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PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker CHAPTER 18 Part 2 RECOMBINANT DNA TECHNOLOGY Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Polymerase Chain Reaction 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-38 The starting material for PCR includes 1. Template DNA 2. Oligonucleotide primers Provide the precursors for DNA synthesis 4. Taq polymerase 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) Contains the region that needs to be amplified 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-39 The polymerase chain reaction (PCR) Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Figure 18.6 Binding of the primers to the DNA is called annealing 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 18-40 Figure 18.6 The sequential process of denaturing-annealingsynthesis is then repeated for many cycles 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 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 220-fold (~ 1 million-fold) After 30 cycles, a DNA sample will increase 230-fold (~ 1 billion-fold) 18-41 18.2 DETECTION OF GENES AND GENE PRODUCTS 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 DNA Libraries 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-45 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings This is termed a hybrid vector Figure 18.2 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 18-15 Cleave DNA with restriction enzyme Figure 18.7 18-46 Figure 18.7 18-47 In most cloning experiments, the ultimate goal is to clone a specific gene For example, suppose that a geneticist wishes to clone the rat b-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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-48 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 b-globin gene is then added Figure 18.8 18-49 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) Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Screening DNA Libraries 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-50 Southern Blotting 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-51 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-52 a) The steps in Southern blotting b) The transfer step An alternative type of transfer uses a vaccuum or nylon Figure 18.9 18-53 a) The steps in Southern blotting A common labeling method is the use of the radioisotope 32P Conditions of high temperature or high salt concentrations Probe DNA and chromosomal fragment must be nearly identical to hybridize 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 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 Figure 18.9 Gene is member of a gene family composed of three distinct members 18-54 Northern Blotting 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 2. It can determine if a specific gene is transcribed at a particular stage of development Nerve vs. muscle cells Fetal vs. adult cells 3. It can reveal if a pre-mRNA is alternatively spliced Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-55 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-56 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 18-57 Western Blotting 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-58 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 The secondary antibody is also conjugated to alkaline phosphatase The colorless dye XP is added 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 This denatures proteins and coats them with negative charges Alkaline phosphatase converts the dye to a black compound Thus proteins of interest are indicated by dark bands 18-59 Figure 18.11 shows the results of a Western blot for the b-globin polypeptide This experiment indicates that b-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 18-60 18.3 ANALYSIS & ALTERATION OF DNA SEQUENCES 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 18-66 DNA Sequencing 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-67 DNA Sequencing 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 Figure 18.14 2’, 3’-dideoxyadenosine triphosphate if a dideoxynucleotide is added to a growing DNA strand, the strand can no longer grow: chain termination Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-68 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-69 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 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 Sequencing ladder 18-70 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 differentcolored fluorescent label attached After incubation and polymerization, the sample is loaded into a single lane of a gel Figure 18.16 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18-71 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 Figure 18.16 18-72