BIOCHEMICAL SYSTEMATIC BY USING DNA FINGERPRINT

DATA

Name : Pratiwi Kusuma Kurniawati

NIM : B1B017007
Entourage : II
Group :2
Assistant : Abdi Rahman Nursyamsi

MICROBIAL SYSTEMATICS LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2019
 I. INTRODUCTION

The science of chemotaxonomy or chemical taxonomy is used for the

classification of plants on the basis of their chemical constituents. All the living
organisms produce secondary metabolites that are derived from primary metabolites.
The chemical structure of the secondary metabolites and their biosynthetic pathways
is often specific and restricted to taxonomically related organisms and hence useful
in classification.The concept of chemotaxonomy has been elaborated in the past
century. The rise of chemotaxonomy is mainly due to the advancement in analytical
techniques for chemical analysis that can detect even trace amount of chemical
compounds. The phenolics, alkaloids, terpenoids and non-protein amino acids, are
the four important and widely exploited groups of compounds utilized for
chemotaxonomic classification (Singh, 2016).
Chemotaxonomy as the method of biological classification based on similarities
in the structure of certain compounds among the organisms. Chemotaxonomy has
been used in all the groups of the kingdom. Chemical characters are particularly of
high taxonomic value when they are stable, unambiguous and not easily, if at all,
changeable (Chen et al., 2015). The phenolics, alkaloids, terpenoids and non-protein
amino acids, are the four important and widely exploited groups of compounds
utilized for chemotaxonomic classification. These groups of compounds exhibit a
wide variation in chemical diversity, distribution and function. The system of
chemotaxonomic classification relies on the chemical similarity of taxon. Three
broad categories of compounds are used in chemotaxonomy: primary metabolites;
secondary metabolites and semantics (Shultz et al., 2004).
DNA testing is increasingly used to analyze biological materials because
DNA has several advantages, including specific DNA (every living thing has a
unique DNA sequence), is relatively stable, can be propagated in vitro. DNA
fingerprinting methods can be applied to all living things, both prokaryotic and
eukaryotic (Madigan et al., 2009). DNA fingerprinting is a major tool in identifying
individuals and in evidence matching. However, this technique can be difficult to
reproduce in practical classes (Henriques & De Marco, 2018). DNA fingerprint
analysis can be done in various ways, one of them using the PCR method. DNA
fingerprinting by PCR method is easier to do because it does not require restriction
enzymes, does not require hybridization with certain tracers, does not require the
transfer of DNA fragments from the gel to the membrane, and can be done in small
amounts of DNA samples (Yuwono, 2006).
A technique for comparing DNA patterns that allow the genomic relatedness
among organisms and classify them is called DNA fingerprinting. There are multiple
DNA fingerprinting techniques. The choice of which of it is depend on their
applications, it could be for medical diagnosis, forensic science, parentage testing,
food industry, agriculture, and many others. The interpretation of banding patterns by
visual observation is a time consuming and arduous task, especially when comparing
distant, different and multiple patterns, and it can be highly dependent on the
researcher. There are several commercial and freely available software tools that can
help to simplify this task and to eliminate the possible suggestibility derived of the
human eye (Heras et al., 2015). Chemical microbial classification using data
fingerprinting has advantages and disadvantages. The advantage of microbial
classification using fingerprinting is that it has a higher level of accuracy to see the
level of fading of several bacterial isolates tested, the process is simple, practical,
efficient and applicable. While the drawback is that it requires a more expensive tool,
and needs more accuracy and understanding of fingerprinting procedures (Wulansari
et al., 2015).
The Rep-PCR technique is a technique for producing bacterial genome
fingerprints, which can be used to differentiate microbial diversity based on the
number and size of bacterial repetitive sequences. This technique is often used so as
not to examine bacterial isolates that turn out to be based on their genetic fingerprints
(Rademaker & Bruijn, 2005). Application of DNA fingerprints with rep-PCR can be
used to distinguish bacterial isolates into species, subspecies, and strains. This
method can also be applied to various types of microorganisms from Gram-negative
and Gram-positive bacteria, even in eukaryotic microorganisms (Versalovic &
Lupski, 1996). One drawback of the DNA fingerprint taxonomy method is that DNA
sequence determination usually takes a long time, because sample preparation must
be pure and there is no availability of DNA sequencing equipment in a simple
laboratory (Shabarni-Gaffar et al., 2014).
Applications of the DNA fingerprinting method include Paintshop Pro, PFE
(Programmer File Editor), MVSP, and Microsoft Excel. Paintshop Pro works to see
representative bands of DNA fingerprinting obtained from scientific publication
journals. PFE (Programmer File Editor) functions to organize DNA fingerprinting
data and convert it in the form of number 1 for positive results and 0 for negative
results. MVSP functions to construct dendrogram which reflects OTU classification
based on similarity index (SSM) and (SJ) values with UPGMA algorithm. Microsoft
Excel functions as a data analysis of the similarity index (SSM) and (SJ) values
(Sembiring, 2003).
The objective of this laboratory activity is students can find out the chemical
taxonomic procedure based on DNA fingerprint data or total cellular protein.
 II. MATERIALS AND METHODS

A. Material

The tools used in this practicum are laptop with several application such Paint
Shop Pro, PFE, MVSP, and Ms. Excel.
The materials used in the practical class is scientific publication journals that
contain DNA fingerprinting band data.
B. Method

The method used in this laboratory activity are:

1. Data Collection
Each practitioner is expected to determine their own microbial group that
will be used in this practicum. DNA fingerprint or protein data from microbial
groups can be obtained from scientific journal publications which can be
downloaded from internet sites.
2. Data Processing Band Fingerprinting
Fingerprinting images from a scientific publication are stored in directories
and named. The Paint Shop Pro program is opened then click file > New, click
OK. A new page appears. Click File > Open. The fingerprint image file is
searched in the directory where the file is stored. After the fingerprint opens, the
image is used as "Background". Create a layer above Background by clicking
Layer > New > OK. After the screen opens then the bands in the background
(fingerprint) can be made a representative image on the layer above by clicking
the "Shape" icon on the left toolbar so that the band can be represented as empty
boxes. The band representation box is colored by clicking the "Flood Fill” icon
on the left toolbar then clicking on each band so that all bands are clearly
colored. To display the results of a representative diagram, the background must
be deleted by: click Layer > Background so that the active page is the
background. Then click Layer > Delete. Then the background will be erased so
that it displays a representative diagram. The fingerprint data representation file
is stored and given a file name.
3. Transformation/Interpretation of Fingerprinting Data into Numerical Data
 The fingerprint data representation file is printed out. Each band formed is a
character that is owned by the strain. Test strains that have the same band and are
in a parallel position are coded (+), whereas strains that do not have a band in the
horizontal position are then given a code (-) on the number of the characters
being compared. The character data is then included in Table n x t in the Excel
program.
4. Data Entry into n x t Table
Genotypic character data that has been given a score (+) or (-) is entered into
the computer using the Excel program. The data is then copied into the PFE
program (Programmer Editor File), then the data (+) is converted to 1 and data (-)
is converted to 0. The data is then processed in the MVSP program to construct
the dendrogram that reflects the OTU classification based on the similarity index
value (SSM ) and (SJ) with the UPGMA algorithm.
5. Classification Presentation Results
The dendrogram generated by the cluster analysis in the MVSP program is
then converted from the file.plg format to the file format. Hgl, this is so that it
can be opened with the PAINTSHOP PRO program for editing dendrogram. The
edited program is then inserted into the document file in the WORDS program
(text file). The MVSP program is opened. Select Cluster Analysis, choose
Jaccard Coeficient or Simple Matching Coeficient, on the advance tab, check
everything, then click OK. Select file name pattern: * .mvd (Enter). Select M
(Clustring method: UPGMA Default). Select R (Run Analysis). Enter the output
file name: * .OT2 (matrix similarity and clustring steps) (Enter). Enter the tree
description file name: * .plg (Enter). (Cluster Analysis) > Finish > Press any key.
Quit MVSP.
6. Determination of Taxonomic Structures
Determination of the taxonomic structure described by dendrogram refers to
the standard rule that is defining fena with a level of similarity of correlation
coefficient is > 70%.
 III. RESULT AND DISCUSSION

Figure 3.1 The DNA Fingerprinting pattern of

Bachillus cereus Group Strain
DNA fingerprinting is a technique for comparing DNA patterns that allows
the analysis of genome links between different samples, as well as for typing and
classifying them. There are several DNA fingerprinting techniques, and which choice
we should use depends on the application (medical diagnosis, forensic science,
heredity testing, food industry, agriculture, and many others) (Heras et al., 2015).
Electrophoresis is a technique of separating and purifying DNA, RNA, or protein
fragments. The basic principle of electrophoresis is to separate molecules based on
intrinsic electrical charge. DNA electrophoresis is usually used to separate DNA
based on differences in size. DNA separation in this case is using agarose gel.
Agarose is a polysaccharide extracted from seaweed. The pore size of agarose is
suitable for the separation of nucleic acid polymers composed of hundreds of
nucleotides (Langga et al., 2012).
 Figure 3.2 The Representation Data from Electrophoresis Result

The electrophoresis data that already got is next modified by using PaintShop
Pro to make the bands are easier to read or to analyze. The modification is done by
tracing on the bands. The extra lines are also drawn to classify bands that have the
same character (Davey, 2004).
Tabel 3.1 Characteristics Table of DNA Fingerptinting Result
  A B C D E F G
1 + - - - - - -
2 - - - - - + -
3 - + - + - - -
4 - - - + - - -
5 - - - - - + -
6 + - - - - + -
7 + - + - - - -
8 + + + + - - +
9 - + + - - - -
10 + + + + - - +
11 + + - + - - +
12 + - + + + + -
13 + - - + + - +
14 + + + + + - +
15 - - + - - - +
16 - - - + + + +
17 - - - - - + -
18 - - - - - + -
19 - - - + - - -
20 + + - - - + +
21 + + - - - + -
22 - - - + + - -
The data obtained in the form of characters are presented in table n x t. The sign
(+) in the table indicates the nature / character of the strain being tried and the sign
(-) states the absence of these properties which will be processed to get a similarity
index value by several methods of calculation approach. If there are two microbial
strains that have a similarity index > 70%, then the two microbial strains can be said
to be one species (Johnson et al., 1975).

Tabel 3.2 Result of Similarity Matrix Using Simple Matching Coefficient

A B C D E F G
A 1.000
B 0,682 1.000
 C 0,636 0,682 1.000
D 0,545 0,591 0,545 1.000
E 0,545 0,5 0,636 0,727 1.000
F 0,455 0,409 0,364 0,273 0,545 1.000
G 0,682 0,727 0,682 0,682 0,682 0,409 1.000
The table shows the correlation among strains that analyzed. The highest
value is 0.727 and the lowest is 0.273. The data that available here is needed to
construct the corelation of coefficent table. The data here is needed for the unsorted
column (Archer & Marble, 1987).

Tabel 3.3 Result of Similarity Matrix of Nodes Using Simple Matching

Coefficient

Simil
Node Group 1 Group 2 in group
.
1 B G 0,727 2
2 D E 0,727 2
3 A Node 1 0,682 3
4 Node 3 C 0,667 4
5 Node 4 Node 2 0,591 6
6 Node 5 F 0,409 7

The table shows the correlation among nodes that analyzed. The highest value
is 0.727 and the lowest is 0.409. The data that available here is needed to construct
the corelation of coefficent table. The data here is needed for the sorted column
(Archer & Marble, 1987).

Table 3.4 Result of Coefficient Analysis (Simple Matching Coefficient)

Unsorte
Node Member Sorted d Coeff
1 BG 0,727 0,727 75,09161
2 DE 0,727 0,727
3 AB 0,682 0,682
AG 0,682 0,682
4 AC 0,667 0,636
BC 0,667 0,682
GC 0,667 0,682
5 AD 0,591 0,545
BD 0,591 0,591
GD 0,591 0,682
CD 0,591 0,545
AE 0,591 0,545
BE 0,591 0,5
GE 0,591 0,682
CE 0,591 0,636
 6 AF 0,409 0,455
BF 0,409 0,409
GF 0,409 0,682
CF 0,409 0,364
EF 0,409 0,545
DF 0,409 0,273
The correlation coefficient between the similarity of unsorted and sorted
similarity values obtained result is the correlation coefficient in the Jaccard’s
Coefficient with average linkage algorithm / UPGMA is 75,09161. This suggests that
the similarity distortions that occur after using average linkage algorithm / UPGMA
still statistically acceptable, because the value of the correlation coefficient obtained
indicates the number is greater than 70 (Yulianti & Rakhmawati, 2017).

UPGMA
F
E
D
C
G
B
A
0,4 0,5 0,6 0,7 0,8 0,9 1
‘
Simple Matching Coefficient

Figure 3.3 Dendogram of Simple Matching Coefficient

Based on dendogram result using Simple Matching Coefficient, the distance of
the node approaching 100% is found in node 1 (BG) and node 2 (DE), which is 72%.
Whereas the furthest relationship is found in node 6 (AF, BF, CF, DF, EF, GF)
which is equal to 40%. From the data matrix n x t, after clustering with MVSP, then
the obtained similiarity value is drawn in the dendogram. Based on the result by
using Simple Matching Coefficient, the result of coefficient is 75.09161%. The node
table consists of 6 nodes. The results of this method also show that 7 strains has a
similar resemblance to the others and associated. Accroding to Priest & Austin
(1999), that the dendogram obtained, the dendogram was evaluated and the
evaluation results were included in the dendogram similarity matrix. Then the
calculation of the cophenetic correlation between the initial similarity matrix and the
similarity matrix results from the evaluation of the dendogram. From this cophenetic
correlation obtained correlation coefficients. The value of the correlation coefficient
is accepted if it is at the level > 60%. If the result of the correlation coefficient
calculation (r) is more than or equal to 60%, it means that the classification carried
out can be trusted and accounted for. The closer the distance of node to the number 1
on the dendogram indicates the more similar.

Tabel 3.5 Result of Similarity Matrix Using Jaccard’s Coefficient

A B C D E F G
A 1
B 0,462 1
C 0,385 0,364 1
D 0,375 0,357 0,286 1
E 0,231 0,083 0,2 0,455 1
F 0,25 0,133 0,067 0,111 0,167 1
G 0,462 0,455 0,364 0,462 0,3 0,133 1
The table shows the correlation among strains that analyzed. The highest
value is 0.462 and the lowest is 0.111. The data that available here is needed to
construct the corelation of coefficent table. The data here is needed for the unsorted
column (Archer & Marble, 1987).

Tabel 3.6 Result of Similarity Matrix of Nodes Using Jaccard’s Coefficient

Simil
Node Group 1 Group 2 in group
.
1 A B 0,462 2
2 D G 0,462 2
3 Node 1 Node 2 0,412 4
4 Node 3 C 0,349 5
5 Node 4 E 0,254 6
6 Node 5 F 0,144 7
The table shows the correlation among nodes that analyzed. The highest value
is 0.462 and the lowest is 0.144. The data that available here is needed to construct
the corelation of coefficent table. The data here is needed for the sorted column
(Archer & Marble, 1987).

Table 3.7 Result of Coefficient Analysis (Jaccard’s Coefficient)

Unsorte
Node Member Sorted d Coeff
1 AB 0,462 0,462 84,37353
2 DG 0,462 0,462
3 AD 0,412 0,375
BD 0,412 0,357
AG 0,412 0,462
BG 0,412 0,455
 4 AC 0,349 0,385
BC 0,349 0,364
DC 0,349 0,286
GC 0,349 0,364
5 AE 0,254 0,231
BE 0,254 0,083
DE 0,254 0,455
GE 0,254 0,3
CE 0,254 0,2
6 AF 0,144 0,25
BF 0,144 0,133
DF 0,144 0,111
GF 0,144 0,133
CF 0,144 0,067
Based on calculation of similarities using Jaccard’s Coefficient, coefficient result
is amount 84,4% these results indicate that the calculation of similarities data is
acceptable or valid. This is accordance with Ertekin & Con (2011), that similarity
coefficient is used to control whether the data are qualified enough to use in
hierarchy clustering at numerical taxonomy. Similarity values between 60%-95% are
good indicator of success of taxonomic classification.
UPGMA
F
E
C
G
D
B
A
0,04 0,2 0,36 0,52 0,68 0,84 1

Jaccard's Coefficient

Figure 3.4 Dendogram of Jaccard’s Coefficient

Based on dendogram result using Simple Matching Coefficient, the distance of
the node approaching 100% is found in node 1 (BG) and node 2 (DE), which is 46%.
Whereas the furthest relationship is found in node 6 (AF, BF, CF, DF, EF, GF)
which is equal to 14%. Coeficient is one method used to calculate the similarity
between two objects (items). In general the calculation of this method based on
vector space similarity measure. The result indicates that the data is valid because
according Ramadhani (2015), Jaccard’s Coeficient (SJ) is calculated, without taking
into account the characters that are not owned by the two organisms which revealed
that in general an organism within a prokaryotic species can be grouped into the
same strain if it has a 70% or greater similarity.
Accroding to Priest & Austin (1999), that the dendogram obtained, the
dendogram was evaluated and the evaluation results were included in the dendogram
similarity matrix. Then the calculation of the cophenetic correlation between the
initial similarity matrix and the similarity matrix results from the evaluation of the
dendogram. From this cophenetic correlation obtained correlation coefficients. The
value of the correlation coefficient is accepted if it is at the level > 60%. If the result
of the correlation coefficient calculation (r) is more than or equal to 60%, it means
that the classification carried out can be trusted and accounted for. The closer the
distance of node to the number 1 on the dendogram indicates the more similar.
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

The conclusion that based on the results and discussion are from Simple
Matching Coefficient, the closest kinship relationship is B, G, D and E. The farthest
relationship is indicated by the node that connects the strain F with strain A. From
Jaccard’s Coefficient the result its same with Simple Matching Coefficient. The
coefficient value obtained from Simple Matching Coefficient is 75,09161%
(acceptable), the coefficient value obtained from Jaccard’s Coefficient is 84,37353%
(acceptable).
B. Suggestion

Suggestions that can be given for this laboratory activity is it will better to
carefully pay attention to the steps when making the laboratory activity and note it so
it is easier to remember..
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