Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis

Türker Bodur; Meryem Cansu Yeşiltaş; Bahar Eldemir

doi:10.33988/auvfd.1893780

Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis

Abstract

This study introduces and validates dynamic multiplex PCR, a newly developed multiplex PCR method designed to overcome the inherent limitations of conventional multiplexing, particularly the challenges associated with heterogeneous primer melting temperatures (Tm) among multiple primer pairs in a single reaction. While traditional protocols rely on a single, fixed annealing temperature (Ta) per cycle, the dynamic multiplex PCR approach applies four sequential annealing temperatures (52°C, 54°C, 57°C, and 60°C) within each individual PCR cycle. This novel intra-cycle multi-temperature strategy was evaluated using an 8-plex microsatellite panel in European sea bass (Dicentrarchus labrax) samples and compared against three established protocols: multiplex touchdown PCR and two conventional PCR setups at fixed temperatures of 54°C and 57°C. The performance of the methods was assessed through a dual-platform quantitative analysis. Amplification yields were first quantified via densitometric analysis of agarose gel electrophoresis, while capillary fragment analysis was utilized to evaluate peak height, total peak area, quality scores, and fragment sizing precision. Sizing accuracy was rigorously determined as the absolute deviation between observed fragment sizes and assigned allele values. Statistical evaluations were conducted using a General Linear Model (GLM) and ANOVA framework to examine the effects of the PCR method, operator, locus, and biological sample on amplification performance and genotyping reliability. The results demonstrate that dynamic multiplex PCR significantly outperforms both conventional and multiplex touchdown PCR protocols by providing more balanced and robust amplification across all eight loci. By accommodating diverse primer requirements within a single cycle, this method eliminates the need for extensive trial-and-error optimization, thereby substantially reducing the consumption of laboratory consumables and total processing time. These findings establish dynamic multiplex PCR as a highly reliable, cost-effective, and reproducible multiplex PCR approach, offering a significant methodological advancement for high-quality multi-locus genotyping and fragment analysis workflows.

Keywords

Project Number

121N529

References

Allawi HT, Santa Lucia J (1997): Thermodynamics and NMR of internal G-T mismatches in DNA. Biochemistry, 36, 10,581–10,594. DOI:10.1021/bi962590c
Berg K, Glaser CL, Thompson RE, et al (2000): Detection of microsatellite instability by fluorescence multiplex polymerase chain reaction. J Mol Diagn, 2, 20-28. DOI:10.1016/S1525-1578(10)60611-3
Bodur T, Çiftçi Y (2026): Genetic Diversity and Morphometric Discrimination of Native and Introduced Pikeperch (Sander lucioperca) Populations in Türkiye. Turk J Fish Aquat Sci, 26, TRJFAS28911. DOI:10.4194/TRJFAS28911.
Bodur T, Tsigenopoulos C, Cagatay IT (2017): Genetic Structure of Wild European Sea Bass (Dicentrarchus labrax L, 1758) Populations in Aegean and Levantine Sea Using Microsatellite Markers. Turk J Fish Aquat Sci, 17, 7-14. DOI:10.4194/1303-2712-v17_1_02
Brondani RP, Grattapaglia D (2001): Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing. BioTechniques, 31, 793-800. DOI:10.2144/01314st06.
Caragine T, Mikulasovich R, Tamariz J, et al (2009): Validation of testing and interpretation protocols for low template DNA samples using AmpFLSTR® Identifiler®. Croat Med J, 50, 250-260. DOI:10.3325/cmj.2009.50.250.
Chistiakov D.A, Tsigenopoulos CS, Lagnel J, et al (2008): A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L.. Anim Genet, 39, 623-634. DOI:10.1111/j.1365-2052.2008.01786.x.
Ciftci Y, Castilho R, McAndrew BJ (2002): More polymorphic microsatellite markers in the European sea bass (Dicentrarchus labrax L.). Mol Ecol Notes, 2, 23-25. DOI:10.1046/j.1471-8286.2002.00327.x.

Clarke LA, Rebelo CS, Gonçalves J, et al (2001): PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences. J Clin Pathol Mol Pathol, 54, 351-353. DOI:10.1136/mp.54.5.351.
Drobinskaya I, Gabbert HE, Moeslein G, et al (2005): A new method for optimizing multiplex DNA microsatellite analysis in low quality archival specimens. Anticancer Res, 25, 3251-3258.
Edwards MC, Gibbs RA (1994): Multiplex PCR: advantages, development, and applications. Genome Res, 3, S65-S75. DOI:10.1101/gr.3.4.S65.
Evett IW, Gill PD, Lambert JA (1998): Taking account of peak areas when interpreting mixed DNA profiles. J Forensic Sci. 43, 62-69. DOI: 10.1520/JFS16091J.
Gibson-Daw G, Crenshaw K, McCord B (2018): Optimization of ultrahigh-speed multiplex PCR for forensic analysis. Anal Bioanal Chem, 410, 235-245. DOI:10.1007/s00216-017-0715-x.
Gill P, Sparkes R, Pinchin R, et al (1998): Interpreting simple STR mixtures using allele peak areas. Forensic Sci Int, 91, 41-53. DOI:10.1016/S0379-0738(97)00174-6.
Guichoux J, Lagache L, Wagner S, et al (2011): Current trends in microsatellite genotyping. Mol Ecol Resour, 11, 591-611. DOI:10.1111/j.1755-0998.2011.03014.x.
Guinand B, Dujardin E, Dufour V, et al. (2008): Characterisation of genetic structure of Dicentrarchus labrax larvae in two nurseries of the Gulf of Lions (NW Mediterranean). Aquat Living Resour, 21, 81–87. DOI:10.1051/alr:2008020.
Henegariu O, Heerema NA, Dlouhy SR, et al (1997): Multiplex PCR: critical parameters and step-by-step protocol. BioTechniques, 23, 504-511. DOI:10.2144/97233rr01.
Heras J, Domínguez C, Mata E, et al (2016): A survey of tools for analysing DNA fingerprints. Brief Bioinform, 17, 903-911. DOI:10.1093/bib/bbv016.
Leclair B, Frégeau CJ, Bowen KL, et al (2004): Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples. J Forensic Sci, 49, 961-973. DOI:10.1520/JFS2003312.
Mansfield ES, Vamer M, Hams DW, et al (1997): Rapid sizing of polymorphic microsatellite markers by capillary array electrophoresis. J Chromatogr A. 781, 295-305. DOI:10.1016/S0021-9673(97)00542-6.
McDonald C, Taylor D, Brinkworth, R, et al (2025): A novel approach to combine qPCR and STR amplification for DNA profiling. Forensic Sci Int Genet, 80, 103332. DOI:10.1016/j.fsigen.2025.103332.
McDonald C, Taylor D, Linacre A (2024): PCR in forensic science: a critical review. Genes (Basel), 15, 438. DOI:10.3390/genes15040438.
Mullis KB, Faloona FA (1987): Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol, 155, 335-350. DOI:10.1016/0076-6879(87)55023-6.
Novel P, Porta JM, Porta J, et al (2010): PCR multiplex tool with 10 microsatellites for the European seabass (Dicentrarchus labrax) – applications in genetic differentiation of populations and parental assignment. Aquaculture, 308, 534-538. DOI:10.1016/j.aquaculture. 2010.06.032.
Quéré N, Desmarais E, Tsigenopoulos CS, et al (2012): Gene flow at major transitional areas in sea bass (Dicentrarchus labrax) and the possible emergence of a hybrid swarm. Ecol Evol, 2, 3061-3078. DOI:10.1002/ece3.406.
Rithidech K, Dunn JJ (2003): Combining multiplex and touchdown PCR for microsatellite analysis. 295–299. In: Bartlett JMS, Stirling D (Ed.). PCR Protocols. Methods Mol Biol. Humana Press. New Jersey, USA. DOI:10.1385/1-59259-384-4:295.
Rychlik W, Spencer WJ, Rhoads RE (1990): Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res, 18, 6409-6412. DOI:10.1093/nar/18.21.6409.
Saiki RK, Gelfand DH, Stoffel S, et al (1988): Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491. DOI:10.1126/science.2448875.
Santa Lucia J (1998): A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA, 95, 1460-1465. DOI:10.1073/pnas.95.4.1460.
Vallone PM, Hill CR, Butler JM (2008): Demonstration of rapid multiplex PCR amplification involving 16 genetic loci. Forensic Sci Int Genet, 3, 42-45. DOI:10.1016/j.fsigen.2008.09.005.
Vartia S, Collins PC, Cross TF, et al (2014): Multiplexing with three-primer PCR for rapid and economical microsatellite validation. Hereditas, 151, 43-54. DOI:10.1111/her.12056.
Vartia S, Villanueva-Cañas JL, Finarelli J, et al (2016): A novel method of microsatellite genotyping-by-sequencing using individual combinatorial barcoding. R Soc Open Sci, 3, 150565. DOI:10.1098/rsos.150565.
Verheij S, Harteveld J, Sijen T (2012): A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples. Forensic Sci Int Genet, 6, 167-175. DOI:10.1016/j.fsigen.2011.03.014.
Walsh PS, Fildes NJ, Reynolds R. (1996): Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA. Nucleic Acids Res. 24, 2807-2812. DOI:10.1093/nar/24.14.2807.

Details

Primary Language

English

Subjects

Animal Science, Genetics and Biostatistics

Journal Section

Research Article

Authors

Türker Bodur ^*
0000-0002-5549-8200
Türkiye

Meryem Cansu Yeşiltaş
0000-0003-0794-3151
Türkiye

Bahar Eldemir
0000-0002-2272-9471
Türkiye

Early Pub Date

April 21, 2026

Publication Date

-

Submission Date

February 20, 2026

Acceptance Date

April 8, 2026

Published in Issue

Year 2026 Number: Advanced Online Publication

DOI

https://doi.org/10.33988/auvfd.1893780

IZ

https://izlik.org/JA36DT45EX

Cite

RIS / Bibtex

APA

Bodur, T., Yeşiltaş, M. C., & Eldemir, B. (2026). Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis. Ankara Üniversitesi Veteriner Fakültesi Dergisi, Advanced Online Publication. https://doi.org/10.33988/auvfd.1893780

AMA

1.Bodur T, Yeşiltaş MC, Eldemir B. Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis. Ankara Univ Vet Fak Derg. 2026;(Advanced Online Publication). doi:10.33988/auvfd.1893780

Chicago

Bodur, Türker, Meryem Cansu Yeşiltaş, and Bahar Eldemir. 2026. “Dynamic Multiplex PCR: A Pioneering Approach to Rapid, Cost-Effective, and High-Quality Microsatellite Fragment Analysis”. Ankara Üniversitesi Veteriner Fakültesi Dergisi, no. Advanced Online Publication. https://doi.org/10.33988/auvfd.1893780.

EndNote

Bodur T, Yeşiltaş MC, Eldemir B (April 1, 2026) Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis. Ankara Üniversitesi Veteriner Fakültesi Dergisi Advanced Online Publication

IEEE

[1]T. Bodur, M. C. Yeşiltaş, and B. Eldemir, “Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis”, Ankara Univ Vet Fak Derg, no. Advanced Online Publication, Apr. 2026, doi: 10.33988/auvfd.1893780.

ISNAD

Bodur, Türker - Yeşiltaş, Meryem Cansu - Eldemir, Bahar. “Dynamic Multiplex PCR: A Pioneering Approach to Rapid, Cost-Effective, and High-Quality Microsatellite Fragment Analysis”. Ankara Üniversitesi Veteriner Fakültesi Dergisi. Advanced Online Publication (April 1, 2026). https://doi.org/10.33988/auvfd.1893780.

JAMA

1.Bodur T, Yeşiltaş MC, Eldemir B. Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis. Ankara Univ Vet Fak Derg. 2026. doi:10.33988/auvfd.1893780.

MLA

Bodur, Türker, et al. “Dynamic Multiplex PCR: A Pioneering Approach to Rapid, Cost-Effective, and High-Quality Microsatellite Fragment Analysis”. Ankara Üniversitesi Veteriner Fakültesi Dergisi, no. Advanced Online Publication, Apr. 2026, doi:10.33988/auvfd.1893780.

Vancouver

1.Türker Bodur, Meryem Cansu Yeşiltaş, Bahar Eldemir. Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis. Ankara Univ Vet Fak Derg. 2026 Apr. 1;(Advanced Online Publication). doi:10.33988/auvfd.1893780