Rekombinant DNA Teknolojisi
Özet
Rekombinant DNA teknolojisi, farklı kaynaklardan elde edilen DNA parçalarının laboratuvar koşullarında kontrollü biçimde birleştirilmesini sağlayan yöntemler bütünüdür ve genetik mühendisliğinin temel aracı konumundadır. Bu bölüm, alanın 1970'li yıllardaki doğuşundan (tip II restriksiyon endonükleazların keşfi, ilk rekombinant DNA molekülü, Cohen ve Boyer'in klonlama çalışmaları, rekombinant insülinin piyasaya sürülmesi) başlayarak günümüz uygulamalarına uzanan bütüncül bir çerçeve sunmaktadır. Gen klonlamanın altı temel adımı tanımlandıktan sonra teknolojinin moleküler araçları ayrıntılı biçimde ele alınmaktadır: restriksiyon enzimlerinin sınıflandırılması, isimlendirilmesi ile küt ve yapışkan uç oluşumu, T4 DNA ligazın çalışma mekanizması, insert:vektör oranı ve alkalin fosfataz uygulaması, klonlama vektörlerinin özellikleri ve plazmitlerden mekik vektörlere, lambda ve M13 fajlarından kozmid, BAC ve YAC sistemlerine uzanan karşılaştırmalı değerlendirilmesi. Hedef DNA'nın eldesi, transformasyon, elektroporasyon, transfeksiyon ve mikroenjeksiyon gibi aktarım yöntemleri ile antibiyotik seçilimi, mavi-beyaz koloni taraması, koloni PCR ve hibridizasyon temelli tarama stratejileri açıklanmaktadır. Genomik ve cDNA kütüphaneleri karşılaştırılmakta; PCR, RT-PCR ve qPCR'ın ilkeleri ile klonlamadaki işlevleri tartışılmaktadır. Bölüm, tıp, tarım, hayvancılık ve endüstriyel biyoteknolojideki uygulama örnekleriyle tamamlanmaktadır.
Recombinant DNA technology comprises the set of methods that allow DNA fragments from different sources to be joined in a controlled manner under laboratory conditions, and it remains the central tool of genetic engineering. This chapter provides an integrated framework extending from the field's origins in the 1970s, the discovery of type II restriction endonucleases, the construction of the first recombinant DNA molecule, the cloning experiments of Cohen and Boyer, and the marketing of recombinant insulin through to contemporary applications. After outlining the six essential steps of gene cloning, the molecular toolkit is examined in detail: the classification and nomenclature of restriction enzymes and the generation of blunt and sticky ends; the mechanism of T4 DNA ligase, insert:vector ratios and alkaline phosphatase treatment, and the properties of cloning vectors, with a comparative survey spanning plasmids and shuttle vectors, lambda and M13 phages, cosmids, BACs and YACs. Target DNA preparation, delivery methods including transformation, electroporation, transfection and microinjection, and selection strategies such as antibiotic resistance, blue-white screening, colony PCR and hybridisation are described. Genomic and cDNA libraries are compared, and the principles and cloning applications of PCR, RT-PCR and qPCR are discussed. The chapter concludes with applications in medicine, agriculture, animal production and industrial biotechnology.
Referanslar
Brown TA. Gene cloning and DNA analysis: An introduction. 7th ed. Oxford: Wiley-Blackwell; 2016.
Smith HO, Wilcox KW. A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol. 1970;51(2):379–91.
Kelly TJ Jr, Smith HO. A restriction enzyme from Hemophilus influenzae. II. Base sequence of the recognition site. J Mol Biol. 1970;51(2):393–409.
Jackson DA, Symons RH, Berg P. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc Natl Acad Sci USA. 1972;69(10):2904–9.
Cohen SN, Chang AC, Boyer HW, Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA. 1973;70(11):3240–4.
Johnson IS. Human insulin from recombinant DNA technology. Science. 1983;219(4585):632–7.
Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, et al. Molecular biology of the cell. 6th ed. New York: Garland Science; 2015.
Arber W. Restriction endonucleases. Angew Chem Int Ed Engl. 1978;17(2):73–9.
Smith HO, Nathans D. A suggested nomenclature for bacterial host modification and restriction systems and their enzymes. J Mol Biol. 1973;81(3):419–23.
Roberts RJ. How restriction enzymes became the workhorses of molecular biology. Proc Natl Acad Sci USA. 2005;102(17):5905–8.
Primrose SB, Twyman RM. Principles of gene manipulation and genomics. 7th ed. Oxford: Blackwell Publishing; 2006.
Winnacker EL. From genes to clones: introduction to gene technology. Weinheim: VCH Publishers; 1987.
Lodish H, Berk A, Kaiser CA, Krieger M, Bretscher A, Ploegh H, et al. Molecular cell biology. 9th ed. New York: W. H. Freeman and Company; 2021.
Lewin B, Krebs JE, Goldstein ES, Kilpatrick ST. Lewin's genes XI. Burlington: Jones & Bartlett Learning; 2014.
Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2001.
Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990;262(4):56–65.
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239(4839):487–91.
Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol. 1976;127(3):1550–7.
Walsh G. Biopharmaceuticals: biochemistry and biotechnology. 2nd ed. Chichester: John Wiley & Sons; 2003.
Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science. 2000;287(5451):303–5.
Glick BR, Pasternak JJ, Patten CL. Molecular biotechnology: principles and applications of recombinant DNA. 4th ed. Washington DC: ASM Press; 2010.
