Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CHAPTER 17

DNA Manipulation Restriction endonucleases revolutionized molecular biology Enzymes that cleave DNA at specific sites –Used by bacteria against viruses Restriction enzymes significant –Allow a form of physical mapping that was previously impossible –Allow the creation of recombinant DNA molecules (from two different sources) 2

3 types of restriction enzymes Type I and III cleave with less precision and are not used in manipulating DNA Type II –Recognize specific DNA sequences –Cleave at specific site within sequence –Can lead to “sticky ends” that can be joined Blunt ends can also be joined 3

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5 DNA ligase –Joins the two fragments forming a stable DNA molecule –Catalyzes formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides –Same enzyme joins Okazaki fragments on lagging strand in replication

Gel Electrophoresis Separate DNA fragments by size Gel made of agarose or polyacrylamide Submersed in buffer that can carry current Subjected to an electrical field Negatively-charged DNA migrates towards the positive pole Larger fragments move slower, smaller move faster DNA is visualized using fluorescent dyes 6

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Transformation Introduction of DNA from an outside source into a cell Natural process in many species –E. coli does not Temperature shifts can induce artificial transformation in E. coli Transgenic organisms are all or part transformed cells 9

Molecular Cloning Clone – genetically identical copy Molecular cloning – isolation of a specific DNA sequence (usually protein-encoding) –Sometimes called gene cloning The most flexible and common host for cloning is E. coli –Vector – carries DNA in host and can replicate in the host –Each host–vector system has particular uses 10

Vectors Plasmids –Small, circular chromosomes –Used for cloning small pieces of DNA –Selectable marker – allows presence of plasmid to be easily identified 11

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DNA Libraries A collection of DNAs in a vector that taken together represent the complex mixture of DNA Genomic library – representation of the entire genome in a vector –Genome is randomly fragmented –Inserted into a vector –Introduced into host cells –Usually constructed in BACs 13

Southern blotting –Sample DNA is digested by restriction enzymes and separated by gel electrophoresis –Double-stranded DNA denatured into single- strands –Gel “blotted” with filter paper to transfer DNA –Filter is incubated with a labeled probe consisting of purified, single-stranded DNA corresponding to a specific gene 14

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Northern blotting –mRNA is separated by electrophoresis and then blotted onto the filter Western blotting –Proteins are separated by electrophoresis and then blotted onto the filter –Detection requires an antibody that can bind to one protein 18

RFLP analysis –Restriction fragment length polymorphisms –Generated by point mutations or sequence duplications –Restriction enzyme fragments are often not identical in different individuals –Can be detected by Southern blotting 19

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DNA fingerprinting –Identification technique used to detect differences in the DNA of individuals –Population is polymorphic for these markers –Using several probes, probability of identity can be calculated or identity can be ruled out –First used in a U.S. criminal trial in 1987 Tommie Lee Andrews was found guilty of rape –Also used to identify remains 21

22 DNA sequencing –Determination of actual base sequence of DNA –Basic idea is nested fragments –Each begin with the same sequence and end in a specific base –By starting with the shortest fragment, one can then read the sequence by moving up the ladder

Automated DNA sequencing –Enzymatic technique is powerful but is labor- intensive and time-consuming –Automation made sequencing faster and more practical –Fluorescent dyes are used instead of radioactive labels –Reaction is done in one tube –Data are assembled by a computer 23

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Fundamentally new method for DNA sequencing –DNA is cleaved into smaller pieces –Both ends are ligated to adapters that are complementary to specific primers –DNA fragments are injected into a flow cell –Each of 7 channels contains a solid substrate with primers that complement the ligated ends of the DNA fragments –Uses fluorescent tag on dNTPs –Camera records colors after each round of extension 25

26 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NH 2 O P O–O– –O–O OCH 2 5´ 4´ 3´2´ 1´ OH NN N O A Reversible terminator A A T T G G C A T G C C g.h. First round of synthesis with labeled dNTPs Image capture for each round of synthesis

27 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. b: © 2007, Illumina Inc. All rights reserved Adapter Attached Clusters Adapters a.b. c.d. e. f. Flow cell 1 cm DNA DNA fragment Dense primer lawn in flow cell 35 cycles of bridge amplification Bridge amplification with unlabeled dNTPs Free end binds to primer Fragments become double- stranded Free terminus Denature double- stranded molecules Attached

Polymerase chain reaction (PCR) –Developed by Kary Mullis Awarded Nobel Prize –Allows the amplification of a small DNA fragment using primers that flank the region –Each PCR cycle involves three steps: 1.Denaturation (high temperature) 2.Annealing of primers (low temperature) 3.DNA synthesis (intermediate temperature) –Taq polymerase 28

29 After 20 cycles, a single fragment produces over one million (2 20 ) copies!

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Applications of PCR –Allows the investigation of minute samples of DNA –Forensics – drop of blood, cells at base of a hair –Detection of genetic defects in embryos by analyzing a single cell –Analysis of mitochondrial DNA from early human species 31

Medical Applications Medically important proteins can be produced in bacteria –Human insulin –Interferon –Atrial peptides –Tissue plasminogen activator –Human growth hormone –Problem has been purification of desired proteins from other bacterial proteins 32

Genetically engineered mouse with human growth hormone 33

Vaccines –Subunit vaccines Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome Injection of harmless recombinant virus leads to immunity –DNA vaccines Depend on the cellular immune response (not antibodies) 34

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Gene therapy –Adding a functional copy of a gene to correct a hereditary disorder –Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems On the positive side, 15 children treated successfully are still alive On the negative side, three other children treated have developed leukemia (due to therapy) 36

Agricultural Applications Ti (tumor-inducing) plasmid –Most used vector for plant genetic engineering –Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants –Part of the Ti plasmid integrates into the plant DNA and other genes can be attached to it –However, bacterium does not infect cereals such as corn, rice, and wheat 37

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Other methods of gene insertion –Gene guns Uses bombardment with tiny gold particles coated with DNA Possible for any species Copy number of inserted genes cannot be controlled –Modification of Agrobacterium system –Use of other bacteria like Agrobacterium 40

Herbicide resistance –Broadleaf plants have been engineered to be resistant to the herbicide glyphosate –Benefits Crop resistant to glyphosate would not have to be weeded Single herbicide instead of many types Glyphosate breaks down in environment –In the United States, 90% of soy currently grown is GM soy 41

Bt crops –Insecticidal proteins have been transferred into crop plants to make them pest-resistant –Bt toxin from Bacillus thuringiensis –Use of Bt maize is the second most common GM crop globally Stacked crops –Both glyphosate-resistant and Bt-producing 42

Golden rice –Rice that has been genetically modified to produce  -carotene (provitamin A) –Converted in the body to vitamin A –Interesting for 2 reasons Introduces a new biochemical pathway in tissue of the transgenic plants Could not have been done by conventional breeding as no rice cultivar known produces these enzymes in endosperm –Available free with no commercial entanglements 43

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Adoption of genetically modified (GM) crops has been resisted in some areas because of questions –Crop safety for human consumption –Movement of genes into wild relatives No evidence so far but it is not impossible 45

Biopharming –Transgenic plants are used to produce pharmaceuticals –1990 – Human serum albumin produced in genetically engineered tobacco and potato plants –In development Recombinant subunit vaccines against Norwalk and rabies viruses Recombinant monoclonal antibodies against tooth decay-causing bacteria 46

Transgenic animal technology has not been as successful as that in plants Molecular techniques combined with the ability to clone domestic animals could produce improved animals for economically desirable traits Main use thus far has been engineering animals to produce pharmaceuticals in milk (also biopharming) 47