基因组24DNA研究.docVIP

4. Studying DNALearning outcomes When you have read Chapter 4, you should be able to: Give outline descriptions of the events involved in DNA cloning and the polymerase chain reaction (PCR), and state the applications and limitations of these techniques Describe the activities and main applications of the different types of enzyme used in recombinant DNA research Identify the important features of DNA polymerases and distinguish between the various DNA polymerases used in genomics research Describe, with examples, the way that restriction endonucleases cut DNA and explain how the results of a restriction digest are examined Distinguish between blunt- and sticky-end ligation and explain how the efficiency of blunt-end ligation can be increased Give details of the key features of plasmid cloning vectors and describe how these vectors are used in cloning experiments, using pBR322 and pUC8 as examples Describe how bacteriophage λ vectors are used to clone DNA Give examples of vectors used to clone long pieces of DNA, and evaluate the strengths and weaknesses of each type Summarize how DNA is cloned in yeast, animals and plants Describe how a PCR is performed, paying particular attention to the importance of the primers and the temperatures used during the thermal cycling the toolkit of techniques used by molecular biologists to study DNA molecules was assembled during the 1970s and 1980s. Before then, the only way in which individual genes could be studied was by classical genetics, using the procedures that we will examine in Chapter 5. Classical genetics is a powerful approach to gene analysis and many of the fundamental discoveries in molecular biology were made in this way. The operon theory proposed by Jacob and Monod in 1961 (Section 9.3.1), which describes how the expression of some bacterial genes is regulated, was perhaps the most heroic achievement of this era of genetics. But the classical approach is limited because it does not involve the direct