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Lecture 2

BIOL 5060 Lecture 2: 1:17_RecombinantDNATechnology_Hoffman

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BIOL 5060

What will be learning? 1/18/2017 2:46:00 AM Call him Charles; Go to Office Hours OH: MONDAY: 9:30-10:30; WEDNESDAY: 9:30- 10:30 About the course • Focused to set you up to do research • Methods oriented • Errors in experiments and how to overcome them Are input and output always linear? • No: you can hit a plateau/ saturated o Example: increasing sound ▪ More about sound… plateaus up to a certain decibel and plateaus after a certain decibel… ▪ We are to insensitive up to a certain decibel ▪ We are too sensitive to high decibel o More isn’t always more • No: Hook graph—more is often not better (esp. in terms of experiments) Example: transforming E. Coli • Plasmid + competent E. Coli transformation to colonies on a plate • Variables= how competent are the E. Coli cells?; How clean is your plasmid DNA? Recombinant DNA tech? What is the purpose in focusing on this? • Central Dogma: DNA  RNA  Protein o If you isolate DNA for a gene o Isolate DNA by cloning (copying)- Putting a species of DNA into a form to generate purified copies o Make many copies of one species of material •  value of cloning o gives you a small piece of DNA that you can purify into E. Coli o gives you access to DNA to produce RNA to produce protein • To improve experiment o we can try to purify from a protein extract o assay o In order to do this we need (in order to find which fraction has our protein)… ▪ Antibodies- detect protein ▪ An assay to detect biochemical activity—relatively few proteins can be assayed for • ****If we can alter DNA we can change the protein without altering its activity***** o We can alter DNA to make a purify-able protein by introducing novel**** • You cannot specifically isolate an RNA species Definition… ASSAY: improves ability to produce output • Example: hearing aid 5 Pillars of Recombinant DNA work 1. Understanding DNA structure (polarity) 2. Purifying enzymes that modify DNA 3. Gene transfer technology a. Ability to take DNA and put it in different types of cells (different organisms) 4. Electrophoresis 5. Ability to synthesize DNA and RNA molecules … 1) Understanding DNA structure Three species of nucleotides • 1) Phosphate group and 2) Base on sugar backbone with… 3) 3 Variations on carbon 3’ and 2’ o 1) Hydroxide groups (-OH and –OH) on 3’ and 2’ = RNA o 2) -OH and –H group on 3’ and 2’ = DNA o 3) -H and –H on 3’ and 2’ = d DNA • 1) Phosphate group- required for ligation (joining of nucleic acid by DNA ligase) Watson strand 5’ 3’ Vs Crick strand= 3’ 5’ • Watson 5’ ATCCG 3’ • Crick 3’ TAGGC 5’ • Know this 2) DNA modifying enzymes—all allow for cloning of DNA or altering of clones • Restriction endonucleases- break DNA • Exonucleases- modify ends • Polynucleotide kinase- add phosphate to 5’ end • Alkaline phosphatase- remove phosphate from 5’ end • T4 DNA ligase- joins • Reverse transcriptase- convert RNA to DNA • Various types of DNA polymerases- replicate DNA in test tube or add nucleotides 3) Gene transfer technology • Transformation- plasmids or animal cells o Change the phenotype of the cell (changing its behavior) o Upon receiving DNA it changes its transformation • Transduction- phage or viral vectors o Putting DNA into a cell • 1/18/2017 2:46:00 AM What is the Purpose of Rec. DNA technology? • Recombinant DNA (rDNA) is an artificially made DNA strand that is formed by the combination of 2 or more gene sequences; is engineered specifically for a purpose • Joining together of DNA molecules from two different species that is inserted into a host organism to produce new genetic combinations, to be used to make new discoveries in medicine, agriculture, industry, and science in general DNA modifying Enzymes • Restriction endonuclease- enzyme that cuts DNA at a specific recognition nucleotide sequence • Exonuclease- o 3’ 5’proof-reading activity o 5’ 3’ repair activity o Pr
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