Biotechnology DNA technology and its applications Biotechnology and Molecular Biology Concepts: Polymerase chain reaction (PCR) Plasmids and restriction digests Recombinant protein production UV spectrophotometry Column chromatography Mitrotiter plate format protein assay SDS-PAGE Agarose gel electrophoresis Scientific Inquiry Use of the scientific method to test and modify ideas about our understanding of molecular biology concepts Critical examination and analysis of data Review labs 1-5! Ch 17: Genomes Ch 18: Recombinant DNA and Biotechnology
Discussion Summary: Week 14 Circadian Rhythms- linking the loops Team Discussion: Related Topics: Interpret the fiture Take home message Questions Group discussion Take home messages - one from each group Explain the figure
Biotechnology, Molecular Biology Skills and Techniques Read over lab reports Make a list of specific skills Make a list of types of thought/ analysis you can do Put this on your resume! Interview preparation Look at resume Think about how you d explain your knowledge of things on it Practice your stories Detailed examples (specific question, use of technique) Check for accuracy Hmmm is this like exam questions?
PCR explanation Dr. Vistrate, University of Ghent h4p://users.ugent.be/~avierstr/principles/pcr.html Main points Template is DNA from individual you are sampling Minimally destructive PRIMERS are complementary to target sequences Two needed! Just a specialized use of DNA replication to target a sequence Small length of sequence, multiple copies regular DNA replication targets the whole genome, produces2 copies Each step of PCR mimics a step/component of natural DNA replication in cells
Plasmid structure and function Prokaryotic structure, used in biotechnology to handle and manipulate DNA sequences Circular Specific sequences needed Origin of replication Something to use for selection Sequences useful for manipulating DNA General functions hold and make many copies of a DNA fragment Express a coding sequence under tight (human) control Modify a sequence before making a transgenic organism Move DNA between organisms Thieman and Palladino h4p://wps.aw.com/ bc_palladino_biotech_2/91/23335/5973880.cw/index.html
Restriction sites Restriction enzymes Thieman and Palladino h4p://wps.aw.com/bc_palladino_biotech_2/91/23335/5973880.cw/index.html Be clear on the difference and the relationship: they have the same name! Restriction site is DNA sequence Palindromic accessed via major groove (usually) Rare in bacteria that produces related enzyme Restriction enzyme is a protein Often acts as a dimer Binds to restriction site Cleaves sugarphosphate backbone at specific bases in or near restriction site Sticky end Natural use is defense against viruses and phage We use to: build DNA constructs analyze DNA constructs
Restriction sites Restriction enzymes Thieman and Palladino h4p://wps.aw.com/bc_palladino_biotech_2/91/23335/5973880.cw/index.html Be clear on the difference and the relationship: they have the same name! Restriction site is DNA sequence Palindromic accessed via major groove (usually) Rare in bacteria that produces related enzyme Restriction enzyme is a protein Often acts as a dimer Binds to restriction site Cleaves sugarphosphate backbone at specific bases in or near restriction site Sticky end Natural use is defense against viruses and phage We use to: build DNA constructs analyze DNA constructs
Restriction sites Restriction enzymes Thieman and Palladino h4p://wps.aw.com/bc_palladino_biotech_2/91/23335/5973880.cw/index.html Be clear on the difference and the relationship: they have the same name! Restriction site is DNA sequence Palindromic accessed via major groove (usually) Rare in bacteria that produces related enzyme Restriction enzyme is a protein Often acts as a dimer Binds to restriction site Cleaves sugarphosphate backbone at specific bases in or near restriction site Sticky end Natural use is defense against viruses and phage We use to: build DNA constructs analyze DNA constructs
Restriction sites Restriction enzymes Restriction enzymes and genetic engineering Ligase is part of DNA replication machinery Seals the sugar-phosphate backbone aper sqcky ends H-bond Requires ATP for energy See by Seeing h4p://dna-ligase.seebyseeing.net/ Northern Ilinois University h4p://www.bios.niu.edu/johns/bios103/dna_files/ v3_document.htm Thieman and Palladino h4p://wps.aw.com/bc_palladino_biotech_2/91/23335/5973880.cw/index.html
Differential and Selective media Ways to observe genetic identity of organisms, limit growth of undesirable organsims Differential Does not kill organism, but phenotypes are different when this media is used Selective Kills organisms without particular characteristic (based on genotype) Streak plate dilute culture by streaking Single colonies started from one founder cell h4p://delliss.people.cofc.edu/virtuallabbook/ DrugRes/AnQbioQcRes.html
Genome editing Based on Bacterial immunity to virus Piece of Viral DNA inserted into bacterial genome Little bit expressed to help target virus next time it infects CRISPR/ Cas9 h4ps://www.horizondiscovery.com/resources/webinars/
CRISPR/ Cas9 Genome editing Produces double-stranded DNA breaks Based on 20 bp sequence and PAM (3bp in genome) h4ps://www.horizondiscovery.com/resources/webinars/
CRISPR/Cas9 h4ps://www.addgene.org/crispr/guide/ Genome editing Based on Bacterial immunity to virus Piece of Viral DNA inserted into bacterial genome Little bit expressed to help target virus next time it infects Produces double-stranded DNA breaks Based on 20 bp sequence and PAM (3bp in genome) DNA repair systems in cell generate genomic changes Homologous sequence used in repair can create specific insertions/deletions Non-homologous end-joining can produce insertions/deletions Target CDS, Promoters, etc. Use plasmid manipulation to generate tools
CRISPR/Cas9 h4ps://www.addgene.org/crispr/guide/ Genome editing Based on Bacterial immunity to virus Piece of Viral DNA inserted into bacterial genome Little bit expressed to help target virus next time it infects Produces double-stranded DNA breaks Based on 20 bp sequence and PAM (3bp in genome) DNA repair systems in cell generate genomic changes Homologous sequence used in repair can create specific insertions/deletions Non-homologous end-joining can produce insertions/deletions Target CDS, Promoters, etc. Use plasmid manipulation to generate tools
Genome editing CRISPR/Cas9 Use plasmid manipulation to generate tools CRISPR-Cas9 Knockin Mice for Genome Edi9ng and Cancer Modeling. h4ps://www.addgene.org/crispr/zhang/
Genome editing CRISPR/Cas9 Use plasmid manipulation to generate tools CRISPR-Cas9 Knockin Mice for Genome Edi9ng and Cancer Modeling. h4ps://www.addgene.org/crispr/zhang/
CRISPR/Cas9 h4ps://www.horizondiscovery.com/resources/webinars/ Genome editing DNA repair systems in cell generate genomic changes Homologous sequence used in repair can create specific insertions/ deletions Nonhomologous end-joining can produce insertions/ deletions Target CDS, Promoters, etc.
STRETCH BREAK!
This week s lab: Lab 5b (week 2) Intro PCR Detecting 3 different genes 3 primer sets Standard cocktail of other ingredients Each DNA extract with each primer set GEL MAP PCR products detected by size, amount on an agarose gel DISEASE RESULTS Compare to expected results
Next week s lab: Lab 5c intro Plasmid DNA production, extraction, analysis E. col inoculation Selective media Differential media Liquid culture versus agar Plasmid DNA maps Plasmid DNA extracts Restriction Digests Agarose gels