BIOTECHNOLOGY

BIOTECHNOLOGYCampbell and ReeceChapter 20DNA Technologymade possible:Human Genome Project completed in 9 yrs (2001)by 2010 genomes of > 7,000 species

Recombinant DNADNA formed when segments of DNA from 2 different species are combined in vitro (in test tube)useful for analyzing genes & gene expressionBiotechnologymanipulation of organisms or their components to produce useful productsincludes selective breeding, using bacteria in fermentation

Genetic Engineeringthe direct manipulation of genes for practical purposes

DNA Cloninguseful for researcher to have just that portion of DNA working with1 gene may be as small as 1/100,000th of a chromosomebacterial plasmids: circular DNA molecules that replicate separately from bacterial chromosomeused by bacterium when environment changesGene Cloning

Restriction Enzymesused to cut DNA w/in short, specific nucleotide sequences (restriction sites) set of dbl stranded DNA fragments with single stranded sticky ends

DNA Ligasesticky ends form H-bonds with sticky ends of C bases from other DNA: temporary bondsDNA ligase make bonds permanent: makes covalent bonds in sugar-phosphate backbone

Restriction Enzymes Animationshttp://www.dnalc.org/resources/animations/restriction.html

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio37.swf::Restriction%20Endonucleases

http://www.dnalc.org/view/15476-Mechanism-of-Recombination-3D-animation-with-with-basic-narration.html

Cloning Vectorsname given original plasmiddfn: a DNA molecule that can carry foreign DNA into host cell & replicate therebacterial plasmids mostly used becausereadily available from supplierscan insert foreign DNA in vitro bacterial cellmultiply rapidlyCloning Vectors Animationhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_2.html

Storing Cloned Genes in DNA Libraries

Bacterial Artificial Chromosome (BAC)large plasmids trimmed down so they contain just the genes necessary for replicationcarry 100 300 kb (kilo base pairs), normal plasmid can insert vectors no larger than 10 kbcDNAcan make libraries using cDNA:

Genomic & cDNA Librarieseach have advantagesGenomic library good if: looking for a gene but not sure where it is in a genome, or what kind of cell to look inif looking for introns or regulatory sequences asc w/genecDNA library good if studying:specific proteinsets of genes expressed in particular cell typeschanges in patterns of genes over life of cell (during development of organism)

Screening Library for Clones Carrying Gene of InterestNucleic Acid hybridization: process of base pairing between a gene & a C sequence on another nucleic acid moleculeC molecule = ssDNA or ssRNA = nucleic acid probeprobe is made that is C to known sequence in geneNucleic Acid Hybridization

Detecting a Specific DNA Sequence by Hybridization with a Nucleic Acid Probe

Expressing Cloned Eukaryotic Genesseveral technical difficulties hinder the expression of cloned eukaryotic genes in bacterial hostscan substitute eukaryotic hosts: yeasts, some insect cell, some mammalian cells that have appropriate expression vectors: a cloning vector that contains a highly active bacterial promoter just upstream of restriction site where eukaryotic gene can be inserted allowing gene to be expressed in bacterial cell or have been genetically engineered to use in specific eukaryotic cellsPCRPolymerase Chain Reactionamplifies specific target segment of DNA in vitro using primers that bracket the derived sequence, & a heat-resistant DNA polymerase, & nucleotides

PCR

http://www.dnalc.org/view/15475-The-cycles-of-the-polymerase-chain-reaction-PCR-3D-animation-with-no-audio.html

What DNA Technology Allowsonce you have many copies of a gene you can ask questions about its functions, where & when the gene is expressed, or how important is it to the organism

Gel Electrophoresisuses a gel made of a polymer (agarose commonly)gel acts like molecular sieveseparates nucleic acids or proteins based on charge and sizenucleic acids carry (-) charges on phosphate group so (+) end of gelas move the longer molecules impeded more by sieveGel Electrophoresis

Restriction Fragment Analysisfragments produced be restriction enzymes put thru gel electrophoresis band pattern characteristic of starting molecule & restriction enzyme usedable to identify viruses & plasmids by their band patternsthen recover DNA from gels 1 way of getting pure samples of DNA wont work with DNA from eukaryotic cells: gel electrophoresis smear not bands

RFLPRestriction Fragment Length Polymorphism: single nucleotide polymorphism (SNP) that exists in the restriction site for a particular enzyme, making the site unrecognizable by that enzyme & changing lengths of the restriction fragments formed by digestion with that enzymefound in coding & noncoding DNA

Using restriction fragment analysis to distinguish the normal & sickle cell alleles of the human -globin gene

Southern Blottingused to detect certain nucleotide sequences w/in a complex DNA sample compares the restriction fragments produced from different samples of genome DNASouthern Blotting

Dideoxy Chain Termination method used to sequence relatively short DNA fragmentsdone by automated sequencing machines

Dideoxy Chain Terminationtechnique: synthesizes a set of DNA strands C to original DNA fragmenteach strand starts with same primer & ends with dideoxyribonucleotide (ddNTP)incorporation of ddNTP terminates growing DNA strand because it lacks 3 OH group (site of attachment of next nucleotide)each ddNTP tagged with distinct fluorescent label so identity of nucleotide at end of each strand (ultimately entire strand) is identified

Northern Blottingin vitro hybridization with labeled probes looking for specific mRNAscould be used to look at how expression of a gene changes during the embryonic development of organismcarry out gel electrophoresis on mRNA

RT-PCRReverse Transcriptase- Polymerase Chain Reactionquicker & more sensitive than Northern blottingisolates mRNA from different developmental stages of organism then add reverse transcriptase to make cDNA which serves as template for PCR amplification using primers from gene being studiedbands will be in samples that originally contained the gene being studiedin situ hybridizationalternative method used to determine which cells are expressing certain genesdone in living organismprobes labeled with fluorescent dyes

DNA Microarray Assays

DNA Microarray Analysisuncover gene interactionssuggest correct therapeutic route in cancer treatments

in vitro Mutagenesismost common method used to determine function of gene: disable it & observe what happensspecific mutations introduced to cloned gene & then mutated gene returned to cell knocking out normal gene in the processRNAiRNA interferencemethod for silencing expression of selected genesuses synthetic dsRNA molecules matching the sequence of gene to trigger breakdown of the genes mRNA or to block its translationRNAiimportant when studying groups of genes to determine how multiple genes interact (basis of systems biology, chap 21)in humans considered unethical to block activity of genes

Genome-Wide Association Studiesused to analyze genomes of large #s of humans with certain phenotype or diseasetest for genetic markers: DNA sequences that vary in a populationuses SNPs (single nucleotide polymorphisms)single base pair site where variation is found in at least 1% of populationfew million in human genome

Using SNPs

Cloning as advances being made in DNA technology also working on technology to make multicellular organism from 1 cell producing genetically identical organism1st attempted late 1950s

Cloning Genomic Equivalence: an organisms cells have the same genomeproved when able to generate new organism from 1 cellused carrot (root) cells cultured adult plant

Single-Cell Culturestotipotent: describing a cell that can give rise to all parts of the embryo & adult, as well as extraembryonic membranes in species that have themNuclear Transplantationused in cloning animalstransplant nucleus from a differentiated animal cell enucleated ova can sometimes give rise to cloneCloning Animals

Stem CellsEmbryonic Stem Cells (ES) or Adult Stem Cells from animal embryos or adult tissues can reproduce & differentiate in vitro and in situES cells are pluripotent: cell that can give rise to many but not all parts of an organismdifficult to acquireStem CellsES cells currently donated by patients undergoing infertility treatment or from long term cell cultures established with cells isolated from donated embryos

when main objective is to produce ES to treat disease process called therapeutic cloningStem Cellsnow scientists can de-differentiate cells returning them to pluripotent cells: called iPS: induces Pluripotent stem cellscan do anything ES cells can doiPS Cells2 major usesreprogram cells from patients with disease to become iPS cellsthen act as model cells for studying the disease & potential treatmentsParkinsons disease, type 1 diabetesfield of regenerative medicinepatients own cells used to regenerate damaged tissues

Gene Therapyintroducing genes into afflicted individual (into somatic cells) for therapeutic purposesuseful for disorders caused by single gene defect (overall, relatively small # of all diseases)for it to be permanent, treated cells must be the ones that continue to divide thru out patients lifeGene Therapy

Forensic Uses of DNA Technologyusing Short Tandem Repeats (STRs) in DNA isolated from crime scenes leads to genetic profilestrong evidence to prove suspect innocent or guiltyused in paternity disputesidentification of remainsTransgenic Organismsgene for desired protein inserted into bacterial genome and become tiny factory for making proteinInsulinDigestive enzymesGrowth Hormone

Environmental Clean Upgenetically engineered microorganisms developed for oil spills or to degrade toxic waste materialsBacteriaAlgaePlants Transgenic Plants & Animalsto improve productivity & food quality