1. PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 18 Part 2 RECOMBINANT DNA TECHNOLOGY

2. 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 18.6 Polymerase Chain Reaction 18-38

3. 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 18-39

4. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The polymerase chain reaction (PCR)

5. 18-40 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 Binding of the primers to the DNA is called annealing

6. 18-41 Figure 18.6 With each successive cycle the relative amount of this type of DNA fragment increases. Therefore, after many cycles, the vast majority of DNA fragments only contain the region that is flanked by the two primers 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 DNA sample will increase 2 20 -fold (~ 1 million-fold) After 30 cycles, a DNA sample will increase 2 30 -fold (~ 1 billion-fold)

7. 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 18.2 DETECTION OF GENES AND GENE PRODUCTS 18-44

8. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A DNA library is a collection of thousands of cloned fragments of DNA When the starting material is chromosomal DNA, the library is called a genomic library A cDNA library contains hybrid vectors with cDNA inserts DNA Libraries 18-45

9. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA Libraries A genomic library – Contains fragments of chromosomal DNA – Includes gene exons/introns and nongene sequences A complementary DNA (cDNA) library – Is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell

10. 18-15 Figure 18.2 Note: In this case, the  -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

11. 18-46 Figure 18.7 Cleave DNA with restriction enzyme

12. 18-47 Figure 18.7

13. 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 18.8 18-48

14. 18-49 Figure 18.8 The filter is treated with detergent (SDS) to permealize the bacteria NaOH is added to denature the DNA A radioactively labeled probe that is complementary to the  -globin gene is then added

17. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Screening DNA Libraries screening a DNA library for a particular nucleotide sequence – Fragment of a gene – Related gene screening a DNA library based on detection of protein expression from cloned gene – Antibody probes – Protein probes – DNA binding sites (transcription factors)

18. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display What if a scientist is looking for a novel type of gene that no one else has ever cloned from any species? If the protein of interest has been previously isolated, amino acid sequences are obtained from it The researcher can use these amino sequences to design short DNA probes that can bind to the protein’s coding sequence 18-50 Screening DNA Libraries

19. 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 mixture of many It was developed by E. M. Southern in 1975 Southern blotting has several uses 1. determine copy number of a gene in a genome 2. detect variations in gene structure 3. identify gene families 4. identify homologous genes among different species 5. characterize structure of cloned genes Southern Blotting 18-51

20. 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 a 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 18-52

21. 18-53 An alternative type of transfer uses a vaccuum a) The steps in Southern blotting b) The transfer step or nylon Figure 18.9

22. 18-54 Figure 18.9 a) The steps in Southern blotting The filter is placed in a solution containing a labeled probe The binding can be done under conditions of low or high stringency Excess probe is washed away and the filter is exposed to X-ray film A common labeling method is the use of the radioisotope 32 P Conditions of high temperature or high salt concentrations Probe DNA and chromosomal fragment must be nearly identical to hybridize Temperature and/or ionic strength are lower 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

23. 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! 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 18-55

24. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Northern blotting is similar to Southern blotting It is carried out in the following manner RNA is extracted from the cell(s) 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 DNA 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 18.10 shows the results of a Northern blot for mRNA encoding a protein called tropomyosin 18-56

25. 18-57 Figure 18.10 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

26. 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 18-58

27. Western blotting is carried out as such Proteins are extracted from the cell(s) and purified 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 18-59

28. 18-60 Figure 18.11 shows the results of a Western blot for the  -globin polypeptide This experiment indicates that  -globin is made in red blood cells but not in brain or intestinal cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

29. 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 18.3 ANALYSIS & ALTERATION OF DNA SEQUENCES 18-66

30. 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 Alan 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 18-67

31. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5’–P of one and the 3’–OH of the other (Refer to Fig. 11.10) Nucleotides missing that 3’–OH can be synthesized 18-68 2’, 3’-dideoxyadenosine triphosphate if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow: chain termination Figure 18.14 DNA Sequencing

32. 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 If double-stranded DNA is used as the template, it must be denatured at the beginning of the experiment 18-69

33. 18-70 Figure 18.15 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 bands when the gel is exposed to X-ray film Sequencing ladder Many copies of the primer, template DNA and radiolabeled dNTPs are mixed together They are then divided into four tubes, each containing a low concentration of a different dideoxynucleotide

36. 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 18-71 Figure 18.16

38. The procedure is automated using a laser and fluorescent detector The fragments are separated by gel electrophoresis 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 18-72 Figure 18.16