1. Chapter 18 *Lecture Outline Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. *See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes.

2. INTRODUCTION Recombinant DNA technology is the use of in vitro molecular techniques to isolate and manipulate fragments of DNA In the early 1970s, researchers at Stanford University were able to construct chimeric molecules called recombinant DNA molecules –Shortly thereafter, it became possible to introduce such molecules into living cells where they are replicated to make many identical copies –This achievement ushered in the era of gene cloning Recombinant DNA technology and gene cloning have been fundamental to our understanding of gene structure and function 18-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. 18.1 GENE CLONING The term gene cloning refers to the technique of isolating and making many copies of a gene The laboratory methods that are necessary to clone a gene were devised during the early 1970s –Since then, many technical advances have enabled gene cloning to become a widely used procedure in science Table 18.1 summarizes some of the more common uses of gene cloning Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-3

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

5. Cloning experiments usually involve two kinds of DNA molecules Chromosomal DNA Serves as the source of the DNA segment of interest Vector DNA Serves as the carrier for the DNA segment that is to be cloned Can replicate independently of the host chromosomal DNA To prepare chromosomal DNA, the scientist has to Obtain cellular tissue from the organism of interest Break open the cells Extract and purify DNA using a variety of biochemical techniques Cloning Experiments Involve Chromosomal and Vector DNA 18-5

6. 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 sequence of the origin of replication determines whether a vector can replicate in a particular host cell The vectors commonly used in gene cloning were originally derived from two natural sources 1. Plasmids 2. Viruses Many naturally occurring 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

7. 18-7

8. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Insertion of chromosomal DNA into a vector requires the cutting and joining 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 Cloning Experiments Involve Enzymes that Cut and Join DNA 18-8

9. A“sticky end” Incubate both DNAs with EcoRI, which cuts the DNA backbone between G and A. C GAA G CT T T A T A C GAA G CT T T A T A 3′5′ 3′ C GAA G CT T T A T A C GAA G CT T T A T A 5′ 3′ G CT T AA C AA G TT 5′ 3′ G CT T AA C AA G TT 5′3′ DNA from 2 different sources Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ A “sticky end” EcoRI recognition sequence 18-9 Figure 18.1 (partial) Cleavage by restriction enzymes is the first step to making recombinant DNA. In this case, the ends are ‘sticky’ in that they are short, single-stranded regions of DNA that can base- pair with another piece of DNA with complementary sequence (e.g. other DNA cut with the same enzyme)

10. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Restriction enzymes were discovered in the 1960s and 1970s by Werner Arber, Hamilton Smith and Daniel Nathans 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

11. 18-11

12. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Restriction enzymes bind to specific DNA sequences These are typically palindromic The sequence is identical when read in the opposite direction in the complementary strand For example, the EcoRI recognition sequence is 18-12 5’ GAATTC 3’ 3’ CTTAAG 5’ Some restriction enzymes digest DNA into fragments with “sticky ends” (see figure 18.1) These DNA fragments will hydrogen bond to each other due to their complementary sequences Other restriction enzymes generate blunt ends The enzyme NaeI (Refer to Table 18.3)

13. A“sticky end” Incubate both DNAs with EcoRI, which cuts the DNA backbone between G and A. Incubate the DNAs together, allowing sticky ends to hydrogen bond. Add DNA ligase, which covalently links the DNA backbones. C GAA G CT T T A T A C GAA G CT T T A T A 3′5′ 3′ C GAA G CT T T A T A C GAA G CT T T A T A 5′ 3′ G CT T AA C AA G TT 5′ 3′ G CT T AA C AA G TT 5′3′ G CTT AA C AA G TT 5′ G CTT AA C AA G TT 3′ G CT T AA C AA G TT 5′ G CT T AA C AA G TT DNA from 2 different sources Covalent bond A recombinant DNA molecule Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ 3′ 5′ A “sticky end” EcoRI recognition sequence This interaction is not stable because it involves only a few hydrogen bonds Figure 18.1 To establish a permanent connection, the sugar-phosphate backbones of the two DNA fragments must be covalently linked A recombinant DNA molecule 18-13

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

15. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. amp R gene Plasmid DNA Origin of replication lacZ gene Unique restriction site Cut the DNAs with the same restriction enzyme. Mix the DNAs together. Allow time for sticky ends to base-pair. Add DNA ligase to covalently link the DNA backbones. Gene of interest Chromosomal DNA from human cells Vector with the gene of interest Recombinant vectors Vector with another fragment of chromosomal DNA Recircularized vector or 18-15 Figure 18.2 This is termed a hybrid vector Digestion of DNA from a human cell would actually produce tens of thousands of fragments.

16. Mix DNA with many E.coli cells that have been treated with agents that make them permeable to DNA. Plate cells on media containing X-Gal, IPTG, and ampicillin. Incubate overnight. Vector with the gene of interest Recombinant vectors Vector with another fragment of chromosomal DNA Recircularized vector Note: This shows a bacterial cell with the plasmid carrying the gene of interest. Other bacterial cells would have other recombinant vectors or a recircularized vector. Each bacterial colony is derived from a single cell; so all the cells in a colony are genetically identical. Blue colony Recircularized vector without an insert Recombinant vector with an insert White colony E. coli cell without a plasmid or 18-16 Figure 18.2 Cells that are able to take up DNA are called competent cells This step of the procedure is termed transformation when plasmid vectors are used, and transfection when a viral vector is introduced into a host cell Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

17. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display All bacterial colonies growing on the plate had to have picked up the vector and its amp R gene Now need to differentiate between the colonies that have a recircularized vector from those with a hybrid vector This is where the lacZ gene comes into play In the hybrid vector, the chromosomal DNA inserts into the lacZ gene, thereby disrupting it By comparison, the recircularized vector has a functional lacZ gene But how is the functionality of the lacZ gene determined? 18-17

18. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The growth media contains two relevant compounds: IPTG (isopropyl-  -D-thiogalactopyranoside) A lactose analogue that can induce lacZ gene expression X-Gal (5-bromo-4-chloro-3-indolyl-  -D-galactopyranoside) A colorless compound that is cleaved by  -galactosidase into a blue dye The color of bacterial colonies will therefore depend on whether or not the  -galactosidase enzyme 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

19. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 a vector Amplification of a cloned gene occurs in two ways: 1. The vector gets replicated by the host cell many times This will generate a lot of copies per cell (25-50 for plasmids) 2. The bacterial cell divides approximately every 20 minutes This will generate a population of many millions of cells overnight 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

20. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 Used by retroviruses to copy their RNA genome to 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 18.3 cDNA 18-20

21. A T A T A T A T A T A T TTTTTT 5′ 3′ 5′ A T A T A T A T A T A T 3′ AAAAAA 5′ 3′ 5′ A T A T A T A T A T A T 3′ 5′ 3′ 5′ Add reverse transcriptase + dNTPs to synthesize a complementary DNA strand. Add a poly-dT primer that binds to the polyA tail of mRNA. Add RNaseH to cut up the RNA and generate RNA primers. Add DNA polymerase and DNA ligase to synthesize the second DNA strand. Double-stranded cDNA mRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18-21 Figure 18.3 polyA tail

22. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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) 18-22

23. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 18.4 outlines the restriction mapping of a bacterial plasmid, pBR322 Restriction Mapping 18-23

24. 18-24 Figure 18.4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cut the DNA with different restriction enzymes. Place samples in separate tubes. Isolate plasmid DNA from host cells. Plasmid DNA Plasmid DNA (pBR322) Bacterial host cell (all cells carry the same plasmid)

25. 18-25 Used for fragment size comparison Figure 18.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display EcoRI PstI EcoRI BamHI PstI EcoRI BamHI PstI BamHI PstI Markers EcoRIBamHIPstI EcoRIBamHIPstIEcoRI BamHI EcoRI PstI Lane Restriction enzyme(s) added (bp) 4360 4000 3600 3200 2300 1600 1100 750 400 200 12345678 Separate the DNA fragments by gel electrophoresis. EcoRI BamHI PstI Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

26. EcoRI BamHI PstI ~3210 bp ~770 bp ~380 bp Figure 18.4 4,363 bp 18-26 The restriction map can be deduced by comparing the sizes of DNA fragments obtained from the single, double and triple digestions Another way to obtain a restriction map is via DNA sequencing Once the DNA sequence of a vector has been determined, computer programs can scan the sequence and identify restriction enzyme sites Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

27. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Another way to copy DNA is a technique called polymerase chain reaction (PCR) It was developed by Kary Mullis in 1985 Unlike gene cloning, PCR can copy DNA without the aid of vectors and host cells The PCR method is outlined in Figure 18.5 18.2 Polymerase Chain Reaction 18-27

28. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-28 Figure 18.5 5′3′ 5′ 3′ 5′ Site where reverse primer binds Many copies of the gene of interest, flanked by the regions where the primers bind. Gene of interest Site where forward primer binds Template DNA Chromosomal DNA A different primer binding near the other end of the gene Primer binding near one end of the gene Forward primer Reverse primer Denaturation: Separate DNA strands with high temperature. Primer annealing: Lower temperature, which allows primers to bind to template DNA. Many PCR cycles 5′ 3′ 5′ Primer extension: Incubate at a temperature that allows DNA synthesis to occur. 5′ 3′ 5′ (b) The 3 steps of a PCR cycle (a) The outcome of a PCR experiment C G TC A G C G C G C G C G C G C A T A T A T A T AG C G T A G CT 3′5′ 3′ Reverse primer Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

29. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The starting material for PCR includes 1. Template DNA Contains the region that needs to be amplified 2. Oligonucleotide primers Complementary to sequences at the ends of the DNA fragment to be amplified These are synthetic and about 15-20 nucleotides long 3. Deoxynucleoside triphosphates (dNTPs) Provide the precursors for DNA synthesis 4. Taq polymerase DNA polymerase isolated from the bacterium Thermus aquaticus This thermostable enzyme is necessary because PCR involves heating steps that inactivate most other DNA polymerases Refer to Figure 18.6 18-29

30. 18-30 PCR is carried out in a thermocycler, which automates the timing of each cycle All the ingredients are placed in one tube The experimenter sets the machine to operate within a defined temperature range and number of cycles Figure 18.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display During each cycle, the DNA strands are separated via heating. The temperature is then lowered to allow the primers to bind, and a complementary strand is made. Region of interest that will be copied Mix together template DNA, present in low amounts, with dNTPs, Taq polymerase, and 2 primers present in high amounts. Cycle 1 Cycle 2 Cycle 3 Template DNA + + + + Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

31. 18-31 Figure 18.6 The sequential process of denaturing-annealing- synthesis is then repeated for many cycles A typical PCR run is likely to involve 20 to 30 cycles of replication This takes a few hours to complete After 20 cycles, a target DNA sequence will increase 2 20 -fold (~ 1 million-fold) After 30 cycles, a target DNA sequence will increase 2 30 -fold (~ 1 billion-fold) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display With each successive cycle, the relative amount of this type of DNA fragment increases. Therefore, after many cycles, the vast majority of DNA fragments contain only the region that is flanked by the 2 primers. + + + + + + + Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

32. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The PCR reaction shown in Figure 18.5 seeks to amplify a specific DNA segment For this type of experiment, a researcher must have prior knowledge about the sequence of the template DNA Required to construct the synthetic primers PCR can also be used to amplify chromosomal DNA semispecifically or nonspecifically 1. Semispecific approach Primers recognize a repetitive DNA sequence found at several sites within the genome Therefore, many different DNA fragments will be amplified 2. Nonspecific approach A mixture of primers with many different random sequences is used These will anneal randomly throughout the genome and amplify most of the chromosomal DNA 18-32

33. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display PCR is also used to detect and quantitate the amount of an RNA in living cells The method is called reverse transcriptase PCR (RT-PCR) RT-PCR is carried out in the following manner RNA is isolated from a sample It is mixed with reverse transcriptase and a primer that will anneal to the 3’ end of the RNA of interest This generates a single-stranded cDNA which can be used as template DNA in conventional PCR Refer to Figure 18.7 RT-PCR is extraordinarily sensitive It can detect the expression of small amounts of RNA in a single cell 18-33

34. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-34 Figure 18.7 RNA isolated from a sample of cells Double-stranded cDNAs derived from the RNA of interest Primer RNA of interest Add reverse transcriptase, a primer that binds near the 3′ of the RNA of interest, and deoxyribonucleotides 5′ 5’ ′ 5′ 3′ Subject to PCR as described in Figures 18.5 and 18.6 5′ 3′ 5′ 3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

35. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Real-time PCR is carried out in a thermocycler that can measure changes in fluorescence emitted by detector molecules in the PCR reaction mix The TaqMan system uses a detector oligonucleotide that has a fluorescent reporter molecule at one end and a quencher molecule at the other end Due to their proximity, the quencher molecule blocks the fluorescence of the reporter molecule on the oligonucleotide During primer extension, Taq polymerase 5’-3’ exonuclease activity digests the detector oligonucleotide, separating reporter and quencher Fluorescence will increase in proportion to the amount of PCR product produced See Figure 18.8 and 18.9 18-35 Real-Time PCR is used to Quantitate the Amount of a Specific Gene or mRNA

36. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-36 During the primer extension step, the detector is digested by Taq polymerase, which separates the reporter from the quencher. Oligonucleotide that is complementary to one strand of the PCR product During the primer annealing step, both a primer and TaqMan detector bind to the template DNA. (a) TaqMan detector Forward primer TaqMan detector Template DNA that is being amplified Taq polymerase ReporterQuencher (b) Use of a TaqMan detector in real-time PCR 3′ 5′ 3′ Reporter is not quenched 3′ 5′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 18.8

37. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-37 Plateau Linear CtCt Exponential Cycle number 0 15105452520303540 CtCt CtCt CtCt Fluorescence PCR product Cycle (a) Phases of PCR Cycle number 0 15105452520303540 CtCt Fluorescence (c) A comparison between an unknown sample and standards of known concentrations (b) Real-time PCR at high, medium, and low concentrations of the starting template DNA HighMediumLow Cycle threshold Standard at a known high concentration Standard at a known lower concentration Unknown sample CtCt CtCt Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cycle threshold

38. Experiment 18A: Early Attempts at Monitoring the Course of PCR This was accomplished by Russel Higuchi in the early 1990s. Researchers chose the chemical Ethidium Bromide (EtBr) –Highly fluorescent when exposed to UV light –Intercalates between bases in double-stranded DNA –Fluorescence increases when bound to DNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-38

39. Higuchi and colleagues used primers that recognize sequences found on the human Y chromosome Tubes contained a low or high concentration of DNA from a male. As controls, one tube contained female DNA and one had no DNA 18-39 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

40. The Hypothesis –As PCR products are made, the level of fluorescence increases because EtBr is more fluorescent when bound to double-stranded DNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure 18.10 18-40

41. Figure 18.10 18-41 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Experimental level Conceptual level 1. Into each of four tubes, add Taq polymerase, dNTPs, and primers that recognize DNA sequences on the human Y chromosome. Also add ethidium bromide (EtBr) to the tubes. These components are needed to carry out PCR. EtBr will intercalate into double-stranded DNA. 2. Into tubes 1 and 2, add a low and high amount of human male DNA, respectively. Into tube 3, add a high amount of human female DNA. Tube 4 is a control that does not contain any template DNA. 1234 1 Male DNA (2 ng) 2 Male DNA (60 ng) 3 Female DNA (60 ng) 3. Allow PCR to proceed as described in Figures 18.5 and 18.6. The template DNA should be amplified only in the tubes containing human male DNA because the primers are complementary to DNA sequences on the Y chromosome. 4 No DNA Fragments of human male DNA Fragment of Y chromosome Many copies of a DNA region on the Y chromosome Primers binding to a fragment of the Y chromosome PCR

42. Figure 18.10 18-42 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 4. At cycles 17, 21, 25, and 29, remove the PCR tubes from the thermocycler and measure the level of fluorescence with a spectrofluorometer. Note: the tubes remained capped while the fluorescence was being measured. See figures 18.5 and 18.6. EtBr (orange) intercalating in the DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

43. The Data Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-43 No amplification occurred in the control tubes. Fluorescence increased faster with a higher concentration of starting DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cycle number 8 6 4 12 0 10 2 201030 2 ng male 60 ng male 60 ng female No DNA Fluorescence

44. 18.3 DNA LIBRARIES AND BLOTTING METHODS Molecular geneticists usually want to study particular genes within the chromosomes of living species –This presents a problem, because chromosomal DNA contains thousands of different genes –The term gene detection refers to methods that distinguish one particular gene from a mixture of thousands of genes Scientists have also developed techniques to identify gene products –RNA that is transcribed from a particular gene –Protein that is encoded in an mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-44

45. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A DNA library is a collection of thousands of different fragments of DNA, each of which is inserted into a vector When the starting material is chromosomal DNA, the library is called a genomic library A cDNA library contains hybrid vectors with cDNA inserts Should represent the genes expressed in the cells from which the RNA was isolated The construction of a DNA library is shown in Figure 18.11 DNA Libraries 18-45

46. 18-46 Figure 18.11 Mix vectors and DNA fragments under conditions that favor base pairing. Plasmid vectors with a single restriction site Fragment with gene of interest Opened vectors Cleave DNA with restriction enzyme. Chromosomal DNA with many restriction sites Different fragments of chromosomal DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

47. 18-47 Figure 18.11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Plate on petri plates containing the selected antibiotic. Select for bacteria that have taken up a plasmid. (Note: In this experiment, only 1 plasmid is taken up by a bacterium.) Transform bacteria. Treat with DNA ligase to covalently join pieces together. Each bacterial colony contains millions of cells that were derived from a single transformed cell. A collection of many colonies is a DNA library. (a) Making a genomic library Each hybrid vector contains a different fragment of chromosomal DNA. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

48. 18-48 Figure 18.11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A cDNA library can be made from mRNA Make cDNAs as described in Figure 18.3. Isolate mRNAs from a sample of cells. Attach oligonucleotide linkers to ends of cDNAs using DNA ligase. Cut cDNAs and plasmid DNA with a restriction enzyme and ligate the cDNAs into vectors. Transform bacteria. Place on petri plates containing the selected antibiotic. (b) Making a cDNA library Recombinant plasmid with a cDNA insert cDNA mRNA Linker DNA, which has a sequence that is recognized by a particular restriction enzyme Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

49. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In most cloning experiments, the ultimate goal is to clone a specific gene For example, suppose that a geneticist wishes to clone the rat  -globin gene Only a small percentage of the hybrid vectors in a DNA library would actually contain the gene Therefore, geneticists must have a way to distinguish those rare colonies from all the others This can be accomplished by using a DNA probe in a procedure called colony hybridization Refer to Figure 18.12 18-49

50. 18-50 Figure 18.12 Master plate Nylon membrane A nylon membrane is gently laid onto the master plate and lifted, yielding a replica of the master plate. The membrane is treated with detergent to permeabilize the bacteria, and the DNA is fixed to the membrane. NaOH is added to denature the DNA. The membrane is submerged in a solution containing a radiolabeled probe that is complementary to the β-globin gene. Based on the orientation of the membrane and X-ray film (see X), the colonies containing the β-globin gene are identified on the master plate. The membrane is washed to remove unbound probe and then placed next to X-ray film. X-ray film Master plate (see above) Colonies containing the cloned β-globin gene Radiolabeled probe Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. β-globin gene in a bacterial colony

51. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display But how does one obtain the probe? If the gene of interest has been already cloned, a piece of it can be used as the probe If not, one strategy is to use a probe that likely has a sequence similar to the gene of interest For example, use the rat  -globin gene to probe for the  -globin gene from another rodent What if a scientist is looking for a novel gene that no one has ever cloned from any species? If the protein of interest has been previously isolated, amino acid sequence is obtained from it The researcher can use the amino acid sequence to design short DNA probes that can bind to the protein’s DNA coding sequence 18-51

52. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Southern blotting can detect the presence of a particular gene sequence within a complex genetic background It was developed by E. M. Southern in 1975 Southern blotting has several uses 1. It can determine copy number of a gene in a genome 2. It can detect small gene deletions that cannot be detected by light microscopy 3. It can identify gene families 4. It can identify homologous genes among different species 5. It can determine if a transgenic organism is carrying a new or modified gene Southern Blotting 18-52

53. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Prior to a Southern blotting experiment, the gene of interest, or a fragment of the gene, has been cloned This cloned DNA is labeled (e.g., radiolabeled) and used as a probe The probe will be able to detect the gene of interest within a mixture of many DNA fragments The technique of Southern Blotting is shown in Figure 18.13 18-53

54. The fragments are separated by gel electrophoresis, and then denatured. A sample of chromosomal DNA is digested into small fragments with a restriction enzyme. As shown in parts b and c, the DNA bands are transferred (blotted) to a nylon membrane. After transfer, the DNA is permanently attached to the membrane. The membrane is placed in a solution containing a radiolabeled probe. The binding can be done under conditions of low or high stringency. Excess probe is washed away, and the membrane is exposed to X-ray film. Gel Nylon membrane X-ray film (a) The steps in Southern blotting(c) The transfer step via electrophoresis (b) Transfer step (traditional method) Low stringency High stringency Lid Cathode plate Blotting paper Gel Nylon membrane Blotting paper Anode plate Base + – Weight Dry paper towels Glass plate Blotting paper Nylon membrane Gel Transfer solution Support for blotting paper and gel Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18-54 An alternative type of transfer uses a vaccuum Figure 18.13

55. 18-55 Figure 18.13 a) The steps in Southern blotting A common labeling method is the use of the radioisotope 32 P Conditions of high temperature and/or low salt concentration Probe DNA and chromosomal fragment must be nearly identical to hybridize Conditions of low temperature and/or high salt concentration Probe DNA and chromosomal fragment must be similar but not necessarily identical to hybridize Gene of interest is found only in single copy in the genome Gene is member of a gene family composed of three distinct members Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The membrane is placed in a solution containing a radiolabeled probe. The binding can be done under conditions of low or high stringency. Excess probe is washed away, and the membrane is exposed to X-ray film. Nylon membrane X-ray film Low stringency High stringency

56. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Northern blotting is used to identify a specific RNA within a mixture of many RNA molecules It was not named after anyone called Northern! Originally known as ‘Reverse-Southern’ which became Northern. Northern blotting has several uses 1. It can determine if a specific gene is transcribed in a particular cell type Nerve vs. muscle cells 2. It can determine if a specific gene is transcribed at a particular stage of development Fetal vs. adult cells 3. It can reveal if a pre-mRNA is alternatively spliced Northern Blotting 18-56

57. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Northern blotting is rather similar to Southern blotting It is carried out in the following manner RNA is extracted from the cells and purified It is separated by gel electrophoresis It is then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a radioactive probe The filters are then exposed to an X-ray film RNAs that are complementary to the radiolabeled probe are detected as dark bands on the X-ray film Figure 18.14 shows the results of a Northern blot for mRNA encoding a protein called tropomyosin 18-57

58. 18-58 Figure 18.14 Smooth and striated muscles produce a larger amount of tropomyosin mRNA than do brain cells This is expected because tropomyosin plays a role in muscle contraction The three mRNAs have different molecular weights This indicates that the pre-mRNA is alternatively spliced Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 123 Lane 1: Smooth muscle cells Lane 2: Striated muscle cells Lane 3: Brain cells

59. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Western blotting is used to identify a specific protein within a mixture of many protein molecules Again, it was not named after anyone called Western! Western blotting has several uses 1. It can determine if a specific protein is made in a particular cell type Red blood cells vs. brain cells 2. It can determine if a specific protein is made at a particular stage of development Fetal vs. adult cells Western Blotting 18-59

60. Western blotting is carried out as follows: Proteins are extracted from the cells They are then separated by SDS-PAGE They are first dissolved in the detergent sodium dodecyl sulfate This denatures proteins and coats them with negative charges The negatively charged proteins are then separated by polyacrylamide gel electrophoresis They are then blotted onto nitrocellulose or nylon filters The filters are placed into a solution containing a primary antibody (recognizes the protein of interest) A secondary antibody, which recognizes the constant region of the primary antibody, is then added The secondary antibody is also conjugated to alkaline phosphatase The colorless dye XP is added Alkaline phosphatase converts the dye to a black compound Thus proteins of interest are indicated by dark bands 18-60

61. 12 (b) Results from a Western blotting experiment (a) Interactions between the protein of interest and antibodies 3 Lane 1: Red blood cells XP (colorless) X (black) Primary antibody +PiPi Secondary antibody Alkaline phosphatase Protein of interest Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Lane 2: Brain cells Lane 3: Intestinal cells 18-61 The results of a Western blot for the  -globin polypeptide The experiment indicates that  - globin is made in red blood cells but not in brain or intestinal cells

62. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Researchers often want to study the binding of proteins to specific sites on a DNA or RNA molecule For example, the binding to DNA of transcription factors To study protein-DNA interactions, the following two methods are used 1. Gel retardation assay Also termed gel mobility shift assay 2. DNA footprinting Techniques that Detect the Binding of Proteins to DNA or RNA 18-62

63. 18-63 Figure 18.16 The technical basis for a gel retardation assay is this: The binding of a protein to a fragment of DNA retards its rate of movement through a gel Gel retardation assays must be performed under nondenaturing conditions Buffer and gel should not cause the unfolding of the proteins nor the separation of the DNA double helix Higher mass and therefore slow migration Lower mass and therefore fast migration Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

64. DNA footprinting was described originally by David Galas and Albert Schmitz in 1978 They identified a DNA site in the lac operon that is bound by the lac repressor This DNA site is, of course, the operator The technical basis for DNA footprinting is this: A segment of DNA that is bound by a protein will be protected from digestion by the enzyme DNase I Figure 18.17 shows a DNA footprinting experiment involving RNA polymerase holoenzyme 18-64

65. 18-65 Figure 18.17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Did not contain RNA pol holoenzyme Tube ATube B Labeled end RNA polymerase holoenzyme 150-bp fragment A site where DNase I randomly cuts the fragment A single cut can occur anywhere in the DNA fragment. A single cut can only occur where the protein is not bound. Load onto a gel. Expose the gel to X-ray film. Only the pieces of DNA with a labeled end are detected.

66. Tube ATube B 150 bases 105 bases 25 bases 1 base +75 +50 +30 +1 –30 –50 –75 Promoter numbering Fragment size Region where RNA polymerase binds Transcription start site 18-66 Figure 18.17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the absence of RNA pol holoenzyme, a continuous range of fragment sizes occurs No bands in this range RNA pol holoenzyme is bound to this DNA region, and thus protects it from DNase I Thus RNA pol holoenzyme binds to an 80-nucleotide region (from -50 to +30) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

67. 18.5 DNA SEQUENCING AND SITE- DIRECTED MUTAGENESIS Analyzing and altering DNA sequences is a powerful approach to understanding genetics –A technique called DNA sequencing enables researchers to determine the base sequence of DNA It is one of the most important tools for exploring genetics at the molecular level –Another technique known as site-directed mutagenesis allows scientists to change the sequence of DNA This too provides information regarding the function of genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-67

68. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display During the 1970s two DNA sequencing methods were devised One method, developed by Allan Maxam and Walter Gilbert, involves the base-specific cleavage of DNA The other method, developed by Frederick Sanger, is known as dideoxy sequencing The dideoxy method has become the more popular and will therefore be discussed here DNA Sequencing 18-68

69. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The dideoxy method is based on our knowledge of DNA replication but uses a clever twist DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’–P of one to the 3’–OH another (Refer to Fig. 11.12) Nucleotides missing that 3’–OH can be synthesized 18-69 Sanger reasoned that if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow This is referred to as chain termination If ddATP is used, termination will always be at an A in the DNA Figure 18.18 3′2′ 1′ 4′ 5′ H H HH CH 2 2′, 3′-Dideoxyadenosine triphosphate (ddA) Adenine Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. HH O OPOOP OOO P O–O– O–O– O–O– O–O–

70. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Prior to DNA sequencing, the DNA to be sequenced must be obtained in large amounts This is accomplished using cloning or PCR techniques In many sequencing experiments, the target DNA is cloned into the vector at a site adjacent to a primer annealing site In the experiment shown in Figure 18.19, the recombinant vector DNA is heat denatured into single strands 18-70

71. 18-71 Figure 18.19 The newly-made DNA fragments can be separated according to their length by running them on an acrylamide gel They can then be visualized as fluorescence peaks as the bands run off the bottom of the gel (a) Automated DNA sequencing(b) Output from automated sequencing Sequence deduced from gel Laser beam G T C A G G A A T G C C A C CACCGTAAGGACTG Annealing site Primer Sequence to be analyzed (target DNA) 5′ C C C C G G G G A A T T T T Recombinant vector Many copies of the recombinant vector, primer, dNTPs, fluorescently labeled dideoxynucleotides, and DNA polymerase are mixed together. Incubate to allow the synthesis of DNA. Fluorescence detector CACCGTAAGGACTddG CACCGTAAGGACddT CACCGTAAGGAddC CACCGTAAGGddA CACCGTAAGddG CACCGTAAddG CACCGTAddA CACCGTddA CACCGddT CACCddG CACddC CAddC CddA ddC Separate newly made strands by gel electrophoresis. Nucleotides added to primer Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

72. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display An important innovation in the method of dideoxy sequencing is automated sequencing It uses a single tube containing all four dideoxyribonucleotides However, each type (ddA, ddT, ddG, and ddC) has a different- colored fluorescent label attached After incubation and polymerization, the sample is loaded into a single lane of a gel 18-72 Figure 18.19 (a) Automated DNA sequencing G T C A G G A A T G C C A C Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

73. The procedure is automated using a laser and fluorescence detector The fragments are separated by gel electrophoresis Indeed, the mixture of DNA fragments are electrophoresed off the end of the gel As each band comes off the bottom of the gel, the fluorescent dye is excited by the laser The fluorescence emission is recorded by the fluorescence detector The detector reads the level of fluorescence at four wavelengths 18-73 Figure 18.19 (b) Output from automated sequencing CACCGTAAGGACTG Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

74. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Analysis of mutations can provide important information about normal genetic processes Therefore, researchers are constantly looking for mutant organisms Mutations can arise spontaneously, or be induced by mutagens Researchers have recently developed techniques to make mutations within cloned DNA Site-Directed Mutagenesis 18-74

75. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display One widely-used method is known as site-directed mutagenesis It allows the alteration of a DNA sequence in a specific way The site-directed mutant can then be introduced into a living organism This will allow the researchers to see how the mutation affects The expression of a gene The function of a protein The phenotype of an organism Mark Zoller and Michael Smith developed a protocol for the site-directed mutagenesis of DNA cloned in a viral vector Refer to Figure 18.20 18-75

76. or A site-directed mutant is made.The DNA is repaired back to the original sequence. T A G C C G C G G C C G C G C C G Add dNTPs, DNA polymerase, and DNA ligase. Mismatch Oligonucleotide primer with a mismatch Gene in a vector (template DNA) Vector The DNA is introduced into a living cell, where the mismatch is repaired. C G C C G Mismatch Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A A 18-76 Figure 18.20 The vector and insert are denatured into single- stranded DNA prior to the experiment Depending on which base is replaced, the mutant or original sequence is produced Can be identified by DNA sequencing and used for further studies