08 genetic engineering

08 genetic engineering - CEE 266 ENVIRONMENTAL...

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Unformatted text preview: CEE 266 ENVIRONMENTAL BIOTECHNOLOGY Lecture 8 (Genetic Engineering and “Omics”) Restriction and Modification Enzymes   Genetic engineering: using in-vitro techniques to alter genetic material in the laboratory   Basic techniques include   Restriction enzymes   Gel electrophoresis   Nucleic acid hybridization   Nucleic acid probes   Molecular cloning Restriction and Modification Enzymes   Restriction enzymes: recognize specific DNA sequences and cut DNA at those sites   Widespread among prokaryotes   Rare in eukaryotes   Recognize inverted repeat sequences (palindromes)   Typically 4–8 base pairs long; EcoRI recognizes a 6 basepair sequence   Sticky ends or blunt ends Restriction and Modification of DNA Figure 12.1 Recognition Sequences of a Few Restriction Endonucleases Restriction and Modification Enzymes Restriction enzymes: protect cell from invasion from foreign DNA   Destroy foreign DNA   Must protect their own DNA from inadvertent destruction Modification enzymes: protect cell’s DNA for restriction enzymes   Chemically modify nucleotides in recognition sequence   Modification generally consists of methylation of DNA Restriction and Modification Enzymes   Gel electrophoresis: separates DNA molecules based on size   Electrophoresis uses an electrical field to separate charged molecules   Nucleic acids migrate through gel toward the positive electrode due to their negatively charged phosphate groups   Gels can be stained with ethidium bromide and DNA can be visualized under UV light Agarose Gel Electrophoresis of DNA Figure 12.2 Restriction and Modification Enzymes   The same DNA that has been cut with different restriction enzymes will have different banding patterns on an agarose gel   Size of fragments can be determined by comparison to a standard   Restriction map: a map of the location of restriction enzyme cuts on a segment of DNA Essentials of Molecular Cloning   Molecular cloning: isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated   Three main steps of gene cloning 1)  Isolation and fragmentation of source DNA 2)  Inserting DNA fragment into cloning vector 3)  Introduction of cloned DNA into host organism Essentials of Molecular Cloning 1) Isolation and fragmentation of source DNA   Source DNA can be genomic DNA, RNA, or PCR amplified fragments   Genomic DNA must first be restriction digested 2) Inserting DNA fragment into cloning vector   Most vectors are derived from plasmids or viruses   DNA ligase: enzyme that joins two DNA molecules   Works with sticky or blunt ends Essentials of Molecular Cloning 3) Introduction of cloned DNA into host organism   Transformation is often used to get recombinant DNA into host   Some cells will contain desired cloned gene, while other cells will have other cloned genes   Gene library: mixture of cells containing a variety of genes   Shotgun cloning: gene libraries made by cloning random genome fragments Major Steps in Gene Cloning Figure 12.5 Plasmids as Cloning Vectors   Plasmids are natural vectors and have useful properties as cloning vectors   Small size; easy to isolate DNA   Independent origin of replication   Multiple copy number; get multiple copies of cloned gene per cell   Presence of selectable markers   Vector transfer carried out by chemical transformation or electroporation Plasmids as Cloning Vectors   Blue/ White Screening   Blue colonies do not have vector with foreign DNA inserted   White colonies have foreign DNA inserted   Insertional activation: lacZ gene is inactivated by insertion of foreign DNA   Inactivated lacZ cannot process Xgal; blue color does not develop Cloning into the Plasmid Vector pUC19 Figure 12.7 Sequencing DNA   Sequencing: determining the precise order of nucleotides in a DNA or RNA molecule   Sanger dideoxy method   Invented by Nobel prizewinner Fred Sanger   Dideoxy analogs of dNTPs used in conjunction with dNTPs   Analog prevents further extension of DNA chain   Bases are labeled with radioactivity   Gel electrophoresis is then performed on products Dideoxynucleotides and Sanger Sequencing Figure 12.8 DNA Sequencing Using the Sanger Method Figure 12.9 DNA Sequencing Using the Sanger Method Figure 12.9 Sequencing DNA   454 sequencing system   Recent technological advance   Generates data 100 X faster than Sanger method   454 relies on two major advances   Massively parallel liquid handling and pyrosequencing   Light is released each time a base is added to DNA strand   Instrument actually measures release of light   Can only handle short stretches of DNA Sequencing and Annotating Entire Genomes   Virtually all genomic sequencing projects use shotgun sequencing   Entire genome is cloned and resultant clones are sequenced   Much of the sequencing is redundant   Generally 7 to 10-fold coverage   Computer algorithms used to look for replicate sequences and assemble them Sequencing and Annotating Entire Genomes   Annotation: converting raw sequence data into a list of genes present in the genome   Great majority of genes encode proteins   Functional ORF: an open reading frame that encodes a protein   Computer algorithms used to search for ORFs   Look for start/ stop codons and Shine-Dalgarno sequences   ORFs can be compared to ORFs in other genomes Amplifying DNA: The Polymerase Chain Reaction   Polymerase chain reaction (PCR): method that produces multiple copies of DNA in vitro   PCR can amplify a target DNA fragment 1,000,000,000-fold from a small amount of template   Uses DNA polymerase   Conceived by Kary Mullis   PCR is performed in a thermocycler   Three steps: denaturation, annealing, extension   Process takes only a few hours The Polymerase Chain Reaction (PCR) Figure 12.11 Amplifying DNA: The Polymerase Chain Reaction   Thermostable DNA polymerase is used (Taq polymerase)   Isolated from thermophilic bacterium Thermus aquaticus   Stable at 90 degrees Celsius   No proofreading activity   Pfu polymerase isolated from Pyrococcus furiosus   More thermostable than Taq   Has proofreading activity Amplifying DNA: The Polymerase Chain Reaction  During each round of PCR the amount of product doubles   Leads to an exponential increase in DNA   Only a few molecules of target DNA are needed   PCR has been used to amplify DNA from mummified remains, fossilized plants and animals Amplifying DNA: The Polymerase Chain Reaction  RT-PCR (Reverse transcriptase-PCR)   Reverse transcriptase is used to convert RNA into DNA   PCR is then performed on cDNA  qPCR (quantitative PCR)   Allows researcher to determine the initial number of target genes in a sample A Short History of Omics Revolution  Genome   Entire complement of genetic information, includes genes, regulatory sequences, and non-coding DNA  Genomics   Discipline of mapping, sequencing, analyzing, and comparing genomes  Bioinformatics   Science that applies powerful computational tools to nucleic acid and protein sequences Microarrays and the Transcriptome   Transcriptome   The entire complement of RNA produced under a given set of conditions   Hybridization techniques can be used in conjunction with genomic sequence data to measure gene expression   Microarrays   Small solid-state supports to which genes or portions of genes are fixed and arrayed spatially in a known pattern Making and Using DNA Chips Figure 13.7 Using DNA Chips to Assay Gene Expression Figure 13.8 Microarrays and the Transcriptome   What can be learned from microarray experiments?   Global gene expression   Expression of specific groups of genes under different conditions   Expression of genes with unknown function; can yield clues to possible roles   Comparison of gene content in closely related organisms   Identification of specific organisms Proteomics  Proteomics   Genome-wide study of the structure, function, and regulation of an organism’s proteins  Two-dimensional (2-D) polyacrylamide gel electrophoresis   Technique for the separation, identification, and measurement of all proteins present in a sample 2-D Polyacrylamide Gel Electrophoresis of Proteins Figure 13.9 Proteomics   Proteins with > 50% sequence identity typically have similar functions   Proteins with > 70% sequence identity almost certainly have similar functions   Protein domains   Distinct structural modules within proteins   Have characteristic functions that can reveal much about a protein’s role, even in the absence of complete sequence homology Metabolomics  Metabolome   The complete set of metabolic intermediates and other small molecules produced in an organism Detecting Uncultured Microorganisms  Metagenome   The total gene content of the organisms present in an environment  Several environments have been surveyed by large-scale metagenome projects   E.g., acid mine run-off waters, deep sea sediments, fertile soils ...
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