1) 20-1 DNA cloning yields multiple copies of a gene or other DNA segment a) Genetic engineering: direct manipulation of genes

for practical purposes b) There is a challenge in studying a particular gene because DNA is very long and gene is only a tiny portion c) DNA cloning and Its Applications: i) Plasmids: small circular DNA molecules that replicate separately from bacterial chromosome ii) To clone pieces of DNA: (1) gene from another source inserted into plasmid (results in recombinant DNA molecule) (2) plasmid put back into bacterial cell (produces recombinant bacterium) (3) host cell grown in culture to form clone of cells containing the inserted gene (4) basic research and various applications iii) Gene cloning: production of multiple copies of a single gene (1) Useful to make many copies of a particular gene and to produce a protein product (2) Resistance gene in one species of plants might be cloned and transferred to another species of plants d) Using Restriction Enzymes to make Recombinant DNA i) Restriction enzymes: enzymes that cut DNA molecules at specific locations; protect bacterial cell by cutting up foreign DNA from other organisms or phages ii) Each restriction enzyme recognizes a particular restriction site: DNA sequence (1) Cuts both DNA strands at precise points within restriction site iii) DNA of bacterial cell protected from cells own restriction enzymes (1) Most restriction sites are symmetrical (sequence is the same on both strands) iv) Restriction enzyme will make many cuts in DNA molecules, yielding restriction fragments // all copies of a DNA molecule will yield the same restriction fragments when exposed to the same restriction enzymes v) Sticky end: occurs when restriction enzyme cleaves sugar phosphate backbone in a staggered manner so that there is at least one single stranded end (1) Can form hydrogen-bonded base pairs w. complementary sticky ends on others (2) Associations can be made permanent by DNA ligase e) Cloning a Eukaryotic Gene in Bacterial Plasmid i) Cloning vector: original plasmid, a DNA molecule that can carry foreign DNA into host and replicate (1) Can be easily isolated from bacteria // manipulated to form recombinant plasmids // multiply fast ii) Producing Clones of Cells Carrying Recombinant Plasmids (1) One way to clone hummingbird genes using bacterial plasmid as cloning vector (a) (1) Isolate plasmid DNA from bacterial cells and DNA from hummingbird cells (hummingbird DNA contains gene of interest) (2) cut both DNA samples w. same restriction enzyme (3) mix cut plasmids and DNA fragments to allow base pairing; add DNA ligase to seal them together, produces some recombinant and many nonrecombinant plasmids (4) mix DNA w. bacterial cells, some take up recombinant plasmid (5) plate bacteria on agar containing ampicillin and X-gal, incubate until colonies grow (b) Only cell that took up plasmid will reproduce, nonrecombinant plasmid colonies will be blue // recombinant plasmid colonies will be white iii) Storing Cloned Genes in DNA libraries (1) Genomic library: complete set of plasmid-containing cell clones, each w. copies of particular segment from initial genome (2) Certain bacteriophages have been used as cloning vectors for making genomic libraries (3) Bacterial artificial chromosome (BAC): another type of vector used in library construcition (a) Large plasmids trimmed down to contain only necessary genes to ensure replication (b) Usually stored in multiwelled plastic plates; one clone per wall efficient for screening (4) Complementary DNA (cDNA: double stranded DNA that came from: reverse transcriptase makes singlestranded DNA from mRNA, and DNA polymerase synthesizes a second DNA strand (a) cDNA modified by addition of restriction enzyme recognition sequences at each end // inserted into vector DNA // cDNAs that are cloned make up a cDNA library (5) genomic library better when the cell type that expresses gene is unknown or to find the regulatory sequences or introns associated w. gene (6) cDNA library better when finding coding sequence of a gene or to study set of genes responsible for specialized functions of a cell type iv) Screening Library for Clones Carrying a Gene of Interest (1) Nucleic acid hybridization: used to detect genes DNA by its ability to base-pair (a) Nucleic acid probe: complementary, a short, single-stranded nucleic acid, DNA/RNA (i) Each probe is labeled w. radioactive isotope or fluorescent tag, traceable

(ii) Can screen a large number of clones at the same time for presence of DNA complementary to DNA probe f) Expressing Cloned Eukaryotic Genes i) Bacterial Expression Systems (1) Expression vector: cloning vector that contains active bacterial promoter, used to overcome differences in promoters and other DNA control sequences btw bacteria and eukaryotes (2) Using cDNA form of gene means not having to deal with introns (b/c cDNA consists of exons) ii) Eukaryotic Cloning and Expression Systems (1) Yeast have two advantages: (1) easy to grow as bacteria (2) have plasmids (rare in eukaryotes) (2) Yeast artificial chromosomes (YAC): combine essentials of eukaryotic chromosome w. foreign DNA (a) Cloned fragment most likely to contain entire gene rather than just a portion (3) Eukaryotic proteins will not function unless they are modified after translation; and bacteria cells cannot carry out modifications (4) Electroporation: brief electrical pulse applied to solution containing cells create temporary holes in their plasma membrane DNA can enter // scientists can inject DNA directly into single eukaryotic cells g) Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) i) PCR is quicker and more selective when source of DNA is scanty or impure; any segment within one or many DNA molecules can be amplified in test tube (1) Three-step cycle brings about chain reaction that produces identical DNA molecules // during each cycle reaction mixture is heated to denature DNA strands and then cooled to allow annealing of DNA primers ii) PCR is very specific, b/c of the primers which hydrogen-bond only to sequences at opp. ends of target segment iii) Occasional errors limit number of good copies made by PCR 2) DNA Technology allows us to study sequence, expression, and function of a gene a) Gel Electrophoresis and Southern Blotting i) Gel electrophoresis: polymer gel separates nucleic acids/proteins based on size, electrical charge, etc. (1) Nucleic acid molecules carry negative charges, the larger they are the more slower they travel to positive side (2) Restriction fragment analysis: can rapidly provide useful info about DNA sequences; compares two diff. DNA molecules (two alleles of a gene) (a) Provides way to prepare pure samples of individual fragments ii) Southern blotting: combines gel electrophoresis and nucleic acid hybridization // identifies carriers of mutant alleles associated w. genetic diseases (pg. 407) (1) Probe is radioactive single-stranded DNA molecule that is complementary to gene of interest b) DNA Sequencing i) Dideoxyribosenucleotide (dideoxy) chain termination method sequences a clones complete nucleic acid sequence (1) researchers compare sequence directly w. genes in other species, where function of gene is known (2) sequence comparison provide clues to a genes function c) Analyzing Gene Expression i) Studying the Expression of Single Genes (1) (To find how expression of B-globin gene changes during embryonic development of hummingbird) (2) First way: Northern Blotting: method where we carry out gel electrophoresis on mRNA at diff. stages of developmenttransfer samples to nitrocellulose membraneallow mRNA on membrane to hybridize w. labeled probe recognizing B-globin mRNA (a) If band is seen at particular stages, then the protein functions during events at that stage (3) Second way: reverse transcriptase polymerase chain reaction (RT-PCR): isolation of mRNAs from diff stagesreverse transcriptase added to make cDNA, serves as template for PCR amplification using primers from B-globin genewhen products are run on gel, copies of amplified region will be bands only in samples that originally contained B-globin mRNA (a) Can also be used to figure out which tissue is producing specific mRNA // alternative to this is tracking down location of specific mRNAs using labeled probes in place (in situ) in intact organism (b) In situ hybridization: carried out with probes labeled by fluorescent dyes ii) Studying the Expression of Interacting Groups of Genes (1) Genome sequences used as probes to find which genes are transcribed in diff. situations or coordinated (2) Basic strategy is to isolate mRNA and use them as templates for cDNA by reverse transcription, use nucleic acid hybridization to compare this w. DNA fragments representing all/part of genome (a) Results identify subset of genes in genome that are expressed at given time or under certain conditions

(3) DNA microarray assays: consists of tiny amounts of a large number of single-stranded DNA fragments fixed
to glass slide in tight grids // fragments represent all genes of an organism (a) Figure on pg 410 shows how it works (b) 60% of genes in Caenorhabditis elegans change a lot during development and many genes are expressed in sex-specific pattern (c) Proves that embryonic development involves complex and elaborate program of gene expression d) Determining Gene Function i) In vitro mutagenesis: specific mutations are introduced into cloned gene, and when returned to cell it knocks out normal cellular copies of gene ii) RNA interference (RNAi): newer method for silencing expression of selected genes // uses double-stranded RNA matching a gene to trigger breakdown of genes mRNA or block translation 3) 20-3 Cloning Organisms may lead to production of stem cells for research and other application a) Organismal cloning: cloning multi-cellular organism from single cells b) Genomic equivalence: if all the cells of an organism have the same genes c) Cloning Plants: Single-Cell Cultures i) Successful cloning of whole plants was during 1950s by Steward; worked w. carrots ii) Differentiated cells from roots (carrot) and incubated in culture medium could grow into normal identical plants (1) Totipotent: ability of cells to dedifferentiate and give rise to specialized cell types of organism d) Cloning Animals: Nuclear Transplantation i) Differentiated cells from animals do not divide in culture, or develop into multiple cell types of new organism ii) Researchers removed nucleus of unfertilized/fertilized egg and replace it w. nucleus of differentiated cell, process called nuclear transplantation iii) Robert Briggs and Thomas King showed that it was possible w. tadpoles and frogs, but that the older the donor nucleuc, the lower the percentage of normally developing tadpoles iv) Reproductive Cloning of Mammels: (1) Birth of Dolly in 1997: culturing mammary cells in medium, fusing these cells w. enucleated sheep eggsembryos implanted into surrogate mothers, out of hundreds of embryos only one lived (2) Dollys death showed that cells were not healthy, incomplete reprogramming of original transplanted nucleus (3) Reproductive cloning: the cloning of new individuals (a) Cloned animals do not always look/behave identically v) Problems Associating w. Animal Cloning (1) In nuclei of fully differentiated cells, small subset of genes is turned on & expression of others are repressed (a) Regulation result of epigenetic changes in chromatin (b) During nuclear transfer procedure, many of these changes must be reversed // reprogramming of donor nuclei requires chromatin restructuring, which occurs incompletely during cloning procedures e) Stem Cells of Animals i) Stem cell: unspecialized cell that can both reproduce itself indefinitely and differentiate into specialized cells (1) Replenish their own population and generate cells that travel down specific differentiation pathways ii) Can be isolated from embryos at blastula stage (or in humans, blastocyst stage), called embryonic stem (ES) cells (1) Adult stem cells are not able to give rise to all cell types in organism // limited to certain tissues iii) Ultimate aim is to supply cells for the repair of damaged or diseased organs iv) ES cells are pluripotent: capable of differentiating into many diff. cell types, but only obtained thru embryos v) Therapeutic cloning: the process of cloning to produce ES cells to treat disease vi) ES cells can be acquired by turning back the clock in fully differentiated adult cells by using retroviruses (1) Transformed cells (induced pluripotent stem (iPS) cells can do anything ES cells can do 4) 20-4 The Practical Applications of DNA technology affect our lives in many ways a) Medical Applications i) Use of DNA technology is identification of human genes whose mutation plays a role in genetic diseases, and may prevent conditions (1) Also helps understand nongenetic diseases, like arthritis and AIDS ii) Diagnosis of Diseases (1) The use of PCR and labeled nucleic acid probes to track down pathogens, like HIV (2) For some genetic disorders, scientists detect abnormal disease-causing allele by testing for genetic markers ( DNA sequence that varies in population) that are known to be very close (linked) to allele (3) Polymorphism: variation in DNA sequence // single nucleotide polymorphism (SNP): single base-pair site where variation is found in at least 1% of the population, occurs once in 100-300 base-pairs

(a) SNPs alter sequence recognized by restriction enzyme // restriction fragment length polymorphism
(RFLP): change the lengths of restriction fragments formed by digestion with that enzyme (b) Southern blotting can be used to detect RFLPs // presence of abnormal allele can be diagnosed w. reasonable accuracy if closely linked SNP marker has been found iii) Human Gene Therapy: introducing genes into afflicted individual for therapeutic purposes (1) Holds potential for treating disorders traceable to single defective gene (2) To be permanent, cells that receive normal allele must be ones that multiply throughout patients life (3) When ten kids with SCID were treated, three developed leukemia and one diedan unknown function on cell (4) Gene therapy has many technical challenges and ethical issues (5) Pharmaceutical Products (a) Synthesized using methods of organic chemistry or biotechnology (b) Synthesis of Small Molecules for Use as Drugs (c) development of small molecules tailored to combat certain cancers by blocking crucial proteins cancer cells need to survive ex. imatinib (trade name Gleevec) helps combat CML (6) Protein Production in Cell Cultures (a) host cells secreting a protein by being injected by DNA produces insulin and human growth hormone (i) plasminogen activator (TPA); helps dissolve blood clots and reduces risk of some heart attacks (b) cells in culture can also be used to produce vaccines // pathogen (7) Protein Production by Pharm Animals and Plants (a) Whole animals can be used to produce proteins // scientists introduce gene from animal of one genotype into genome of another individual (the transgenic animal), often of diff. species (b) Remove eggs from female recipient and fertilize them in vitroclone gene from donor and inject it into nuclei of fertilized eggembryos implanted into mother (i) A transgenic goat can produce wanted protein included in its milk // chickens in its eggs (ii) Proteins might be diff, so it must be tested to ensure it will not cause allergic reactions, etc (c) They are now developing pharm plants b) Forensic Evidence and Genetic Profiles i) Forensic laboratories can determine blood type or tissue type by using antibodies to detect cell-surface proteins ii) Genetic profile: individuals unique set of genetic markers iii) Short tandem repeats (STRs): variations in length (2-to5-base sequences in region of genome) of genetic markers (1) Number of repeats present highly variable in each person // PCR used to amplify STRs using primers iv) Genetic profiles used to find the father of a child and identifying victims of mass casualties c) Environmental Cleanup i) Many bacteria can extract heavy metals and incorporate them into copper sulfate or lead sulfate ii) Biofuels can be replacement for fossil fuels//starch in plants converted to sugar and fermented by microorganisms d) Agricultural Applications i) Animal Husbandry: the selective breeding of livestock (1) Transgenic animals is another way of selective breeding (2) Problems such as low fertility or increased susceptibility to disease are not uncommon ii) Genetic Engineering in Plants (1) Single tissue cell can be manipulated and then it can give rise to an adult plant (2) Ti plasmid: most commonly used vector for introducing new genes into plant cells // integrates segment of its DNA (T DNA) into chromosomal DNA of its host plant cells (a) Introduce Ti plasmids into plants by electrophoration (3) Genetic engineering is replacing traditional plant-breeding programs, and can improve nutritional value of crop plants ex. transgenic rice plants that produce beta-carotene e) Safety and Ethical Questions Raised by DNA Technology i) Possibility of hazardous pathogens might be created // set of guidelines adopted to prevent it (1) set of laboratory procedures designed to protect from infection and prevent microbes from leaving lab (2) strains of microorganisms to be used in recombinant DNA are genetically crippled ii) genetically modified (GM) organisms: one that has acquired by artificial means genes from another species // concern of GM crops used as food (1) GM organisms must be identified in exports iii) Worried that GM crops might pass new genes to weeds, etc.