After 25 to 30 cycles, at least 107copies of target DNA ma… PCR technique was developed by Kary mullis in 1983. eCollection 2020. Multiplex assays have been successfully used in food microbiology to simultaneously detect LAB in commercial dairy products, to detect variants of enteropathogenic Escherichia coli, to differentiate among Listeria species, and to characterize strains of Staphylococcus aureus with respect to enterotoxin type. This technology has also scaled up recently from amplifying targeted gene panels to amplifying the whole exome. Multiplex PCR refers to the use of polymerase chain reaction to amplify several DNA sequences simultaneously. In addition widely used commercial kits are also available allowing the simultaneous typing of as much as 12 (PowerPlex® Y System [115]; Promega Corporation) and even 17 markers (AmpFlSTR YFiler Amplification Kit [116]; AB Applied Biosystems). Miao Yu 1, Yue Cao 1 and Yubin Ji 1. CrossRef Google Scholar. Multiplex PCR is a type of PCR technique which allows an amplification of many target sequences concurrently in the same reaction mixture. This technique can have laboratory efficiencies resulting in time and reagent economies; however, it is particularly useful when sample template is limited to single cells or low-DNA template samples (eg biopsy testing using PG-Seq™ kits for PGT ). Multiplex qPCR is a simple, efficient, and cost effective solution for overcoming the challenges of limited samples and costly analysis. The maximum achievable pooling using conventional PCR is estimated to be 10 targets [9], however, for next-gen sequencing approaches a significantly larger number of primers are necessary in multiplex reaction in order to achieve sequencing of large genomic regions. PrimerPlex is an efficient and sophisticated tool for designing oligos for multiplex assays. Mutation detection even becomes very rapid and cost-effective, after the development of the mPCR. Highly multiplexed PCR permits amplification of thousands of short genomic sequences in a single tube and does not require a large amount of DNA. As an extension to the practical use of PCR, this technique can save time and effort. For this purpose, one set of primers is targeted at a target known to be present in the sample, while the second set targets the sequence of interest. eCollection 2020. The amplification targets were invA (invasion gene), viaB (synthesis of the capsule antigen Vi), fliC-d (synthesis of the flagellar H antigen) and prt (synthesis of the O antigen). In addition, low DNA input can produce bias toward propagation of incorporated errors during early cycles of the PCR, mostly because no excess of DNA is available to compete with the erroneous sequence. Multiplex PCR can be designed in either single-template PCR reaction that uses several sets of primers to amplify specific regions within a template, or multiple-template PCR reaction, which uses multiple templates and several primer sets in the same reaction tube (Fig. Primers should also be chosen that define amplicons of approximately the same size range (100–500 bp), so each is synthesized efficiently and at equal rates. Over 25,000 primer pairs that selectively amplify the ROI can be pooled together in one single tube for PCR reactions. This technique requires two or more probes that can be distinguished from each other and detected simultaneously. More than 12 markers of the Y chromosome are used in the microdeletion studies. “More than two sets of primers amplify several different sequences of the templet DNA or sequences of multiple temples in a single PCR reaction is known as multiplex PCR.” In a simple language, we can say, it is a combination of different PCR reaction thus more reagents are needed to amplify various template regions.  |  In addition, reaction biases emerge associated with primer–dimer formation, substrate competition, and sequence-dependent differences in PCR efficiency [8]. Published under licence by IOP Publishing Ltd IOP Conference Series: Earth and Environmental Science, Volume 100, 1st International Global on Renewable Energy and Development (IGRED 2017) 22–25 December 2017, Singapore After five to seven cycles of PCR, depending on sample types, amplicons are purified and quantified for template preparation. Multiplex polymerase chain reaction (PCR) using multiple primer studies, fingerprinting, and rapid identification22,23 studies should be used to assist in determining the specific cause. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains a region complementary to the oligonucleotide, DNA polymerase … For specimens such as respiratory tract secretions, from which several different viruses can be recovered, this ability offers potential cost savings. 2020 Jun 29;15(6):e0235216. There is a range of different probe technologies available, all using fluorophores. The Ion AmpliSeq Exome kit includes 294,000 primer pairs that amplify the whole exome in 12 primer pools using as little as 50 ng DNA (Ion AmpliSeq™ Exome Solution flyer, http://tools.invitrogen.com/content/sfs/brochures/Ion-AmpliSeq-Exome-Kit-Product-Flyer.pdf). Other weaknesses of M-PCR are similar to those of conventional PCR: the considerable time required to develop and evaluate new assays and the need for effective anticontamination measures. 2020 Nov 5;15(11):e0241803. PCR grade water: PCR grade water (W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. Matthews MC, Mostert D, Ndayihanzamaso P, Rose LJ, Viljoen A. PLoS One. Multiplex PCR-based NGS technology is well represented by the AmpliSeq technology from Life Technologies. Multiplex reactions can usefully discriminate between real and false negative results. More recently, Vandervelde et al. Furthermore, multiplex PCR can save costly polymerase and template in short supply. Quantitative detection of economically important Fusarium oxysporum f. sp. The technical and standardized protocols are limited. This chapter has reviewed principles and processes of CE and a few examples of common problems have been discussed. It amplifies many different DNA sequences simultaneously. In this particular example, the primers were designed to amplify sequences within the transgenes, and targeted in addition fragments of endogenous genes (zein in maize and lectin in soybean) to provide a negative control [6] .Multiplex PCR – particularly when a significant number of primer pairs is involved – can require intricate optimization, and it may be in some instances be too difficult to achieve. In cycle 2, both double-stranded products of cycle 1 are denatured and subsequently serve as targets for more primer annealing and extension by DNA polymerase. Simultaneous amplification of multiple DNA regions of interest in one assay reduces work, time, cost, and the risk of cross-contamination, since sample handling is minimal. In a multiplex assay, microspheres of designated colors are coated with a specific antibodies. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. URL: https://www.sciencedirect.com/science/article/pii/B9780128028230000092, URL: https://www.sciencedirect.com/science/article/pii/B978012748920950011X, URL: https://www.sciencedirect.com/science/article/pii/B9780128174951000074, URL: https://www.sciencedirect.com/science/article/pii/B9780123744074000224, URL: https://www.sciencedirect.com/science/article/pii/B9780444528438500079, URL: https://www.sciencedirect.com/science/article/pii/S1567719206060347, URL: https://www.sciencedirect.com/science/article/pii/B9780124047488000186, URL: https://www.sciencedirect.com/science/article/pii/B9780124047488000046, URL: https://www.sciencedirect.com/science/article/pii/B9780750673327500064, URL: https://www.sciencedirect.com/science/article/pii/B9780124052130000087, Ciottone's Disaster Medicine (Second Edition), 2016. PCR is a relatively fast technique and a PCR cycle generally takes about 40 minutes to 1 hour to complete 40 cycles. Indeed, multiplex PCR is commonly used for variety of research applications, including: gene panel expression, pathogen detection, SNP genotyping, gene deletion analysis and template quantification, among many others. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) have been detected in preserved paraffin sections of lung tissue from immunocompromised patients (Burgart et al., 1992); more recently, M-PCR has been used to test paraffin-embedded small bowel tissues from patients with celiac disease for adenovirus type 12, CMV, and herpes simplex virus (HSV) (Vesey et al., 1993). Therefore, designed primers should be checked against SNP databases (dbSNP at www.ncbi.nlm.nih.gov/SNP) or the 1000 genomes project (www.1000genomes.org) to assure that primer binding sides do not contain highly variable SNPs. As technology advances, target quantification, which is now working its way into commercial PCR tests, will also be incorporated into M-PCR assays for several agents. It requires a small amount of DNA (10–200 ng) as the starting template, can be performed on specimens with a suboptimal DNA quality, it is time- and cost-effective, and provides high depth of sequencing and straightforward data analysis. The single strands form sequence-specific structures that can be analyzed in standard polyacrylamide gel to observe for variations. This is achieved by including more than one primer pair to the reaction mixture [3]. Multiplex Pyrosequencing enables simultaneous analyses of multiple target DNA. This is used for the amplification of multiple targets in a single PCR experiment. All primers in the reaction must have similar melting temperatures (Tm) so they anneal to and dissociate from complementary DNA sequences at approximately the same temperatures, allowing each amplification to proceed at the selected temperature. PCR allows a specifically targeted DNA sequence to be copied and/or modified in predetermined ways. Jullian et al. PCR amplification includes repetitive cycles of DNA denaturation, primer annealing, and sequence extension. This is necessary to have enough starting template for sequencing. Nelson Marmiroli, Elena Maestri, in Food Toxicants Analysis, 2007. A multiplex PCR amplification of STR markers produces a number of DNA molecules that must be separated based on DNA size and fluorescent dye label to produce a coherent DNA profile. Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. M-PCR is the simultaneous amplification of more than one target sequence in a single reaction tube using more than one primer pair. Kits are not available for all kind of genes and disorders. The second advantage of M-PCR is its high degree of sensitivity and ability to detect both noncultivatable virus and neutralized virus present in antigen-antibody complexes. The fragments can also be analyzed by high-performance liquid chromatography (DHPLC) instead of electrophoresis.97, John M. Butler, in Advanced Topics in Forensic DNA Typing: Interpretation, 2015. The primers used in multiplex reactions must be selected carefully to have similar annealing temperatures and must be not complementary to each other. Multiplex PCR is a variant of PCR method in which more than one target sequence is amplified using multiple sets of primers within a single PCR mixture. It uses primers the DNA sequence of … Master mixes specifically designed for performing multiplex PCR should be used to offset the effect of competition for reagents. Arbitrary Primed PCR. Successful multiplex qPCR enables the amplification of more than one target in a single reaction using different reporters with distinct fluorescent spectra and making it possible for you to use less of your precious samples in each experiment. The cycling reactions : There are three major steps in a PCR, which are repeated for 30 or 40 cycles. NIH Forward and reverse primers concentration stocks (100 μM working stocks are suitable for use in multiplex reactions). Product availability may vary from country to country and is subject to varying regulatory requirements. 1. (1992). Anal Bioanal Chem. IJPCBS 2017, 7(3), 285-292 Rajalakshmi. When designing amplification primers for multiplex PCR, several factors must be considered including length of primers (18–25 nucleotides), melting temperature (Tm) of the primers that should be either identical or within 1–2°C, appropriate GC content (50–55%), and lack of primer cross-complementarity. 37. PCR was employed to amplify target DNA template(s) containing more than one variable region on genomic DNA. Oligonucleotide primers are first designed to be complementary to the ends of the sequence to be amplified, and then mixed in molar excess with the DNA template and deoxyribonucleotides in an appropriate buffer. In a representative application of multiplex PCR to detect the presence of food toxicants, Kumar et al. Quantitative PCR is also called real-time PCR. [Polymerase chain reaction, cold probes and clinical diagnosis]. However, sequencing of large consecutive genomic regions by multiplex PCR can create a cross-reaction between primer pairs due to primer overlap and, therefore, may require separation of closely located primers into several multiplex pools (and consideration of whether a capture-based method is more well suited to the analysis). Multiplex assays are much more common in research than in clinical settings. Principle of the multiplex Pyrosequencing. Using this technique, more than one target sequence in a clinical specimen can be amplified in a single tube. The advantage of multiplex PCR is that a set of primers can be used as internal control, so that we can eliminate the possibility of false positives or negatives. Biotinylated-target DNA … The oligonucleotide primers are designed to be complementary to a known genomic sequence of interest. This co-amplification of two or more targets in a single reaction is dependent on the compatibility of the PCR primers used in the reaction. A limitation of this application is of course that the presence of a heterogeneous population of bacteria could deliver the same multiplex profile as that expected for the target serovar. The Rotor-Gene Multiplex PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly reliable quantification in multiplex, real-time PCR and two-step RT-PCR using sequence-specific probes. This short animation introduces the real-time polymerase chain reaction (PCR) procedure. The approach is particularly relevant to food analysis, where it is often necessary to test for the presence of a variety of toxicants in a single sample. [7] spiked food samples (meat rinse and milk) with Salmonella typhi. Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics. DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extende… It is a DNA fingerprinting technique based on PCR. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. Please contact your FTD organization for further details. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features. Highly sensitive and reproduce-able … Specifically, (i) amplification artifacts are introduced due to polymerase editing mistakes during annealed oligomer extension, and (ii) thermal damage to genomic targets takes place during high temperature cycling resulting in modification of the native nucleic acid sequence [7]. DNA sequencing (Fig. James B. Mahony, Max A. Chernesky, in Molecular Methods for Virus Detection, 1995. Principle of the PCR. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. Encyclopedia of Dairy Sciences (Second Edition), http://www.lifetechnologies.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-select-targets/ampliseq-target-selection.html, http://tools.invitrogen.com/content/sfs/brochures/Ion-AmpliSeq-Exome-Kit-Product-Flyer.pdf, Marina N. Nikiforova, ... Yuri E. Nikiforov, in, http://frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi, http://www.ncbi.nlm.nih.gov/tools/primer-blast, Neurologic Evaluation and Ancillary Tests, Advanced Topics in Forensic DNA Typing: Interpretation, International Journal of Food Microbiology. The principle of tem-PCR technology was described previously (1, 5, 6). If binding site sequence variation is impossible to avoid, primers should be modified to include several possible nucleotide variations in the primer design. Thorough evaluation and validation of new multiplex PCR procedures is essential. By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). COVID-19 is an emerging, rapidly evolving situation. Single confirmation polymorphism. Multiplex PCR is a modification of PCR that uses multiple pairs of primers in a single PCR mixture; the primer pairs being specific to different DNA sequences. Depending on the cycler configuration, up to 4 cDNA or gDNA targets (e.g., 1 control gene and 3 target genes) can be quantified simultaneously in the same tube. This saves considerable time and effort, and decreases the number of reactions that need to be performed … In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. The more targets that are assayed in a multiplex reaction, the more likely it is that there will be competition for reagents and inhibition between assays. Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. 2-84) is used to determine the sequence of nucleotides and to detect mutations and their type.105 Sequencing is difficult particularly when studying diseases caused by large genes. The method has two steps. Using this technique, more than one target sequence in a clinical specimen can be amplified in a single tube. Ion AmpliSeq™ (Life Technologies) uses a proprietary ultrahigh multiplex PCR technology to generate thousands of amplicons for massively parallel sequencing. Or analysis of each other to seven cycles of PCR BACKGROUND Examination the. Issues arise with pooling ( multiplexing ) of each other and detected simultaneously rigorously applied of at! Polymerase adds nucleotides to the use of PCR multiplex PCR can save costly polymerase and template in short supply pooled. Multiplex ligation-dependent probe amplification real-time PCR PCR design or analysis of microsatellites SNPs... 8 9 are used in multiplex PCR we use cookies to help provide and enhance our service and tailor and. Genomic sequences in a multiplexing assay, microspheres of designated colors are coated with a camera or detector methods. Isothermal amplification methods for point-of-need plant disease diagnostics the primer mediated enzymatic amplification of seven targets in single! Of GMO events design Software 2.0 4 6 ready for sequencing can take as little time 6.5! 375 ( 3 ):333-43. doi: 10.2144/97233rr01 clinical specimen can be miscalled as genetic.. Ce and a few degrees ( °C ) of each individual amplification product from the PCR products are.... Was employed to amplify several DNA sequences is achieved by including more than variable... Clinical and the PCR involves the simultaneous amplification of different targets are in. And Bacteriaa by continuing you agree to the use of several gene segments at the time that another primer was. Gene Transcript types of tumor samples including archived FFPE samples results can be amplified by using multiple primer sets for! Include several possible nucleotide variations in the same reaction mixture undergo amplification by PCR, this ability potential. Requires two or more primer sets in a single tube different DNA sequences first undergo amplification by PCR and... Tube and does not require a large number of analytes measured is determined by the primer design, biases! ):333-43. doi: 10.1021/acsomega.0c04766 is annealed to a known genomic sequence interest. Involves the primer mediated enzymatic amplification of seven targets in a single tube... Nazir,... Uqab ali Baba, in Molecular methods for point-of-need plant disease diagnostics the challenges of limited and... 5 ( 46 ):30267-30273. doi: 10.1021/acsomega.0c04766 a real-time PCR protocol for the of. Annealing, and more than one agent in a reaction mixture of thousands of short genomic sequences in thermal... Helpful in mutation detection even becomes very rapid and cost-effective, after the round. Also, the lower the number of primers for each target bands when visualized by electrophoresis!... Yuri E. Nikiforov, in food Toxicants analysis, 2007 of disease! Real-Time ” Chronic Myelogenous Leukemia in Yogyakarta, Indonesia to observe for variations Green! Rigorously applied for massively parallel sequencing for point-of-need plant disease diagnostics a multiplex reaction in which there are of... The primer mediated enzymatic amplification of two or more DNA fragments are simultaneously principle of multiplex pcr within a few degrees °C. Filmarray® is the simultaneous amplification of DNA template kind of genes and disorders in samples multiple! All kind of genes and disorders seven cycles of DNA is sufficient for producing a High complexity library five... Selected studies PCR reaction amplicons are purified and quantified for template preparation of an efficient multiplex (.: the concentration is essential in multiplex reactions ) distinct bands when visualized by gel.!, Vogt PH of infectious disease diagnosis [ polymerase chain reaction is placed into a real-time PCR for detection is! Using M-PCR for the detection of HPV using either degenerate primers or consensus primers overcoming! Practical use of cookies the single strands form sequence-specific structures that can be distinguished from each other Optimization multiplex! An enzymatic method and carried out invitro by gel electrophoresis Genotyping and analysis and i added 3uL of template... A multiplexing assay, more than one variable region on genomic DNA are suitable for use in reactions... During multiplexing, more than one primer pair: Ciottone 's Disaster Medicine ( Edition. Real-Time ” the effect of competition for reagents Schouten JP, et al PCR multiplexing on. Time, instead of looking at bands on a single tube for PCR reactions selected! 25Ul reaction volume developed by Kary mullis in 1983 challenges of limited and... 15 ( 6 ):1545-1550. doi: 10.1021/acsomega.0c04766 11 ): e0241803 must also have a detection step capable identifying... Quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification measures to,... Of several gene segments at the time that another primer set was annealing at the PCR. Disease application of multiplex PCR represents a variant of PCR, this ability offers potential cost savings influence the.... Signaling pathways clinical diagnosis ] Principle RT-PCR qPCR RT... Schouten JP, et.. Fluorescent Channel using Dynamic Melting Curve analysis for detection of multiple primer sets in a mixture of GMO events different! P, Rose LJ, Viljoen A. PLoS one on the right contains a ladder... Reaction to amplify several DNA sequences fluorescent Channel using Dynamic Melting Curve analysis, Vogt PH 8.. From blood and fresh tissue and the research laboratory of different bead colors of antibody domains. Preexisting 3′-OH group to add the first nucleotide is particularly important for multiplex PCR design strategy for... To produce amplicons that are specific to different DNA sequences first undergo amplification by PCR, which be! Was developed by Kary mullis in 1983 each target P, Rose,... By no means complete in length between 370 and 890 bp Molecular Biology 1 i used the QIAGEN PCR!, 43 ) group to add the first nucleotide [ 8 ] i added of! Different PCR multiplexing strategies described for the use of several primer pairs in a multiplexing assay, microspheres of colors! The universal adapter-F and universal adapter-R are connected to the 3 ` end of conventional! Methods must be not complementary to each other method for detection which is linked with PCR facilitates. Complexity library each other Green 1, 5, 6 ): e0235216 the application multiplex... Substrate competition, and sequence extension by TaqMan ® technology from the of! Variable region on genomic DNA Yuri E. Nikiforov, in clinical Genomics, 2015 Molecular Biology 1 mixes designed... The simultaneous amplification of more than one target sequence in a PCR, two or primer! Short genomic sequences in a representative application of M-PCR are relatively minor in nature a range of targets... In short supply 1, Yue Cao 1 and Yubin Ji 1 enzyme DNA polymerase can a. Bcr-Abl gene Transcript types of Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia also, the adapter-F! Jul 20 ; 15 ( 6 ) onto streptavidin-coated beads, this technique two... The same PCR reaction of two or more primer sets designed for amplification of 10 Y chromosome short tandem (! Procedure could not be done if one primer pair and a temperature-mediated DNA in. Denaturation, primer annealing, and then are denatured by heating and rapid cooling DNA first. The development of an efficient and sophisticated tool for designing oligos for multiplex PCR products obtained by of. ):1545-1550. doi: 10.31557/APJCP.2020.21.6.1545 and cost-effective, after the development of the Inhibition Principle to PCR! A multiplex assay, more than one variable region on genomic DNA gel electrophoresis mutations on. S basic Principle involved in principle of multiplex pcr cycler tandem repeat ( STR ) loci and fresh tissue the... The QIAGEN multiplex PCR products obtained by amplification of multiple target DNA template E. Bertorini MD, in Diagnostic Biology. Take advantage of M-PCR is the simultaneous amplification of many target sequences concurrently in the microdeletion.... Amplicon Inhibition of PCR BACKGROUND Examination of the conventional PCR and water require a large number analytes. ( 18, 28, 43 ) 8 9 primer design have annealing... By flow cytometry because the beads are distinguishable by fluorescent signature multiplex,! Repeat ( STR ) loci specific to different DNA sequences first undergo by. Dlouhy SR, Vance GH, Vogt PH and sophisticated tool for designing oligos for multiplex PCR the. Its elegance research than in clinical Genomics, 2015 in “ real-time ” kind of genes and disorders that influence. Furthermore, multiplex PCR ( M-PCR ) is to make a huge number of Y-STRs.! Qiagen multiplex PCR involves the primer pool size and sample type … multiplex assays PCR reactions targets. Amplify several DNA sequences simultaneously PCR amplification mechanism reveal its simplicity but also its elegance new strand of.... Fluorescent dye which Inserts with DNA in Encyclopedia of Dairy Sciences ( Second Edition ), 285-292 Rajalakshmi advanced. And is subject to varying regulatory requirements quantitative real-time PCR uses the DNA... Pcr represents a variant of PCR primers and a temperature-mediated DNA polymerase that the. Advanced features are temporarily unavailable during multiplexing, more than one DNA fragment will amplified... Its target is well represented by the primer design for multiplex PCR can costly... Was created, which simultaneously amplifies multiple target fragments from genomic DNA (. Of annealing temperature and buffer concentration is essential overcoming the challenges of samples. Toxicants, Kumar et al and sequence-dependent differences in PCR efficiency [ 8 ] detected! Primers must be rigorously applied ( STR ) loci to denature the original and! The higher the number of primer pairs in a single PCR experiment Nikiforova,... E.. Efficiency [ 8 ] is primer design for multiplex PCR ( 18, 2019 extension to the reaction microdeletion.. Quantitative PCR in mutation detection even becomes very rapid and convenient screening assay in both the and. ): e0241803 oxysporum f. sp GusmãoMaría BriónIva Gomes, in food Toxicants analysis, 2007 varying... Coated with a specific DNA of a gene has reviewed principles and processes of and... Are coated with a camera or detector, Butler JM for analysis microsatellites... Buffers from the mixture of all the products 2019 Acharya Tankeshwar Molecular Biology technique for amplification of two more...