Gene Cloning - Advanced

Transcription

1 Gene Cloning - Advanced Douglas Wilkin, Ph.D. Say Thanks to the Authors Click (No sign in required)

2 To access a customizable version of this book, as well as other interactive content, visit AUTHOR Douglas Wilkin, Ph.D. CK-12 Foundation is a non-profit organization with a mission to reduce the cost of textbook materials for the K-12 market both in the U.S. and worldwide. Using an open-source, collaborative, and web-based compilation model, CK-12 pioneers and promotes the creation and distribution of high-quality, adaptive online textbooks that can be mixed, modified and printed (i.e., the FlexBook textbooks). Copyright 2015 CK-12 Foundation, The names CK-12 and CK12 and associated logos and the terms FlexBook and FlexBook Platform (collectively CK-12 Marks ) are trademarks and service marks of CK-12 Foundation and are protected by federal, state, and international laws. Any form of reproduction of this book in any format or medium, in whole or in sections must include the referral attribution link (placed in a visible location) in addition to the following terms. Except as otherwise noted, all CK-12 Content (including CK-12 Curriculum Material) is made available to Users in accordance with the Creative Commons Attribution-Non-Commercial 3.0 Unported (CC BY-NC 3.0) License ( licenses/by-nc/3.0/), as amended and updated by Creative Commons from time to time (the CC License ), which is incorporated herein by this reference. Complete terms can be found at terms-of-use. Printed: December 14, 2015

3 Chapter 1. Gene Cloning - Advanced CHAPTER 1 Gene Cloning - Advanced Describe gene cloning and the processes involved. What does it mean to clone? A genetically exact copy. A clone can be a gene, a cell, an animal or plant. And these clones are produced all the time. Theoretically, a clone could also be a human. But would that be a smart thing to do? Is it ethical? Is it even legal? Gene Cloning You probably have heard of cloning. A clone is a genetically exact copy. It can be a clone of a gene, a cell or an organism. Even a human. However, whereas cloning of humans has many ethical issues associated with it and is illegal throughout most of the world, the cloning of genes has been ongoing for well over 30 or 40 years, with cloning of animals occurring more recently. Gene cloning, also known as molecular cloning, refers to the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. The identical copies are clones. In 1973, Stanley Cohen and Herbert Boyer developed techniques to make recombinant DNA, a form of artificial DNA. They talk about their work in Genes can be moved between species at mation.html. Recombinant DNA is engineered through the combination of two or more DNA strands, combining DNA sequences which would not normally occur together. In other words, selected DNA (or the DNA of "interest") is inserted into an existing organismal genome, such as a bacterial plasmid DNA, or some other sort of vector. The recombinant DNA can then be inserted into another cell, such as a bacterial cell, for amplification and possibly production of the 1

4 resulting protein. This process is called transformation, the genetic alteration of a cell resulting from the uptake, incorporation, and expression of foreign genetic material. Recombinant DNA technology was made possible by the discovery of restriction endonucleases. In The RNA message is sometimes edited at Rich Rioberts and Phil Sharp discuss the development of tools and techniques to analyze DNA. Restriction Enzyme Digestion and Ligation Restriction enzymes or restriction endonucleases are prokaryotic enzymes that recognize and cut DNA at specific sequences, called restriction sites. It is believed that they evolved as a defense mechanism against foreign DNA, such as viral DNA. Over 3,000 restriction enzymes have been identified. Some of the more common restriction enzymes are shown in the table below, where up and down arrows show the sites of cleavage. Restriction enzymes are named based on the prokaryotic organism they are isolated from. For example, those isolated from Escherichia coli would begin with Eco. As the Table 1.1 shows, digestion with the restriction enzymes will result in overlapping or blunt ends. EcoRI produces overlapping "sticky" ends: the enzyme cleaves between the G and A on both strands. On the other hand, SmaII restriction enzyme cleavage produces "blunt" ends. The enzyme cleaves between the G and C on both strands. TABLE 1.1: Common Restriction Endonucleases Enzyme Source Recognition Sequence Restriction Digest EcoRI Escherichia coli 5 GAATTC3 CTTAAG 5 G AATTC 3 3 CTTAA G 5 BamHI Bacillus amyloliquefaciens 5 GGATCC3 CCTAGG 5 G GATCC 3 3 CCTAG G 5 TaqI Thermus aquaticus 5 TCGA3 AGCT 5 T CGA 3 3 AGC T 5 2

5 Chapter 1. Gene Cloning - Advanced TABLE 1.1: (continued) Enzyme Source Recognition Sequence Restriction Digest HinfI Haemophilus influenzae 5 GANTCA3 CTNAGT 5 G ANTC 3 3 CTNA G 5 Sau3A Staphylococcus aureus 5 GATC3 CTAG 5 GATC 3 3 CTAG 5 PvuII Proteus vulgaris 5 CAGCTG3 GTCGAC 5 CAG CTG 3 *3 GTC GAC 5 SmaI Serratia marcescens 5 CCCGGG3 GGGCCC 5 CCC GGG 3 *3 GGG CCC 5 HaeIII Haemophilus aegyptius 5 GGCC3 CCGG 3

6 TABLE 1.1: (continued) Enzyme Source Recognition Sequence Restriction Digest 5 GG CC 3 *3 CC GG 5 Key: N = C or G or T or A = blunt ends Cloning of a segment of DNA of interest can easily be carried out with restriction enzyme digestion, followed by ligation and transformation or transfection. In the classical restriction enzyme digestion and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps: 1. isolation of the DNA of interest (or target DNA), 2. ligation, 3. transfection (or transformation), and 4. a screening/selection procedure. For an overview of cloning, see MEDIA Click image to the left or use the URL below. URL: Isolation of DNA Initially, the DNA fragment to be cloned must be isolated. This DNA of interest may be a gene, part of a gene, a promoter, or another segment of DNA, and is frequently isolated by the polymerase chain reaction (PCR) or restriction enzyme digestion. As discussed above, a restriction enzyme is an enzyme that cuts double-stranded DNA at a specific sequence. The enzyme makes two incisions, one through each strand of the double helix, without damaging the nitrogenous bases. This produces either overlapping ends (also known as sticky ends) or blunt ends. The 1978 Nobel Prize in Medicine was awarded to Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases. The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetics. Ligation Once the DNA of interest is isolated, a ligation procedure is necessary to insert the amplified fragment into a vector to produce the recombinant DNA molecule. Restriction fragments (or a fragment and a plasmid/vector) can be spliced together, provided their sticky ends are complementary. Blunt end ligation is also possible. The plasmid or vector (which is usually circular) is digested with restriction enzymes, opening up the vector to allow insertion of the target DNA. If the isolated DNA of interest and the plasmid or vector are digested with the same 4

7 Chapter 1. Gene Cloning - Advanced restriction enzyme, their sticky ends will be complementary. The two DNAs are then incubated with DNA ligase, an enzyme that can attach together strands of DNA with double strand breaks. This produces a recombinant DNA molecule. Figure 1.1 depicts a plasmid with two additional segments of DNA ligated into the plasmid, producing the recombinant DNA molecule. Figure 1.1 depicts DNA before and after ligation. FIGURE 1.1 (left) This image shows a line drawing of a plasmid. The plasmid is drawn as two concentric circles that are very close together representing the two strands of DNA, with two large segments and one small segment depicted. The two large segments (blue and green) indicate antibiotic resistances usually used in a screening procedure, and the small segment (red) indicates an origin of replication, used in DNA replication. The resulting DNA is a recombinant DNA molecule. (right) Sticky ends produced by restriction enzyme digestion can be joined with the enzyme DNA ligase. Transfection and Selection Following ligation, the recombinant DNA is placed into a host cell, usually bacterial, in a process called transfection or transformation. Finally, the transfected cells are cultured. Many of these cultures may not contain a plasmid with the target DNA as the transfection process is not usually 100% successful, so the appropriate cultures with the DNA of interest must be selected. Many plasmids/vectors include selectable markers - usually some sort of antibiotic resistance ( Figure 1.1). When cultures are grown in the presence of an antibiotic, only bacteria transfected with the vector containing resistance to that antibiotic should grow. However, these selection procedures do not guarantee that the DNA of insert is present in the cells. Further analysis of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of a process PCR or restriction fragment analysis, both of which need to be followed by gel electrophoresis and/or DNA sequencing (DNA sequence analysis). DNA sequence analysis, PCR, or restriction fragment analysis will all determine if the plasmid/vector contains the insert. Restriction fragment analysis is digestion of isolated plasmid/vector DNA with restriction enzymes. If the isolated DNA contains the target DNA, that fragment will be excised by the restriction enzyme digestion. Gel electrophoresis will separate DNA molecules based on size and charge. Examples are shown in Figure 1.2. Gel Electrophoresis Gel electrophoresis is an analytical technique used to separate DNA fragments by size and charge. Notice in Figure 1.2 that the "gels" are rectangular in shape. The gels are made of a gelatin-like material of either agarose or polyacrylamide. An electric field, with a positive charge applied at one end of the gel, and a negative charge at the other end, forces the fragments to migrate through the gel. DNA molecules migrate from negative to positive charges due to the net negative charge of the phosphate groups in the DNA backbone. Longer molecules migrate more slowly through the gel matrix. After the separation is completed, DNA fragments of different lengths can be 5

8 visualized using a fluorescent dye specific for DNA, such as ethidium bromide. The resulting stained gel shows bands correspond to DNA molecules of different lengths, which also correspond to different molecular weights. Band size is usually determined by comparison to DNA ladders containing DNA fragments of known length. Gel electrophoresis can also be used to separate RNA molecules and proteins. FIGURE 1.2 (left) DNA samples in a blue tracking dye are being loaded into wells of an agarose gel prior to electrophoresis. (right) Agarose gel following agarose gel electrophoresis on an UV light box. Recombinant DNA technology is discussed in the following videos and animations: (4:36) MEDIA Click image to the left or use the URL below. URL: (0:50) MEDIA Click image to the left or use the URL below. URL: (7:20) MEDIA Click image to the left or use the URL below. URL: 6

9 Chapter 1. Gene Cloning - Advanced Vocabulary clone: A genetically identical copy; may be a gene, a cell or an organism; an organism that is genetically identical to its parent. DNA ligase: Enzyme that can attach together (ligates) strands of DNA with double strand breaks. gel electrophoresis: An analytical technique used to separate DNA fragments by size and charge; can also be used to separate RNA and proteins. gene cloning: The process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. ligation: The process of forming a bond to join two nucleotide pairs; joining DNA with double strand breaks. plasmid: A small circular piece of DNA that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. polymerase chain reaction (PCR): A repeating series of cycles used to amplify millions of times specific regions of a DNA strand. recombinant DNA: DNA engineered through the combination of two or more DNA strands; combines DNA sequences which would not normally occur together. restriction endonuclease: An enzyme that cuts double-stranded DNA; also known as a restriction enzyme. restriction enzyme: An enzyme that cuts double-stranded DNA; also known as a restriction endonuclease. restriction site: DNA sequence recognized and digested by a restriction enzyme. transfection: The insertion of foreign DNA into a host cell. transformation: The change in genotype and phenotype of a cell/organism due to the assimilation of external DNA (heredity material) by a cell. Summary Gene cloning, also known as molecular cloning, refers to the process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. Classic gene cloning involves the following steps: 1. Restriction enzyme digestion and ligation. 2. Isolation of DNA. 3. Ligation. 4. Transfection and Selection. 5. Gel electrophoresis. 7

10 Practice Use this resource to answer the questions that follow. Gene Cloning at 1. What is gene cloning? 2. What is a gene library? 3. How do restriction enzymes function? 4. What is a plasmid and how is it involved in producing recombinant DNA? 5. Describe how a gene library is screened. Review 1. What are restriction endonucleases? 2. How are gene cloning and recombinant DNA related? 3. Describe the process of gene cloning. 4. How does gel electrophoresis analyze DNA? References 1. Left: Sam McCabe; Right: Zachary Wilson. CK-12 Foundation. CC BY-NC Left: Image copyright phloxii, 2014; Right: Image copyright Jarrod Erbe, Used under licenses from Shutterstock.com 8