Molecular Markers as a Diagnostic Tool

Middle East Journal of Applied Science & Technology (MEJAST)
(Peer Reviewed International Journal) Volume 2, Issue 3, Pages 87-94, July-September 2019
87 | P a g e ISSN (Online): 2582- 0974 Website: www.mejast.com
Molecular Markers as a Diagnostic Tool
Muhammad Asif Raheem1
, Misbah Aslam1
, Muhammad faisal1
, Nimra Izhar1
and Rana Khalid Iqbal1
*
1
*Institute of Molecular Biology & Biotechnology, Bahauddin Zakariya University, Multan-60880, Pakistan.
Article Received: 21 February 2019 Article Accepted: 15 July 2019 Article Published: 30 August 2019
Abbreviation
RFLP (Restriction fragment length polymorphism)
RAPD (Random amplification of polymorphic DNA)
AFLP (Amplified fragment length polymorphism)
SSR(Simple sequence repeats)
ISSR (Inter-Simple Sequence Repeat Amplification)
CAPS (Cleaved Amplified Polymorphic Sequences)
SCAR (Sequence Characterized Amplified Region)
1. INTRODUCTION
During the last few years, the invention and use of the molecular marker have brought a revolution in the field of
diagnosis and identification of diseases. Before this, the following methods were used for the identification of the
diseases. These are Biotyping, ribotyping, antibiogram, resistogram, bacteriocin, protein analysis1
. These methods
were not as accurate in the detection of the diseases so the intention moves towards the molecular marker methods
of diagnostics. A.S.Serebrovskii was the first person who gave the background about the molecular marker as a
diagnostic tool2
.
There are following types of genetic markers(i) Morphological markers that identify the characters based on the
visual bases of the characters such as plant height, color, seed shape, color, flower color, and shape, etc. 3
. (ii)
Biochemical markers, these are also known as the isozymes that are the allelic alternatives of enzymes and these
can be easily detected by using gel electrophoresis. These markers have the ability to identify the variation among
species at the gene level which is functional and these markers show a small level of inheritance. (iii) DNA marker
that identifies the variation among species at the DNA level3
. The identification and the diagnosis of the disease by
the use of these markers are easy and accurate.
ABSTRACT
Molecular markers are the DNA sequences that are used to tag or highlight specific genes or nucleotide sequence in the genome. In the past,
diagnosis, and monitoring of infectious disease, there many conventional methods like Biotyping, Ribotyping and Protein analyses are used but these
methods are not so reliable and exact. So by the invention of the molecular markers, the detection of diseases was started by using these molecular
markers. In this review, we discussed different types of molecular markers that are used to detect diseases and many purposes as diagnostic tools. The
Molecular markers include Restriction fragment length polymorphism (RFLP), Random amplification of polymorphic DNA (RAPD), Amplified
fragment length polymorphism (AFLP), Simple sequence repeats (SSR), Inter-Simple Sequence Repeat Amplification (ISSR), Cleaved Amplified
Polymorphic Sequences (CAPS) and Sequence Characterized Amplified Region (SCAR) are discussed in the article.
Keywords: RFLP, RAPD, AFLP, SSR, ISSR, CAPS.

By the use of molecular markers the accuracy increases of detection. The previous conventional methods that were
used early due to their less reliability and handling problems now the molecular markers are now used. These
markers are used to detect complex traits4
.
2. CONVENTIONAL METHODS
The conventional methods of identification of the microbes were based on their physical appearance of the
microbes such as their growth, shape, their consumption of the food etc. The following methods were used for the
identification of diseases.
2.1.BIOTYPING
This method identifies the microbes on the basis of their physical appearance such as their growth mechanism,
consumption of the food, shape, etc. to construct a profile of any organisms is known as biogram5
. So the
identification of different organisms based on their profile is known as biotyping. The organisms whose biogram
comes same this shows the different strain of the same organism1,6
.
2.2.RIBOTYPING
Ribotyping is the method which identifies the bacteria based on the formation of the rRNA7
. In this method we take
the DNA and then cut with the restriction enzymes and then separate on the gel electrophoresis that is then
transferred to the nylon sheet and then that is dipped in the probes the probe will bind to their complementary
sequence and then we compare the sequence with the data stored in the databases1,6
.
2.3.PROTEIN ANALYSIS
By the use of proteins, we can detect the different antigenic proteins that are produced by the microbes. In this
method, different types of monoclonal antibodies are applied to different organisms and then they are detected. The
protein is then extracted and then separated by SDS PAGE and detected1,8
.
3. TYPES OF MOLECULAR MARKERS
There are mainly two types of marker PCR based and non-PCR based methods. Non-PCR base methods or
Hybridization base Methods include Restriction fragment length polymorphism (RFLP)9,10
and PCR base methods
are Random amplification of polymorphic DNA (RAPD)11,12
, Amplified fragment length polymorphism (AFLP)13
,
Microsatellite polymorphism Simple sequence repeat (SSR )14,15
Variable number tandem repeat (VNTR)16
,
Sequence Tagged Site (STS)17
, Sequence Characterized Amplified Region (SCAR)18
, Inter-Simple Sequence
Repeat Amplification (ISSR)19
, Cleaved Amplified Polymorphic Sequences (CAPS)20,21
, Single nucleotide
polymorphism (SNP)2223
, Short tandem repeat (STR)9
, Retrotransposon Microsatellite Amplified Polymorphism
(REMAP)2425
, Simple sequence length polymorphism (SSLP)26
, Diversity Arrays Technology (DArT)27
, Anchored
Microsatellite Primed PCR (AMP-PCR)28
, Aritrarily Primed Polymerase Chain Reaction (AP-PCR)29
, DNA
Amplification Fingerprinting (DAF)29
, Inverse PCR (IPCR)30
, Inverse Sequence-Tagged Repeats (ISTR)26
. Some
of these markers are similar to each other and some work on the same methods.

3.1.NON-PCR BASE METHODS OR HYBRIDIZATION BASE METHODS
3.1.1. RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
This is a hybridization-based marker which detects the polymorphism. This type of marker is used to detect the
polymorphism between two species or individual. This gives different types of bands even due to a small change in
nucleotides of about 1, 2 base pairs due to mutation. Hence the restriction site can be gain or loss due to the
mutation.9
The procedure of the RFLP marker is the following:
Extract the DNA of the organisms then it is cut by using molecular scissors restriction enzymes that are present in
bacteria and use as their defensive system his enzyme has the ability to cut down the DNA at restriction sites. Then
the DNA fragments are separated on the agarose gel depending upon their size to mass ratio. These DNA bands are
then transferred to the nylon or nitrocellulose membrane by the process of blotting. In this technique, the gel is
placed by the help of sponge in the bath that contains alkaline buffer. The towel stack is placed on the top of the
apparatus. The towel sucks the buffer solution which denatures the double-stranded DNA and these single strands
of DNA will move on the nylon sheet on which they adhere firmly. After this, the nylon sheet is separated from
there and then dipped in a bucket that contains radioactively labeled probes. The probe will bind to their
complement sequence. Then take the sheet and wash it, the DNA that is not attached with the probes will wash out.
At the end, gel is place under UV light and observe the bands26,31
.
3.2.PCR BASED METHODS
3.2.1 RANDOM AMPLIFICATION OF POLYMORPHIC DNA (RAPD)
As the name indicates this type of primer is applied which randomly amplify the DNA strand. This is a PCR based
marker in which we get amplification by using PCR32
.
Figure: 1: DNA template is taken and design primers of short base pairs up to 10. Then PCR the DNA template the
RAPD will attach to the sites complementary to the primer. Run on gel and comparison the Marker with the
template DNA.

We take 2-6 bp short primers. the primer we used should have the following conditions the GC contents should be
minimum of 40% and should not contain the palindromic sequences the reason is that during annealing the primer
if contain more percentage then it will require more temperature to anneal and the RAPD anneal at low
temperature. In RAPD we use only single primer this primer will act as reverse and forward primer. The protocol of
RAPD is designed to short oligonucleotide primer add in the PCR the primer then will anneal at low temperature.
The part of the DNA will be amplified if the reverse and forward primers are not at more distance than 3000
nucleotides12
. The primer then will amplify the band. This marker has major limitations that it is not reproducible
mean when we repeat the sample the result will not be the same. It is a dominant marker it only detects the dominant
traits. It is mainly used to detect the traits that the trait is present or absent. It is also used for paternity tests and help
in plant and animal breeding11
.
3.2.2. AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)
This marker is a combination of two markers RFLP and RAPD. In this, we use restriction enzyme for cutting the
DNA and then PCR used for amplification13
. The protocol of marker is the extraction of the genomic DNA then
cutting the DNA with restriction enzymes. Then to prevent the ligation of the restriction sites of the DNA we design
specific adapters which will bind with the sticky ends of the DNA. This adopter will give the signal of amplification
during the PCR. In this process, we do 2 amplification first pre-amplification in which we extend the adopter length
by 1 bp. Then after pre-amplification, we do the selective amplification in which we extend the adopter up to 3 bp
which gives specified bands instead of 1 bp extension which is very common in the genome. Hence the amplified
region obtained is then separated on gel electrophoresis and then stained by using silver staining and detected by
autoradiography26
.
Figure 2: Take the template DNA digest it by the help of restriction enzymes. Design adopter to prevent the
attachment of the sequences of DNA. Start the preamplification process in the PCR run another PCR for selective
amplification in which 1 or 2 base pairs of adaptor are changed. Then run on the gel and read it.

3.2.3. SIMPLE SEQUENCE REPEATS (SSR)
SSS is abbreviated as short tandem repeats. They are also known as microsatellites. These are the tandem repeats
that have the length of 1-6 bp found in many eukaryotes and prokaryotes. In the plant, AT repeats are mostly
founded but in animals, AC repeats are found. This difference is the general point of polymorphism in the genomes
of plants and animals. The SSR is present all over the genome also in both coding and in non-coding regions. They
show polymorphism in nature due to the different numbers of their repeats. They can be easily reproducible and
they can be detected with the polymerase reaction (PCR). They show high polymorphism and they reproduce able
to have the application in paternity tests, construction of high genome maps and they have also used for the
mapping the beneficial genes, marker-assisted selection, for evolutionary relationship analysis and other diagnostic
purposes14
.
There are 2 types of classification of SSR markers. The first is on the bases of size of repeats. These are di, tri, tetra,
penta which have 2,3,4,5 no. of repeats. The second classification is on the bases of how the repeats are present.
These may be perfect having the same repeated base pairs, imperfect being that is interrupted by other base pair,
compound imperfect that is interrupted by the pair of base pairs but the two repeats have the same length. The
microsatellites are named on the bases of their occurrence in the genomes. If they are present in nucleus they are
termed as nuclear SSR and termed the same if they present in mitochondria chloroplast etc33
.
It is seen that the SSR are mostly present as nuclear SSR. The SSR is found in coding and non-coding regions of the
genome. But it is demonstrated that the SSR is located in the coding regions of the genome34
.
The development process of the SSR is that firstly they are isolated from the genome either coding or non-coding
regions then after isolations, they are cloned. Their cloned are formed by ligated them in the vector and their copies
are generated. After that these clones are hybridized with the probes that consist of repeats, then the probes can bind
to the nylon sheet and then these are viewed under UV light and then the SSR are isolated and collected in
libraries9,31
.
3.2.4. INTER-SIMPLE SEQUENCE REPEAT AMPLIFICATION (ISSR)
ISSR is used for the amplification of the genomic region between two SSR repeats that are present in the opposite
direction in the genome 28
. This method used simple sequence repeats as a primer in the PCR reaction and hence the
region between the repeats can be amplified. The SSR used as a primer can be di, tri or tetra repeat of base pairs.
The primer used is of 15-20bp length and they required high annealing temperature that primarily depends upon the
GC contents. It shows a high level of polymorphism among individuals. It can detect by using PAGE and silver
staining. It has any limitations that it is of dominant inheritance, it is not reproducible35
.
3.2.5. CLEAVED AMPLIFIED POLYMORPHIC SEQUENCES (CAPS)
It is abbreviated as sequence tagged sites. This marker is a combination of two steps one is PCR second is RFLP20
.
In this marker, we first extract the DNA and then we amplify it by using PCR by giving all the requirements needed
for the PCR. Then the DNA we get is then further used for the next step is we cut the amplified DNA by the use of
restriction enzyme. Hence by the difference of the restriction sites, we can check the polymorphism among

individuals. The advantages of the STS are that is co-dominant marker so it can detect the dominant and recessive
traits another advantage is that it is very time-consuming due to the amplification in PCR. It is also known as
PCR-RFLP17
.
3.2.6. SEQUENCE CHARACTERIZED AMPLIFIED REGION (SCAR)
It is a specific genomic sequence that can identify by using two pairs of the primer by the help of PCR. It developed
by the help of cloning of the RAPD marker that is used for some specific purpose of diagnosis. The advantages of
the scar are that it can be changed into co-dominant nature, it is used to detect specific locus of any trait, and the
amplification is not very sensitive17,20
.
4. CONCLUSION
The molecular markers are the best process used for the diagnostic purpose. As in the earlier, the methods used for
the detection of infections were conventional methods such as biotyping, ribotyping, and protein analysis. But the
results of these methods are not clear to be reliable. So by the invention of molecular markers, the methods of
detection become very clear and easy. Every molecular marker is used for specific purposes and has different
methods of detection.
REFERENCES
1. Tang Y, Procop GW, Persing DH. Molecular diagnostics of infectious diseases. 1997;2038:2021-2038.
2. Khlestkina EK. Molecular Markers in Genetic Studies and Breeding. 2014;4(3):236-244.
doi:10.1134/S2079059714030022
3. Kordrostami M, Rahimi M. Molecular markers in plants : Concepts and applications. 2015;(September).
4. Hamburgo N, Alegre P, State S, Hamburgo N, Alegre P. Molecular techniques for the study and diagnosis of
parasite infection. 2011;17(3):239-248.
5. Higuera AD La, Gutihez J, Likbana J, Garcia-mendoxa A. A new biotyping method for Streptococcus mutans
with the API ZYM system. 1999:88-91.
6. Tenover FC, Arbeit R, Archer G, et al. Comparison of Traditional and Molecular Methods of Typing Isolates
of Staphylococcus aureus. 1994;32(2):407-415.
7. Relander P. THE DEMONSTRATION BY RIBOTYPING OF THE STABILITY OF ORAL
STREPTOCOCCUS MUTANS INFECTION OVER 5 TO 7 YEARS IN CHILDREN. 1994;39(6):467-471.
8. Fernández-álvarez C, Torres-corral Y, Santos Y. SC. J Proteomics. 2017. doi:10.1016/j.jprot.2017.09.007
9. Tharachand C, C IS, Mn M. Molecular markers in characterization of medicinal plants : An overview.
2012;2(2):1-12.
10. Finger A, Klank C. Review Molecular Methods : Blessing or Curse ? 2010:309-320.
doi:10.1007/978-3-540-92160-8

11. Bardakci F. Random Amplified Polymorphic DNA ( RAPD ) Markers. 2001;25:185-196.
12. Kumar NS, Gurusubramanian G. Random amplified polymorphic DNA ( RAPD ) markers and its applications.
2011;11(3):116-124.
13. Agarwal M, Shrivastava ÆN, Á SÁRÁP. Advances in molecular marker techniques and their applications in
plant sciences. 2008:617-631. doi:10.1007/s00299-008-0507-z
14. Lucia M, Vieira C, Santini L, Diniz AL, Munhoz CDF. Microsatellite markers : what they mean and why they
are so useful. 2016;328:312-328.
15. Shehata AI, Al-ghethar HA, Al-homaidan AA. Application of simple sequence repeat ( SSR ) markers for
molecular diversity and heterozygosity analysis in maize inbred lines. Saudi J Biol Sci. 2009;16(2):57-62.
doi:10.1016/j.sjbs.2009.10.001
16. Jonah PM, Bello LL, Lucky O, Midau A, Moruppa SM. Review : The Importance of Molecular Markers in.
2011;11(5).
17. Kesawat MS, Das BK. Molecular Markers : It ’ s Application in Crop Improvement. 2009;2009(10):169-181.
18. Adinolfi B, Chicca A, Martinotti E, Breschi MC, Nieri P. Sequence characterized amplified region ( SCAR )
analysis on DNA from the three medicinal Echinacea species. 2007;78:43-45. doi:10.1016/j.fitote.2006.09.012
19. Molecular Markers in Crop Improvement Indian Institute of Pulses Research.
20. Jiang G. Molecular Markers and Marker-Assisted Breeding in Plants. 2010.
21. Elangbam M, Misra AK. Development of CAPS markers to identify Indian tea ( Camellia sinensis ) clones
with high catechin content. 2016;15(2).
22. Lateef DD. DNA Marker Technologies in Plants and Applications for Crop Improvements. 2015;(May):7-18.
23. Grover A, Sharma PC, Grover A, Sharma PC. Critical Reviews in Biotechnology Development and use of
molecular markers : past and present. 2016;8551. doi:10.3109/07388551.2014.959891
24. Schulman AH. Molecular markers to assess genetic diversity. 2007;(September 2006):313-321.
doi:10.1007/s10681-006-9282-5
25. Kalendar R, Schulman AH. PROTOCOL IRAP and REMAP for retrotransposon-based genotyping and
fingerprinting. 2007;1(5):2478-2484. doi:10.1038/nprot.2006.377
26. Semagn K, Bjørnstad Å, Ndjiondjop MN. An overview of molecular marker methods for plants.
2006;5(25):2540-2568.
27. Appleby N, Edwards D, Batley J. Chapter 2 New Technologies for Ultra-High Throughput Genotyping in
Plants. 513:19-39. doi:10.1007/978-1-59745-427-8
28. Reddy MP, Sarla N, Siddiq EA. Inter simple sequence repeat ( ISSR ) polymorphism and its application in
plant breeding. 2002:9-17.

29. Kumar LS. DNA markers in plant improvement : An overview. 1999;17:143-182.
30. Silva D, Santos G, Barroca M, Collins T. Chapter 5 Inverse PCR for Point Mutation Introduction.
1620:87-100. doi:10.1007/978-1-4939-7060-5
31. K MK, Rs F, Ballani A, Vinita T, Yachana J. Potential and application of molecular markers techniques for
plant genome analysis. 2014;2(1):169-188.
32. Tingey S V, Rafalski JA, Hanafey MK. Genetic Analysis with RAPD Markers. 2000;(1990).
33. Varshney RK, Graner A, Sorrells ME. Genic microsatellite markers in plants : features and applications.
2005;23(1). doi:10.1016/j.tibtech.2004.11.005
34. Kalia RK, Rai MK, Kalia S. Microsatellite markers : an overview of the recent progress in plants.
2011:309-334. doi:10.1007/s10681-010-0286-9
35. Profi M, Goulao LF, Oliveira CM. Chapter 11. 2014;1115:211-231. doi:10.1007/978-1-62703-767-9

Molecular Markers as a Diagnostic Tool

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Molecular Markers as a Diagnostic Tool

Similar to Molecular Markers as a Diagnostic Tool (20)

More from Associate Professor in VSB Coimbatore

More from Associate Professor in VSB Coimbatore (20)

Recently uploaded

Recently uploaded (20)

Molecular Markers as a Diagnostic Tool