Journal article Open Access

Basic Principles of Polymerase Chain Reaction Technology

Tekin, Kemal; Aygar, İsmail Selçuk; Hoşbul, Tuğrul

Özet

Kary Mullis’in 1984 yılında bulduğu ve kendisine Nobel ödülü kazandıran polimeraz zincir reaksiyonu (PCR), spesifik DNA sekanslarının in-vitro DNA sentezi yoluyla eksponensiyel (üstel) olarak çoğaltılabildiği hızlı ve duyarlılığı yüksek bir tekniktir. PCR temel olarak bir DNA çoğaltma yöntemidir, bu yöntemle RNA çoğaltılmak istenirse önce "revers transkriptaz" enzimi kullanılarak hedef RNA sekanslarının DNA kopyaları (komplementer DNA, cDNA) çıkartılır ve PCR ile bu cDNA molekülleri çoğaltılır. PCR'ın kullanılmasıyla belirli bir genetik segmentin, birkaç kalıp DNA molekülünden başlayarak milyonlarca kopyası üretilebilmektedir. Kromozomal DNA'nın in-vivo replikasyonu milyonlarca nükleotidin replikasyonunu kapsar, PCR amplifikasyon ürünleri ise genellikle 1000 bp'den (baz çifti, base pair; bp) daha kısa olacak şekilde dizayn edilir. Bununla beraber ekstrem durumlarda özel termostabil DNA bağımlı DNA polimeraz kombinasyonları kullanılarak 35000 bp'den daha uzun PCR amplikonlarının başarılı olarak çoğaltılabildiği bildirilmiştir. Günümüzde bu yöntemin çeşitli varyasyonları mikrobiyoloji, adli tıp ve genetik bilimlerinde araştırma ve tanı amacıyla yaygın olarak kullanılmaktadır. PCR temelli yöntemlerin önemli kullanım alanları arasında dizi analizi, klonlama, kantitatif hasta izlemi, moleküler ilaç direnç testleri, filogenetik analizler ve salgın yönetimi ve doku uyumluluk testleri gibi uygulamalar yer alır. Bu makalede PCR yönteminin temel prensipleri ele alınmıştır.

Abstract

The polymerase chain reaction (PCR) discovered by Kary Mullis in 1984, which earned him the Nobel prize, is a fast and highly sensitive technique in which specific DNA sequences can be exponentially amplified via in-vitro DNA synthesis. PCR is basically a DNA amplification method, if it is desired to reproduce RNA; DNA copies (complementary DNA, cDNA) of the target RNA sequences are firstly produced by using the enzyme "reverse transcriptase" and these cDNA molecules are amplified by PCR. Using PCR, millions of copies of a specific genetic segment can be produced, starting with several target DNA molecules. In-vivo replication of chromosomal DNA involves replication of millions of nucleotides, while PCR amplification products are generally designed to be less than 1000 bp (base pair; bp). However, in extreme cases it has been reported that PCR amplicons longer than 35000 bp can be successfully amplified using special thermostable DNA-dependent DNA polymerase combinations. Today, various variations of this method are widely used in microbiology, forensics and genetics for research and diagnosis purposes. Important uses of PCR based methods include sequence analysis, cloning, quantitative patient monitoring, molecular drug resistance tests, phylogenetic analysis, and epidemic management and tissue compatibility tests. In this article, basic principles of PCR method are discussed.

Polimeraz Zincir Reaksiyonu Teknolojisinin Temel Prensipleri
Files (747.7 kB)
Name Size
6.JMVI-2020-1.1;57-66.pdf
md5:cbc3ba880dd9a804de97767771d0cad1
747.7 kB Download
  • 1. McPherson MJ, Moller SG, An introduction to PCR (Chapter 1). In: McPherson MJ, Moller SG (eds), PCR: The Basics. 2000, BIOS Scientific Publishers Limited, Oxford, UK. pp:1-7.

  • 2. van Pelt-Verkuil E, van Belkum A, Hays JP. A Brief Comparison Between In Vivo DNA Replication and In Vitro PCR Amplification (Chapter 2). In: van Pelt-Verkuil E, van Belkum A, Hays JP (eds), Principles and Technical Aspects of PCR Amplification. 2008, Springer, New York. pp:9-15.

  • 3. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 1987; 155: 335-50.

  • 4. Lübeck PS, Hoorfar J. PCR Technology and Applications to Zoonotic Food-Borne Bacterial Pathogens. In: Sachse K, Frey J (eds), PCR Detection of Microbial Pathogens: Methods and Protocols. 2003, Humana Press Inc, Totowa, NJ, USA. pp:65-86.

  • 5. Sachse K. Specificity and Performance of Diagnostic PCR Assays. In: Sachse K, Frey J (eds), PCR Detection of Microbial Pathogens: Methods and Protocols. 2003, Humana Press Inc, Totowa, NJ. pp:3-29.

  • 6. Rio DC. Reverse transcription-polymerase chain reaction. Cold Spring Harb Protoc. 2014; 2014(11): 1207‐16.

  • 7. Chang D, Tram K, Li B, Feng Q, Shen Z, Lee CH, et al. Detection of DNA Amplicons of Polymerase Chain Reaction Using Litmus Test. Sci Rep 2017; 7(1): 3110.

  • 8. Chan KC, Lo YM. Introduction to the Polymerase Chain Reaction. In: Lo YM, Chiu RW, Chan KC (eds), Clinical Applications of PCR (2nd edition). 2006, Humana Press, Totowa, New Jersey. pp:1-10.

  • 9. van Pelt-Verkuil E, van Belkum A, Hays JP. The Polymerase Chain Reaction (Chapter 1). In: van Pelt-Verkuil E, van Belkum A, Hays JP (eds), Principles and Technical Aspects of PCR Amplification. 2008, Springer, New York. pp:1-7.

  • 10. Thermo Fisher Scientific, Waltham, Massachusetts, USA. Troubleshooting Guide for PCR. Available at: https://www.thermofisher.com/tr/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-troubleshooting.html [Accessed November 18, 2019].

  • 11. Grunenwald H. Optimization of Polymerase Chain Reactions. In: Bartlett JMS, Stirling D (eds), Methods in Molecular Biology 226, PCR Protocols (2nd edition). 2003, Humana Press, Totowa. pp:89-99.

  • 12. Kralik P, Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front Microbiol 2017; 8: 108.

  • 13. Şahiner F, Kubar A, Yapar M, Şener K, Dede M, Gümral R. Detection of major HPVs by a new multiplex real-time PCR assay using type-specific primers. J Microbiol Methods 2014; 97: 44-50.

  • 14. Şahiner F, Gümral R, Yıldızoğlu Ü, Babayiğit MA, Durmaz A, Yiğit N, Saraçlı MA, Kubar A. Coexistence of Epstein–Barr virus and Parvovirus B19 in tonsillar tissue samples: Quantitative measurement by real-time PCR. Int J Pediatr Otorhinolaryngol 2014; 78(8): 1288-93.

  • 15. van Pelt-Verkuil E, van Belkum A, Hays JP. The PCR in Practice (Chapter 3). In: van Pelt-Verkuil E, van Belkum A, Hays JP (eds), Principles and Technical Aspects of PCR Amplification. 2008, Springer, New York. pp:17-24.

  • 16. Bentley HA, Belloni DR, Tsongalis GJ. Parameters involved in the conversion of real-time PCR assays from the ABI prism 7700 to the Cepheid SmartCycler II. Clin Biochem 2005; 38(2): 183-6.

  • 17. Quan PL, Martin Sauzade M, Brouzes E. dPCR: A Technology Review. Sensors (Basel) 2018; 18(4): 1271.

  • 18. Thermo Fisher Scientific, Waltham, Massachusetts, USA. Troubleshooting Guide for PCR. Available at: https://www.thermofisher.com/tr/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-basics.html [Accessed November 18, 2019].

  • 19. van Pelt-Verkuil E, van Belkum A, Hays JP. Deoxynucleotide Triphosphates and Buffer Components (Chapter 6). In: van Pelt-Verkuil E, van Belkum A, Hays JP (eds), Principles and Technical Aspects of PCR Amplification. 2008, Springer, New York. pp:91-102.

  • 20. Nagai H, Murakami Y, Morita Y, Yokoyama K, Tamiya E. Development of a microchamber array for picoliter PCR. Anal Chem 2001; 73(5): 1043-7.

  • 21. Brody JR, Kern SE. History and principles of conductive media for standard DNA electrophoresis. Anal Biochem 2004; 333(1): 1-13.

  • 22. Kreader CA. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl Environ Microbiol 1996; 62(3): 1102-6.

  • 23. Radström P, Knutsson R, Wolffs P, Dahlenborg M, Löfström C. Pre-PCR Processing of Samples. In: Sachse K, Frey J (eds), PCR Detection of Microbial Pathogens: Methods and Protocols. 2003, Humana Press Inc, Totowa, NJ. pp:31-50.

  • 24. Jenkins FJ. Basic methods for the detection of PCR products. PCR Methods Appl 1994; 3(5): S77-82.

  • 25. Liu D. Chapter 1: Introductory Remarks. In: Liu D (ed), Molecular Detection of Human Bacterial Pathogens. 2011, CRC press, USA. pp:1-12.

34
32
views
downloads
Views 34
Downloads 32
Data volume 23.9 MB
Unique views 32
Unique downloads 31

Share

Cite as