Abstract View

Author(s): Naveen Dewangan*1, Khemraj Sahu2, Harish Bhardwaj3

Email(s): 1naveendkumardew2@gmail.com, 2khemrajsahu1197@gamil.com, 3harishbhardwaj808@gmail.com

Address:

    1School of Studies of Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    2School of Studies of Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    33 University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    *Corresponding Author Email- naveendkumardew2@gmail.com

Published In:   Volume - 2,      Issue - 1,     Year - 2020

DOI: 10.52228/NBW-JAAB.2020-2-1-3  

 View HTML        View PDF

Please allow Pop-Up for this website to view PDF file.

ABSTRACT:
Polymerase Chain Reaction (PCR) has revolutionized the field of molecular biology since its invention in 1983. The technique has enabled scientists to amplify specific sequences of the genomic DNA for various applications, from basic research to clinical diagnosis. However, like any other experimental protocol, PCR has its limitations and challenges. Troubleshooting and optimization are crucial steps for achieving consistent and reliable results in PCR. This review provides an overview of the history of PCR, highlighting its evolution over the past four decades. The article also discusses common troubleshooting strategies and optimization methods that researchers use to overcome obstacles in PCR experiments. By understanding the principles and techniques of PCR optimization, scientists can design experiments that efficiently amplify specific DNA sequences, minimize non-specific amplification, and improve the overall accuracy and sensitivity of the results.

Cite this article:
Naveen Dewangan, Khemraj Sahu, Harish Bhardwaj (2020) Revolutionizing Molecular Biology: The Evolution of PCR through Troubleshooting and Optimization. NewBioWorld A Journal of Alumni Association of Biotechnology, 2(1):1-17.DOI: https://doi.org/10.52228/NBW-JAAB.2020-2-1-3


Araujo, R. P., and Franco, C. (2019). Tracking beneficial Streptomyces strains in plant shoot, roots and rhizosphere soils.

Bartlett, J. M., and Stirling, D. (Eds.). (2003). PCR protocols (Vol. 226, pp. 3-525). Totowa, NJ, USA:: Humana Press.

Brandes, J. C., Carraway, H., and Herman, J. G. (2007). Optimal primer design using the novel primer design program: MSPprimer provides accurate methylation analysis of the ATM promoter. Oncogene26(42), 6229-6237.

Brownie, J., Shawcross, S., Theaker, J., Whitcombe, D., Ferrie, R., Newton, C., and Little, S. (1997). The elimination of primer-dimer accumulation in PCR. Nucleic acids research25(16), 3235-3241.

Bustin, S. A., Beaulieu, J. F., Huggett, J., Jaggi, R., Kibenge, F. S., Olsvik, P. A., Penning L.C., and Toegel, S. (2010). MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC molecular biology11(1), 1-5.

Cheung, H. H., Lee, T. L., Rennert, O. M., and Chan, W. Y. (2009). DNA methylation of cancer genome. Birth Defects Research Part C: Embryo Today: Reviews87(4), 335-350.

Chiang, Y. C., Lai, C. H., Lin, C. W., Chang, C. Y., and Tsen, H. Y. (2014). Improvement of strain discrimination by combination of superantigen profiles, PFGE, and RAPD for Staphylococcus aureus isolates from clinical samples and food-poisoning cases. Foodborne pathogens and disease11(6), 468-477.

Corless, C. E., Guiver, M., Borrow, R., Edwards-Jones, V., Kaczmarski, E. B., and Fox, A. J. (2000). Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. Journal of clinical microbiology38(5), 1747-1752.

Demba Diallo, M., Willems, A., Vloemans, N., Cousin, S., Vandekerckhove, T. T., De Lajudie, P., Gillis, M., and Van der Gucht, K. (2004). Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N2‐fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal. Environmental Microbiology6(4), 400-415.

Demeke, T., and Jenkins, G. R. (2010). Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Analytical and bioanalytical chemistry396, 1977-1990.

Dieffenbach, C. W., and Dveksler, G. S. (2003). PCR primer: a laboratory manual (No. Ed. 2). Cold Spring Harbor Laboratory Press.

Green, S. J., Venkatramanan, R., and Naqib, A. (2015). Deconstructing the polymerase chain reaction: understanding and correcting bias associated with primer degeneracies and primer-template mismatches. PloS one10(5), e0128122.

Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. (1993). Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Bio/technology11(9), 1026-1030.

Hoshino, Y. T., and Morimoto, S. (2008). Comparison of 18S rDNA primers for estimating fungal diversity in agricultural soils using polymerase chain reaction-denaturing gradient gel electrophoresis. Soil science and plant nutrition54(5), 701-710.

Huggett, J. F., Cowen, S., and Foy, C. A. (2015). Considerations for digital PCR as an accurate molecular diagnostic tool. Clinical chemistry61(1), 79-88.

Ishino, S., and Ishino, Y. (2014). DNA polymerases as useful reagents for biotechnology–the history of developmental research in the field. Frontiers in microbiology5, 465.

Jain, M., Nijhawan, A., Tyagi, A. K., and Khurana, J. P. (2006). Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochemical and biophysical research communications345(2), 646-651.

Joshi, M., and Deshpande, J. D. (2010). Polymerase chain reaction: methods, principles and application. International Journal of Biomedical Research2(1), 81-97.

Kalendar, R., Lee, D., and Schulman, A. H. (2009). FastPCR software for PCR primer and probe design and repeat search. Genes, Genomes and Genomics3(1), 1-14.

Kaltenboeck, B., and Wang, C. (2005). Advances in real‐time PCR: Application to clinical laboratory diagnostics. Advances in clinical chemistry40, 219.

Kashyap, V. K., Sitalaximi, T., Chattopadhyay, P., and Trivedi, R. (2004). DNA profiling technologies in forensic analysis. International Journal of Human Genetics4(1), 11-30.

Khamlor, T., Pongpiachan, P., Parnpai, R., Punyawai, K., Sangsritavong, S., and Chokesajjawatee, N. (2015). Bovine embryo sex determination by multiplex loop-mediated isothermal amplification. Theriogenology83(5), 891-896.

Kim, H. S., and Smithies, O. (1988). Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Research16(18), 8887-8903.

Kumar, D., and Thakur, S. (2018). Molecular tools to study preharvest food safety challenges. Preharvest Food Safety, 361-382.

Lianidou, E. S. (2016). Gene expression profiling and DNA methylation analyses of CTCs. Molecular oncology10(3), 431-442.

Liu, H., and Naismith, J. H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC biotechnology8(1), 1-10.

Llop, P., Bonaterra, A., Penalver, J., and Lopez, M. M. (2000). Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material. Applied and environmental microbiology66(5), 2071-2078.

Lorenz, T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. JoVE (Journal of Visualized Experiments), (63), e3998.

Malorny, B., Paccassoni, E., Fach, P., Bunge, C., Martin, A., and Helmuth, R. (2004). Diagnostic real-time PCR for detection of Salmonella in food. Applied and environmental microbiology70(12), 7046-7052.

Mao, X., Liu, C., Tong, H., Chen, Y., and Liu, K. (2019). Principles of digital PCR and its applications in current obstetrical and gynecological diseases. American journal of translational research11(12), 7209.

Matsuda, K. (2017). PCR-based detection methods for single-nucleotide polymorphism or mutation: real-time PCR and its substantial contribution toward technological refinement. Advances in clinical chemistry80, 45-72.

Moritz, C., and Cicero, C. (2004). DNA barcoding: promise and pitfalls. PLoS biology2(10), e354.

Mullis, K. B. (1990). The unusual origin of the polymerase chain reaction. Scientific American262(4), 56-65.

Mullis, K. B. (1994). The polymerase chain reaction (Vol. 41, No. 5). Springer science and business media.

Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1992). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Biotechnology Series, 17-17.

Murphy, J., and Bustin, S. A. (2009). Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard. Expert review of molecular diagnostics9(2), 187-197.

Neff, M. M., Neff, J. D., Chory, J., and Pepper, A. E. (1998). dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. The Plant Journal14(3), 387-392.

Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., and Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic acids research28(12), e63-e63.

Orlando, L., Gilbert, M. T. P., and Willerslev, E. (2015). Reconstructing ancient genomes and epigenomes. Nature Reviews Genetics16(7), 395-408.

Overbergh, L., Giulietti, A., Valckx, D., Decallonne, B., Bouillon, R., and Mathieu, C. (2003). The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. Journal of biomolecular techniques: JBT14(1), 33.

Parida, M., Sannarangaiah, S., Dash, P. K., Rao, P. V. L., and Morita, K. (2008). Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Reviews in medical virology18(6), 407-421.

Patton, W. F. (2000). A thousand points of light: The application of fluorescence detection technologies to two‐dimensional gel electrophoresis and proteomics. ELECTROPHORESIS: An International Journal21(6), 1123-1144.

Radstrom, P., Knutsson, R., Wolffs, P., Lovenklev, M., and Lofstrom, C. (2004). Pre-PCR processing: strategies to generate PCR-compatible samples. Molecular biotechnology26, 133-146.

Rand, K. N., Ho, T., Qu, W., Mitchell, S. M., White, R., Clark, S. J., and Molloy, P. L. (2005). Headloop suppression PCR and its application to selective amplification of methylated DNA sequences. Nucleic acids research33(14), e127-e127.

Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T. (1997). Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Analytical biochemistry245(2), 154-160.

Roux, K. H. (2009). Optimization and troubleshooting in PCR. Cold Spring Harbor Protocols2009(4), pdb-ip66.

Rowlands, V., Rutkowski, A. J., Meuser, E., Carr, T. H., Harrington, E. A., and Barrett, J. C. (2019). Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA. Scientific Reports9(1), 12620.

Rychlik, W. J. S. W., Spencer, W. J., and Rhoads, R. E. (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic acids research18(21), 6409-6412.

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis K.B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science239(4839), 487-491.

Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985). Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science230(4732), 1350-1354.

Sanchez-Freire, V., Ebert, A. D., Kalisky, T., Quake, S. R., and Wu, J. C. (2012). Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns. Nature protocols7(5), 829-838.

Schefe, J. H., Lehmann, K. E., Buschmann, I. R., Unger, T., and Funke-Kaiser, H. (2006). Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula. Journal of molecular medicine84, 901-910.

Schoske, R., Vallone, P. M., Ruitberg, C. M., and Butler, J. M. (2003). Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci. Analytical and bioanalytical chemistry375, 333-343.

Soda, M., Isobe, K., Inoue, A., Maemondo, M., Oizumi, S., Fujita, Y., Takeuchi, K., and North-East Japan Study Group and the ALK Lung Cancer Study Group. (2012). A prospective PCR-based screening for the EML4-ALK oncogene in non–small cell lung cancer. Clinical Cancer Research18(20), 5682-5689.

Syvanen, A. C. (2001). Accessing genetic variation: genotyping single nucleotide polymorphisms. Nature Reviews Genetics2(12), 930-942.

Taylor, J. W., Geiser, D. M., Burt, A., and Koufopanou, V. (1999). The evolutionary biology and population genetics underlying fungal strain typing. Clinical Microbiology Reviews12(1), 126-146.

Templeton, J. E., Brotherton, P. M., Llamas, B., Soubrier, J., Haak, W., Cooper, A., and Austin, J. J. (2013). DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification. Investigative genetics4(1), 1-13.

Wu, J., Matthaei, H., Maitra, A., Dal Molin, M., Wood, L. D., Eshleman, J. R., Edil, B.H., and Vogelstein, B. (2011). Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development. Science translational medicine3(92), 92ra66-92ra66.

 

Related Images:



Recent Images



Comparative antibiotic susceptibility profiling of S. haemolyticus recovered from frequency hand-touched surfaces of hospital settings and urban built environments of central India
Eco-Friendly Agricultural Waste Management through Pleurotus sapidus Cultivation
Morphological diversity of some actinomycetes isolated from Durg district of Chhattisgarh
Exploring the antimicrobial properties of essential oils derived from Nyctanthes arbor-tristis on food borne pathogens
Flavonoids as magical bullets: In silico ADMET profile and molecular docking study against hMPX Virus
Isolation and characterization of multiple Drug resistant bacteria isolated from poultry waste water
Growing Beyond Soil: The Future of Farming with Hydroponics
Survey of Indoor Aeromycoflora of Guru Ghasidas Museum in Summer Season
Nature’s Defender: The Antimicrobial Potential of spices– An Overview
A Study on Polyherbal Skin Ointment: Formulation and Evaluation

Tags


Recomonded Articles:

Author(s): Alka Kaushik*; S.K. Jadhav

DOI: 10.52228/NBW-JAAB.2022-4-2-6         Access: Open Access Read More

Author(s): Mona Tandon; Shailesh Kumar Jadhav; Kishan Lal Tiwari

DOI: 10.52228/NBW-JAAB.2019-1-2-6         Access: Open Access Read More

Author(s): Mona Tandon; Veena Thakur; Kunjlata Sao; Shailesh Kumar Jadhav

DOI: 10.52228/NBW-JAAB.2019-1-1-1         Access: Open Access Read More

Author(s): Naveen Dewangan*; Khemraj Sahu; Harish Bhardwaj

DOI: 10.52228/NBW-JAAB.2020-2-1-3         Access: Open Access Read More

Author(s): Tarun Kumar Patel

DOI: 10.52228/NBW-JAAB.2024-6-1-2         Access: Open Access Read More