Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 87-94, July-September 2019
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
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.
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. 87 | P a g e
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Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 87-94, July-September 2019
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.
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Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 87-94, July-September 2019
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. 89 | P a g e
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Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 87-94, July-September 2019
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. 90 | P a g e
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Middle East Journal of Applied Science & Technology (MEJAST) (Peer Reviewed International Journal) Volume 2, Issue 3, Pages 87-94, July-September 2019
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
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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.
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