Principles of Biochemistry Fourth Edition Chapter 23 Recombinant DNA Technology Copyright © 2006 Pearson Prentice Hall, Inc. Horton Moran Scrimgeour Perry

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Principles of Biochemistry Fourth Edition Chapter 23 Recombinant DNA Technology Copyright 2006 Pearson Prentice Hall, Inc. Horton Moran Scrimgeour Perry Rawn Slide 2 23.1 Making Recombinant DNA Recombinant DNA molecules are constructed with DNA from different sources Recombinant DNA molecules are created often in nature Bacteriophage or eukaryotic virus infects a host cell and integrates its DNA into the host creating a recombinant DNA molecule Chapter 23 - Recombinant DNA Technology Slide 3 Fig 23.1 Basic steps in the generation of a recombinant DNA molecule Slide 4 Fig 23.1 Basic steps in the generation of a recombinant DNA molecule Slide 5 Fig 23.1 (cont) Slide 6 Six Basic Steps in a Recombinant DNA Experiment 1. Preparation of DNA. Vector and target DNA 2. Cleavage of DNA at particular sequences. Insert DNA can be added at specific points that have been cleaved 3. Ligation of DNA fragments. Joining of the fragments (continued) Slide 7 Six Basic Steps (cont) 4. Introduction of recombinant DNA into compatible host cells. Genetic transformation is the uptake of foreign DNA by a host cell 5. Replication and expression of recombinant DNA in host cells. Cloning vectors allow insert DNA to be replicated in host cells 6. Identification of host cells that contain recombinant DNA of interest. Screening a large number of DNA clones for desired fragment Slide 8 23.2 Cloning Vectors Cloning vectors can be: plasmids, bacterio- phages, viruses, small artificial chromosomes Some vectors can be replicated autonomously in a host cell, other vectors can be integrated into the host chromosome Vectors have at least one unique cloning site: a sequence cut by a restriction endonuclease to allow site-specific insertion of foreign DNA Slide 9 Fig 23.2 Restriction enzymes can generate recombinant DNA Slide 10 A. Plasmid( ) Vectors Plasmids are small, circular DNA molecules used as vectors for DNA fragments to 20 kb Replicate autonomously within a host cell Carry genes conferring antibiotic resistance, used as marker genes for cells carrying vectors pBR322 was one of the first plasmid vectors Slide 11 Fig 23.3 Plasmid vector pBR322 pBR322 has 4361 base pairs Origin of replication (ori) Antibiotic resistance genes amp and tet Rop gene regulates replication for ~20 copies of the plasmid per cell Slide 12 B. Bacteriophage Vectors Efficient, commonly used vector for delivering DNA into a bacterial cell Advantage over plasmid vectors is that transfection( ) is more efficient than transformation( ) Disadvantage: DNA must be packaged into phage particles in vitro Slide 13 Fig 23.4 Preparation and use of phage vector Slide 14 Fig 23.4 Preparation and use of phage vector Slide 15 Fig 23.4 (cont) (From previous slide) Slide 16 Fig 23.4 (cont) Slide 17 Cosmids Cosmids combine the advantages of plasmid and phage vectors Cosmids can accommodate large DNA fragments and allow efficient transfection Recombinant DNA molecule can be propagated as a plasmid in the host cell Slide 18 C. Shuttle Vectors Shuttle vectors can replicate in either prokaryotic or eukaryotic cells They can be used to transfer recombinant DNA between prokaryotes and eukaryotes Useful for cloning eukaryotic DNA in bacteria, and then expressing the gene products in a eukaryotic cell Slide 19 D. Yeast Artificial Chromosomes as Vectors Large DNA fragments can be inserted into artificial chromosomes that are replicated in eukaryotic cells Such chromosomes must be linear and contain a eukaryotic replication origin Yeast artificial chromosome (YAC) is a shuttle vector Slide 20 Fig 23.5 Yeast artificial chromosome (YAC) Slide 21 23.3 Identification of Host Cells Containing Recombinant DNA After a cloning vector and insert DNA have been joined in vitro, recombinant DNA is introduced into a host cell such as E. coli (transformation) Only a small percentage of cells take up the DNA Selection -cells are grown under conditions in which only transformed cells survive Screening - transformed cells are tested for the presence of the recombinant DNA Slide 22 A. Selection Strategies Use Marker Genes Bacterial plasmid vectors can carry a -lactamase marker gene (marker genes allow detection of cells) -Lactamase hydrolyzes -lactam antibiotics (e.g. ampicillin) Only cells transformed with plasmids expressing the -lactamase gene are ampicillin resistant and can grow in media containing ampicillin (amp R ) Slide 23 Selection or Screening by Insertional Inactivation Insertional inactivation - insertion of a DNA fragment within the coding region of a gene on a vector results in inactivation of that gene If the gene product can be detected, this can be used for selection and screening pBR322 gene for tetracycline resistance (tet R ) can be inactivated by DNA insertion making them tetracycline sensitive (tetS) Slide 24 Fig 23.3 Plasmid vector pBR322 pBR322 has 4361 base pairs Origin of replication (ori) Antibiotic resistance genes amp and tet Rop gene regulates replication for ~20 copies of the plasmid per cell Slide 25 B. Selection in Eukaryotes Yeast shuttle vectors may contain prokaryotic genes for antibiotic resistance and yeast genes for metabolite biosynthesis Yeast gene LEU2 encodes the enzyme -isopropylmalate dehydrogenase, (leucine biosynthesis pathway) Cells transformed with a Leu2-containing plasmid can grow in the absence of leucine Slide 26 Fig 23.6 Yeast shuttle vector is propagated, selected in both E. coli and S. cerevisiae Slide 27 C. Visual Markers: Insertional Inactivation of the -Galactosidase Gene The lacZ gene of E. coli encodes -galactosidase and cleavage of an artificial substrate produces a blue dye (X-gal) Vectors without inserts in the lacZ gene give rise to blue colonies in the presence of X-gal Vectors with DNA inserted in the lacZ gene do not produce the enzyme and yields colonies which are white Slide 28 Fig 23. 7 Blue/white screen Blue colonies: cells transformed with cloning vectors not containing inserts ( -galactosidase is active) White colonies: cells transformed with recombinants. -Galactosidase gene disrupted by insert Slide 29 23.4 Genomic Libraries A method for isolating large quantities of specific DNA fragments from organisms DNA library consists of all the recombinant DNA molecules generated by ligating all the fragments of a particular DNA into vectors Recombinant DNA molecules are then introduced into cells for replication Slide 30 Genomic Library Properties Genomic libraries represent all the DNA from an organisms genome ( ) Partial (rather than total) restriction digestion is used to ensure that every gene is represented Cosmid, YAC and BAC vectors used Genomic libraries include both expressed and non-expressed DNA from the organism Slide 31 23.5 cDNA Libraries Are Made from Messenger RNA cDNA libraries represent all the mRNAs made in a given cell or tissue cDNA (complementary DNA) is double-stranded DNA made with reverse transcriptase Purification of mRNA relies on the polyA tails on mature eukaryotic mRNA The more abundant rRNA and tRNA lack tails Slide 32 Fig 23.8 Preparation of cDNA RNase H: a ribonuclease that cleaves the 3'-O-P-bond of RNA in a DNA/RNA duplex to produce 3'-hydroxyl and 5'-phosphate terminated products. Slide 33 Fig 23.8 Preparation of cDNA Slide 34 Fig 23.8 (continued) Slide 35 Properties of cDNA libraries Using a cDNA library from a specific tissue with abundant protein of interest increases the chances of successfully cloning the gene for that protein Specialized phage vectors and plasmids are used in constructing cDNA libraries cDNA libraries from mRNA do not include introns or flanking sequences (much less complex than genomic libraries) Slide 36 23.6 Screening a Library Isolation of the desired recombinant DNA is difficult (probability that a library of a given size contains the particular clone of interest is): P = 1 - (1-n) N or N = ln(1-P)/ln(1-n) N = number of recombinant clones in library P = probability of finding a particular clone n = frequency of occurrence of the clone Slide 37 Finding a Clone in a cDNA Library Probability of finding desired clone depends on the abundance of the original DNA molecule and not on the genome size n Represents the abundance of the relevant mRNA molecule General procedure for screening a DNA library with a probe (next slide) Slide 38 Fig 23.9 General procedure for screening DNA library Slide 39 Fig 23.9 General procedure for screening DNA library Slide 40 Fig 23.9 (cont) Hybridization Slide 41 23.7 Chromosome Walking A recombinant DNA fragment from a nearby region of the chromosome can be used as a starting point for a chromosome walk to the desired gene (Fig. 23.10, next slide) Overlapping DNA fragments are isolated in successive screenings Larger and larger regions of DNA are cloned in a walk along the chromosome Slide 42 Fig 23.4 Preparation and use of phage l vector Slide 43 Slide 44 23.8 Expression of Proteins Using Recombinant DNA Technology Cloned or amplified DNA can be purified and sequenced or used to produce RNA and protein Such DNA can also be introduced into organisms to change their phenotype Purification of proteins begins with overproduction of the protein in a cell containing the expression vector Slide 45 A. Prokaryotic Expression Vectors Expression vectors - plasmids that have been engineered to contain regulatory sequences for transcription and translation Eukaryotic genes can be expressed in prokaryotes Examples of sequences: strong promoters, ribosome-binding sites, transcription terminators Slide 46 Expression of a eukaryotic protein in E. coli Fig 23.11 Slide 47 Slide 48 Fig 23.11 (cont) Slide 49 B. Expression of Proteins in Eukaryotes Prokaryotic cells may be unable to produce functional eukaryotic genes Some expression vectors are for eukaryotes Recombinant DNA molecules can also be integrated into the genomes of large multicellular organisms Creates transgenic organisms Sli

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