PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker CHAPTER 18 Part 1 RECOMBINANT DNA TECHNOLOGY Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

18-4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Cloning Experiments Involve Chromosomal and Vector DNA Cloning experiments usually involve two kinds of DNA molecules Chromosomal DNA or cDNA Serves as the source of the DNA segment of interest Vector DNA Serves as the carrier of the DNA segment that is to be cloned 18-5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The cell that harbors the vector is called the host cell When a vector is replicated inside a host cell, the DNA that it carries is also replicated The vectors commonly used in gene cloning were originally derived from two natural sources 1. Plasmids 2. Viruses Commercially available plasmids have selectable markers Typically, genes conferring antibiotic resistance to the host cell Table 18.2 provides a general description of several vectors used to clone small segments of DNA 18-6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Cloning Experiments Involve Enzymes that Cut and Paste DNA Insertion of chromosomal DNA into a vector requires the cutting and pasting of DNA fragments The enzymes used to cut DNA are known as restriction endonucleases or restriction enzymes These bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand Figure 18.1 shows the action of a restriction endonuclease 18-8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

To be continued  Figure 18.1 18-9

Restriction enzymes are made naturally by many species of bacteria They protect bacterial cells from invasion by foreign DNA, particularly that of bacteriophage Currently, several hundred different restriction enzymes are available commercially Table 18.3 gives a few examples 18-10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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Restriction enzymes bind to specific DNA sequences These are typically palindromic For example, the EcoRI recognition sequence is 5’ GAATTC 3’ 3’ CTTAAG 5’ Some restriction enzymes digest DNA into fragments with “sticky ends” Other restriction enzymes generate blunt ends 18-12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 19-2 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 19-2 Some common restriction enzymes, with their recognition sequences, cleavage patterns, and sources. Figure 19-2 Copyright © 2006 Pearson Prentice Hall, Inc.

A recombinant DNA molecule This interaction is not stable because it involves only a few hydrogen bonds To establish a permanent connection, the sugar-phosphate backbones of the two DNA fragments must be covalently linked Add DNA ligase which covalently links the DNA backbones A recombinant DNA molecule Figure 18.1 18-13

The Steps in Gene Cloning The general strategy followed in a typical cloning experiment is outlined in Figure 18.2 The procedure shown seeks to clone the human b-globin gene into a plasmid vector The vector carries two important genes ampR  Confers antibiotic resistance to the host cell lacZ  Encodes b-galactosidase Provides a means by which bacteria that have picked up the cloned gene can be identified 18-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 19-6 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 19-6 A diagram of the plasmid pUC18 showing the polylinker region, located within a lacZ gene. DNA inserted into the polylinker region disrupts the lacZ gene, resulting in white colonies that allow direct identification of bacterial colonies carrying cloned DNA inserts. Figure 19-6 Copyright © 2006 Pearson Prentice Hall, Inc.

This is termed a hybrid vector Note: In this case, the b-globin gene was inserted into the plasmid It is also possible for any other DNA fragment to be inserted into the plasmid And it is possible for the plasmid to circularize without an insert This is called a recircularized vector This is termed a hybrid vector Figure 18.2 18-15

18-16 Figure 18.2 This step of the procedure is termed transformation. Cells that are able to take up DNA are called competent cells Figure 18.2 18-16

Nonrecombinant: recircularized Recombinant: vector plus inserted cloned gene Selection for vector: ampicillin resistance Selection for recombinant vs. nonrecombinant vector: b-galactosidase activity Selection for for gene of interest? 18-17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The growth media contains two relevant compounds: IPTG (isopropyl-b-D-thiogalactopyranoside) A lactose analogue that can induce the lacZ gene X-Gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside) A colorless compound that is cleaved by b-galactosidase into a blue dye The color of bacterial colonies will therefore depend on whether or not the b-galactosidase is functional If it is, the colonies will be blue If not, the colonies will be white In this experiment Bacterial colonies with recircularized vectors form blue colonies While those with hybrid vectors form white colonies 18-18 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Recombinant DNA technology is not only used to clone genes The net result of gene cloning is to produce an enormous amount of copies of a gene During transformation, a single bacterial cell usually takes up a single copy of the hybrid vector Amplification of the gene occurs in two ways: 1. The vector gets replicated by the host cell many times 2. The bacterial cell divides approximately every 30 minutes Recombinant DNA technology is not only used to clone genes Sequences such as telomeres, centromeres and highly repetitive sequences can be cloned as well 18-19 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Experiment 18A: The First Gene Cloning Experiment This was accomplished by Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling in 1973 Several important discoveries led to their ability to clone a gene DNA ligase covalently links DNA fragments together EcoRI produces sticky ends when digesting DNA Cohen et al realized that it is possible to create recombinant DNA molecules using EcoRI then DNA ligase 18-20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Tetracycline resistance They chose a plasmid for their vector The plasmid was designated pSC101 The insertion of the gene will occur at the lone EcoRI site As source of the gene, they obtained a second plasmid They called it pSC102 One of the three EcoRI fragments is expected to carry the KanR gene Kanamycin resistance 18-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Testing the Hypothesis A piece of DNA carrying a gene can be inserted into a plasmid vector using recombinant DNA techniques If this recombinant plasmid is introduced into a bacterial host cell, it will be replicated and transmitted to daughter cells, producing many copies of the recombinant plasmid Testing the Hypothesis Refer to Figure 18.3 18-22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 18.3 18-23

Figure 18.3 18-24

Figure 18.3 18-25

Figure 18.3 18-26

Figure 18.3 18-27

Density Gradient Centrifugation Single peak The Data A single plasmid with intermediate density; NOT a mixture of two plasmids Density Gradient Centrifugation Control Experiment pSC102 pSC101 18-28 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

This band corresponds to pSC101 This band is also found in pSC102 The Data Gel Electrophoresis This band corresponds to pSC101 This band is also found in pSC102 18-29

The recombinant plasmid is shown here This experiment showed it is possible to create recombinant DNA molecules and to propagate them in bacterial cells This hallmark achievement ushered in the era of gene cloning 18-30 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

cDNA To clone DNA, one can start with a sample of RNA The enzyme reverse transcriptase is used Uses RNA as a template to make a complementary strand of DNA DNA that is made from RNA is called complementary DNA (cDNA) It could be single- or double-stranded Synthesis of cDNA is presented in Figure 18.4 18-31 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

polyA tail Figure 18.4 18-32

This has two ramifications From a research perspective, an important advantage of cDNA is that it lacks introns This has two ramifications 1. It allows researchers to focus their attention on the coding sequence of a gene 2. It allows the expression of the encoded protein Especially, in cells that would not splice out the introns properly (e.g., a bacterial cell) 18-33 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Gel electrophoresis Nucleic acid electrophoresis separates DNA and RNA fragments by size smaller fragments migrate at a faster rate through a gel than large fragments.

Figure 10-27 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 10-27 Electrophoretic separation of a mixture of DNA fragments that vary in length. The photograph shows an agarose gel with DNA bands corresponding to the diagram. Figure 10-27 Copyright © 2006 Pearson Prentice Hall, Inc.

Restriction Mapping Sometimes, it is necessary to obtain smaller clones from a large chromosomal DNA insert This process is termed subcloning Cloning and subcloning require knowledge of the locations of restriction enzyme sites in vectors and hybrid vectors A common approach to examine the locations of restriction sites is known as restriction mapping Figure 18.5 outlines the restriction mapping of a bacterial plasmid, pBR322 18-34 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 18.5 18-35

Used for fragment size comparison Figure 18.5 18-36

The restriction map can be deduced by comparing the sizes of DNA fragments obtained from the single, double and triple digestions Figure 18.5 4,363 bp 18-37 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 19-22 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 19-22 An agarose gel containing separated DNA fragments stained with a dye (ethidium bromide) and visualized under ultraviolet light. Smaller fragments migrate faster and farther than do larger fragments, resulting in the distribution shown. Figure 19-22 Copyright © 2006 Pearson Prentice Hall, Inc.

Figure 19-23 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 19-23 Constructing a restriction map. Samples of the 7.0-kb DNA fragments are digested with restriction enzymes: One sample is digested with HindIII, one with SalI, and one with both HindIII and SalI. The resulting fragments are separated by gel electrophoresis. The separated fragments are measured by comparing them with molecular-weight standards in an adjacent lane. Cutting the DNA with HindIII generates two fragments: 0.8 kb and 6.2 kb. Cutting with SalI produces two fragments: 1.2 kb and 5.8 kb. Models are constructed to predict the fragment sizes generated by cutting with HindIII and with SalI. Model 1 predicts that 0.4-, 0.8-, and 5.8-kb fragments will result from cutting with both enzymes. Model 2 predicts that 0.8-, 1.2-, and 5.0-kb fragments will result. Comparing the predicted fragments with those observed on the gel indicates that model 1 is the correct restriction map. Figure 19-23 Copyright © 2006 Pearson Prentice Hall, Inc.