.0

S1 0.837 0.459 1.82 41.8

S2 0.764 0.406 1.88 38.2

T2 0.709 0.388 1.83 35.4

U1 0.235 0.111 2.12 11.8

U2 0.138 0.060 2.31 6.9

 13

4.3 PROTOCOL III: GEL ELECTROPHORESIS

Gel 1

Gel 2

Figure 4.3: RFLP analysis of 16s rDNA of three bacteria species (S, T, U) corresponding to
digestion by BamHI (A), EcoRI (B), Hind III (C) and Sma I (D) respectively in Gel 1 and Gel
2. E denotes empty well. 1 kb ladder (L) were placed in well 1 of the first gel and well 5 of gel
2. Arrows indicate raised regions of agarose attributed to poor handling of agarose during
preparation, transportation, up to viewing. Due to that, sizes of fragments cannot be determined
concretely and an approximation is made when comparing to reference picture (Kodachrome)
for bacteria identification. Sample S, T and U were identified as, Escherichia coli (D),
Salmonella typhi (A) and Streptococcus pneumonia (C) respectively.
 14

4.4 PROTOCOL III: REFERENCE PICTURE.

Figure 4.4: Kodachrome (reference picture) of known bacterial species. First alphabet
represents bacteria: Salmonella typhi (A), Staphylococcus aureus (B), Streptococcus
pneumonia (C), Escherichia coli (D) while the second alphabet represents restriction enzymes
utilized: EcoRI(E), BamHI(B), HindIII(H), SmaI (S).
 15

CHAPTER IV

DISCUSSION

5.1 PROTOCOL I: BACTERIAL DNA EXTRACTION, QUANTIFICATION OF DNA

AND DETERMINATION OF DNA PURITY
The bacterial DNA extraction kit produced by BioTeke Corporation were chosen as the
protocol is relatively simple and can be completed within 30 minutes. As mentioned in the kit’s
insert, the kit consists of several reagents which carries out their own specific functions and
interacts in such a way that the end product of high yield purified DNA obtained can be applied
for PCR directly. According to Miller, Bryant, Madsen, Ghiorse, & Al, 1999, the critical
variables in DNA extraction are the purity of the DNA, its molecular weight, its concentration,
and the speed of pellet formation. In the beginning of the protocol, lysozyme was added to
break down the bacterial cell wall integrity by cleaving the linkage between N‐
acetylglucosamine (NAG) and N‐acetylmuramylpentapeptide (NAM pentapeptide) encasing
the bacteria (Primo, Otero, Ruiz, Klinke, & Giordano, 2018). Subsequent addition of RNase
removes RNA from the sample. Proteinase K added in the next step performed several
functions, the first being bacterial DNA liberation through bacterial lysis, prevention of
bacterial DNA autolysis by inactivating cellular nucleases and performing protein digestion
(Shahriar, Haque, Kabir, Dewan, & Bhuyian, 2011). Furthermore, when subjected to high salt
conditions, Proteinase K enables DNA to selectively adsorb to the silica membrane of the spin-
column. Subsequent incubation in temperature of 70ºC helps to denature proteins and thus
eventual autodigestion of Proteinase K. The multiple washing and centrifugation steps in the
protocol were performed to remove cellular metabolites, proteins and other contaminants and
enabled the attainment of high-purified DNA. As for isopropyl alcohol, its addition is required
for precipitation of nucleic acids. Isopropyl alcohol that is not mixed thoroughly to break up
large precipitates may clog column and decrease DNA yield. Low salt solution (buffer EB) is
used to elute the purified genomic DNA from the silica membrane(Biase, Franco, Goulart, &
Antunes, 2002). Throughout the protocol of DNA extraction, pellet formation was formed at a
timely manner. The quantity and quality of the extracted DNA were determined by
spectrophotometry. 2μl of the extracted DNA material was quantified by measuring the optical
density (OD) at 260 nm using NanoDrop™ 2000/2000c Spectrophotometer. The more
 16

concentrated the DNA solution, the more UV light at 260 nm will be absorbed by the DNA
samples thus a high value indicating high concentration of DNA material. Besides directly
extracting DNA concentration values off the data sheet produced by NanoDrop™
spectrophotometer, the concentration of nucleic acids of the samples can be calculated by
multiplying the absorbance A260 with the dilution factor and using the relationship that an
A260 of 1.0 = 50µg/ml pure dsDNA. The formula are as follows:
[dsDNA]µg/mL= A260 x dilution factor x 50
A260 =the absorbance, in optical densities, at 260 nm (OD260), while 50 = The standard
extinction coefficient multiplier for dsDNA (50 μg/OD260) (Robert E. Farrell, 2017). Purity
of DNA materials were determined by the ratio of A260/A280 whereby the absorbance of the
sample at 280 demonstrates proteins absorbing light at 280 nm through tryptophane residues
and at A260 indicates nucleic acids absorbing light at 260 nm through adenine residues
respectively. For DNA samples, a ratio within the range of 1.8-2.0 is considered as pure.
Values above 2.0 indicates the presence of RNA contaminants whereas lower ranges indicate
protein contaminants. A reading of 1.6 however, can still be utilized for analysis although lower
ratios indicates that more contaminants are present. (Khare, Raj, Chandra, & Agarwal, 2014).
As observed from Table 4.1, the purity of DNA acquired represented by the ratio of A260
/A280 for samples S(a), T(b)and U(c) were 1.73, 1.68 and 1.46 respectively. As the values fall
below 1.8, this indicated the contamination of extracted DNA by protein elements. The higher
the A260 values, the higher the DNA concentration as indicated in Table 4.1

5.2 PROTOCOL I: AMPLIFICATION OF 16S RDNA USING PCR

All the DNA samples were subjected to PCR in automated DNA thermocycler. To obtain an
optimal yield in PCR, concentration of the DNA material was determined to be within ranges
of 10-100ng as per recommendation in the PCR protocol. Since µg/ml is equivalent to ng/µl,
the volume of DNA samples that were added to the reaction volumes can be determined on the
basis that 1OD = 50µg/ml (concentration of DNA) (Robert E. Farrell, 2017). The volumes of
DNA template to be used in the reaction mix was calculated and tabulated (Table 3.1.2). The
sequences of forward(16SF) and reverse (16SR) primers are 5'- GAG TTT GAT CCT GGC
TCA G -3' and 5'- CGG CTA CCT TGT TAC GAC TT -3' which amplifies a 1,500-bp product
of the 16S rDNA. Each 50μl reaction mixture contained 1X PCR Master mix, both primers at
10 µM each, and template DNA. PCR Master mix contains 0.06U/µl of Taq DNA Polymerase,
3mM MgCl2, reaction buffer and 400µM of each dNTPs (deoxynucleoside triphosphate).
 17

Buffer maintains constant pH while MgCl2, as a necessary co-factor for enzyme activity. Taq,
a heat tolerant enzyme obtained from the hyperthermophilic organisms Thermus aquaticus
functions to attach nucleotides to DNA template. PCR primers are short fragments of single
stranded DNA (15-30 nucleotides in length) that are complementary to DNA sequences that
flank the target region of interest. PCR primers provide a “free” 3'-OH group to which the DNA
polymerase can add dNTPs. Based on the results, most of the samples selected obtained
sufficient amount of amplicon and with improved purity except sample U2 whereby
concentration is low. PCR, a rapid, automated technique used for the amplification of specific
DNA sequences enables valuable genetic information about the microorganisms to be obtained
quickly. All the DNA samples were subjected to PCR in an automated DNA thermocycler.
Amplification occurs as following diagram. the first target sequences were obtained in the 3rd
cycle of PCR. End product of PCR reaction is 1.5kb (based on primer used, known during
selection of primer). Post PCR, the purity of DNA samples increases to the optimal ranges of
1.8 – 2.0 was obtained. Sample U(C) however was possibly contaminated with RNAs as its
A260/ A280 exceeds 2.

Figure 5.2: PCR product formation.

 18

5.3 PROTOCOL II: RESTRICTION ENZYME (RE) DIGESTION

PCR product with sufficient concentration of DNA and best purity were selected for restriction
enzyme digestion and labelled as S1, S2, T1, T2, U1 and U2. The samples taken from our group
was T2, however it was not selected for gel electrophoresis interpretation due to damaged gel.
During RE digestion, Nucleic acid concentration used were maintained within 100-200ng as
suggested in the PCR protocol for optimal reaction. Total volume used for the assay buffer was
25 µl instead of recommended 50µl to accelerate the reaction. Restriction enzymes must be
placed in an ice bucket immediately after removal from the -20 °C freezer as heat can cause
the enzymes to denature and lose their function. Restriction Enzyme(s) are nucleases that
recognize specific nucleotide sequences (4-6 nucleotides long) and with the ability to cleave
DNA (cleavage of phosphodiester bond) to produce cohesive or blunt ends. The specific
sequence detected by RE are ‘restriction sites’. The following table (5.3)depicts the four
restriction enzymes with 6-bp recognition site utilized in this protocol (BamHI, EcoRI, HindIII,
SmaI) for RFLP.

Table 5.3: Restriction enzymes.

Restriction Enzyme(s) Region Optimal reaction buffer

BamHI(3’overhang cut) 5 ' . . . G↓G AT C C . . . 3 ' V2

3 ' . . . C C TA G↑G . . . 5 '
EcoRI (3’overhang cut) 5'…G↓AATTC…3' V3
3'…CTTAA↑G…5'
HindIII(3’overhang cut) 5 ' . . . A↓A G C T T. . . 3 ' V2
3 ' . . . T T C G A↑A . . . 5 '
SmaI (blunt end) 5 ' . . . C C C↓G G G . . . 3 ' V5
3 ' . . . G G G↑C C C . . . 5 '
 19

5.4 PROTOCOL III: 1% AGAROSE GEL PREPARATION

During gel preparation, ethidium bromide (EtBr) was not the chosen dye due to its
mutagenicity, carcinogenicity and teratogenicity potentials. EtBr is readily absorbed through
the skin, producing fumes that can irritate the eyes, mouth, and respiratory tract. Even though
it is cheaper compared to SYBR®-based dyes for example, its costly decontaminated protocols
and waste management are burdensome leading to opting for a substitute dye (Saeidnia &
Abdollahi, 2013). runSAFE, a non-hazardous alternative to Ethidium Bromide was instead
selected as this fluorescent reagent is currently the most sensitive in detecting double-stranded
DNA (dsDNA) under Blue Light or UV. Supplied in 6X DNA Loading Buffer, it is used to
prepare DNA markers and samples for loading on agarose or polyacrylamide gels thus
performing dual action whereby it acts as the stain and loading dye. Based on manufacturer
insert, runSAFE contains three tracking dyes which are Bromophenol Blue, Xylene Cyanol FF,
and Orange G for visual tracking of DNA migration thus, electrophoresis progression can be
easily monitored. Since Orange G (50bp) runs fastest followed by Bromphenol Blue(400bp)
and Xylene Cyanol FF (4kb), it serves as an indicator to stop and prevents over running of gel.
The colour transferred by runsafe to colourless DNA samples also acts as an added benefit and
ease sample loading into wells and detection of leakage between wells. Loading dye contributes
to the formation of sharp bands as they increase density of samples and cause them to settle at
the bottom of the wells instead of dispersing in the running buffer (Lee, Costumbrado, Hsu, &
Kim, 2012).

In electrophoresis, selection of high-quality buffers is vital as they allow current to be carried

through the sample while resisting pH changes in the overall solution. DNA electrophoresis
commonly utilizes buffers like TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). These
buffers facilitate the separation of the samples into readable gels. During gel electrophoresis,
TBE buffer was selected to be used in our protocol. For TBE preparation: 0.5M EDTA, pH 8.0
by adding 186.1 g of Na2EDTA-2H2O in 700 ml of dH20, adjusting pH to 8.0 (EDTA will not
dissolve otherwise) by adding 10 M NaOH (approximately 50 ml), and topped up to 1 litre
using dH20(Laboratorio de Genomica Viral y Humana, 2017). TBE which is active under basic
conditions is able to protect DNA by keeping it deprotonated and water soluble. In addition to
producing well-defined bands on the gel, due to its greater buffering ability, TBE is able to
withstand longer duration of high-voltage electrophoresis without overheating unlike TAE
(Miura, Wake, & Kato, 1999). The undesirable aspect of TBE is that not recommended for
preparative gels in recovery of nucleic acids attributed to the inability of TBE to stabilize RNA.
 20

TBE buffer is also prone to precipitation over time, however this will not gravely affect its
performance (Buchmueller & Weeks, 2004).

5.5 PROTOCOL III: VISUALIZING THE GEL

The use of restriction endonucleases allows DNA to be cleaved into sequence-specific
fragments. Agarose gel electrophoresis was then performed to view DNA fragments generated
by restriction endonuclease digestion sorted on the basis of size. In the electrophoresis
chamber, the negative terminal (black wire) is located close to the wells and DNA travels in
the direction of positively charged anode (red wire). (Sambrook et al., 1989). After the run was
completed, The agarose gel was visualized under UV light. The ladders in both gels (figure
5.1) as expected, exhibit slightly more intense luminesce at sizes 3000bp and 1000bp
respectively. However due to the raised and uneven condition of the gel as indicated by arrows
in Figure 5.1, the sizes of fragments cannot be determined concretely as sizes of fragments
obtained throughout the wells vary from its own DNA ladder. This is evident when comparing
gel1 and gel 2 with reference picture (Figure 5.2) whereby the largest fragment obtained by
bacterial samples should be about 1500bp, instead all of the samples portrayed sizes of
>1500bp with some up to 2000bp. Therefore, correct sizing of the fragments could not be
performed and an approximation is made when comparing to reference picture (Figure 5.2) for
bacteria identification. Furthermore, since we were informed that three out of the four species
in reference picture is bacteria S, T, or U, estimates were possible in identification of the
potential bacterial species. On the basis of band clarity, separation and least distortion, samples
S1, T2 and U1 (Figure5.1) were selected for comparison with given known reference picture
(Figure 5.2).

In the restriction enzyme(RE) digestion protocol, 4 different RE were utilized. This was done
to increase information for each sample as the probability that two or three different species
analysed (S,T,U) giving rise to an exact restriction site in the 16S rDNA resulting in identical
bands exist (Moeseneder, Arrieta, Muyzer, Winter, & Herndl, 1999). This scenario can be
observed at digestion of Salmonella typhi & Escherichia coli with SmaI and all bacteria
samples with EcoRI. Sample S, T and U were identified as, Escherichia coli (D), Salmonella
typhi (A) and Streptococcus pneumonia (C) respectively.
 21

CHAPTER V

CONCLUSION

6.1 CONCLUSION
As the speed of microbial identification greatly affects proper management of infectious
disease patients by clinicians, development and practice of novel methods for bacterial
identification are vital in preventing emergence of multiresistant bacteria. Accelerated
phenotypic methods in tandem with molecular techniques such as RFLP are necessary as they
offer great promise in increased diagnostic resolution and decreased time-to-result (Mateo et
al., 2005). Restriction fragment length polymorphism (RFLP). methods in identification of the
three bacteria species in this experiment was fruitful due to the simple and effective DNA
extraction kits, appropriate selection of primers based on the conserved sequences of the 16S
rDNA gene and restriction endonuclease digestion of the polymerase chain reaction (PCR)
product. In conclusion, RFLP of 16s rDNA is indeed a valuable tool for analysis of the bacteria
species as small volumes of clinical specimens can be used directly with rapid results delivered
compared to conventional methods.
 22

REFERENCES

Buchmueller, K. L., & Weeks, K. M. (2004). Tris-borate is a poor counterion for RNA: a
cautionary tale for RNA folding studies. Nucleic Acids Research, 32(22), 1–10.
https://doi.org/10.1093/nar/gnh182

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