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Review Sheet BIOL251 Final Exam Wednesday, May 14th, 2008 10:30-12:20

General hints:  While the final is cumulative, I plan to ask questions that address the more important concepts that we have reviewed in class, and the "big picture" of the course. Exam format: exam will include short answer questions (few word to few sentence). I will give you a list of terms to describe. Some "fill in the blanks". In each section, I will allow you to choose a certain number from the total number of questions. Some key concepts: making recombinant DNA, and recombinant DNA techniques. I will ask some questions like last time which ask you to distinguish which technique to use for a given situation (also make sure you know one of the techniques in detail – I WILL ask you this). Although the list here looks long, if you look at your previous sheets, you will see that there are number of topics which have been entirely eliminated, and many that have been pared down considerably. If it is not on the list, do not worry about it! If it is on the list, it is fair game. I will ask more detailed questions on material covered since the last exam. 

I MAY further edit this list when I write the exam--look for an update closer to the exam. 

Structure and Properties of DNA

Terms to Understand:Terms to Understand:

  • Gene
  • Base pair
  • Complimentary
  • antiparallel
  • denaturation
  • renaturation
  • annealing temperature
  • plasmid (high/low copy)

Concepts to know:

  • Understand directionality of DNA and be able to identify 5’ and 3’ ends of the molecule.
  • Review features of the Watson-Crick model of DNA structure
  • Review how DNA is denatured and renatured.

Transcription:

Terms to understand:

  • Structural gene
  • Introns/exon
  • Splicing
  • RNA polymerase

Concepts to know: Know basic mechanism of transcription.  Concepts to know: Know basic mechanism of transcription

Regulation of gene expression:

Terms to understand:

  • Gene expression
  • Promoter
  • Polycistronic
  • Operon
  • Repression
  • Activation
  • Repressor
  • Activator
  • Effector/inducer
  • Lac operon
  • Arabinose operon

 Concepts to know:

  • Know basic structure of prokaryotic and eukaryotic promoter regions.
  • What is the advantage of genes being arranged in an operon?
  • Review repression and activation of prokaryotic genes.
  • Review the lac operon in detail – consider the effect of mutating one or more components of the system.
  • Review the arabinose operon and pGLO plasmid.

Recombinant DNA Technology:

Terms to know:

  • Recombinant DNA technology
  • Clone
  • Restriction enzymes
  • Palindrome
  • Sticky ends
  • Blunt ends
  • restriction site
  • ligase
  • alkaline phosphatase
  • vector
  • plasmid
  • pBR322
  • pUC19
  • polylinker/multi-cloning site
  • directional cloning
  • transformation

 Concepts to understand:

  • Basic steps involved in recombinant DNA technology
  • Be able to "anneal" complimentary ends.
  • Know how to set up a restriction enzyme digestion (see lab notes—what is in the reaction?).
  • Know the function of ligase and alkaline phosphatase in cloning.
  • Review the key features that make plasmids good vectors.
  • How is pBR322 used in cloning? PUC19? Be sure to understand the selection techniques used for both. Does cloning with these vectors allow you to distinguish between re-circularized vector and recombinant plasmid? What about re-circularized insert?
  • Review lacZ’/X-gal/IPTG use with pUC19. – what color colonies would result from re-circularized vector? recombinant plasmid? What about re-circularized insert?

Construction and Cloning of libraries:

Terms to know:

  • Library
  • Partial digestion
  • DNA hybridization
  • Immunological Assay
  • Protein activity Assay
  • probe
  • cDNA
  • genomic library
  • cDNA library
  • Klenow

Concepts to understand:

  • Basic steps involved in creating a prokaryotic DNA library.
  • What is the purpose of creating a library?
  • How can you screen a DNA library?
  • Know the basic method of screening by DNA hybridization, immunological Assay or protein activity.
  • Basic steps involved in creating a eukaryotic DNA library. How and Why is it different than creating a prokaryotic library?

Expression of recombinant proteins (eukaryotic):

Terms to know:

  • Transfection
  • Calcium phosphate
  • Electroporation
  • lipofection
  • Microinjection

 Concepts to understand:

Why would you want to express a protein in a eukaryotic cell?/ How can you get DNA into eukaryotic cells?

Recombinant DNA Techniques – Sequencing, PCR, Southern blotting, Northern blotting, DNA Fingerprinting, ELISA:

Terms to know:

  • Dideoxy nucleotide triphosphate (ddNTP)
  • Primer
  • Thermostable polymerase
  • Nitrocellulose/nylon membrane
  • probe
  • RFLP analysis
  • VNTRs
  • STRs
  • primary Ab
  • Secondary Ab
  • antigen
  • epitope

Expression of recombinant proteins in eukaryotic cells:

Terms to know Post-translational modification, G418

Concepts to understand:

  • Why would you want to express a protein in a eukaryotic cell?
  • Dominant selectable marker --APH gene/G418 selection in particular.
  • Review the key features that make good eukaryotic expression vectors. (Figure 12-5, plus euk and prok ori)

 

 Concepts to understand:

  • Basic steps involved in a sequencing reaction. What is necessary for the reaction? How does the reaction work? What is the purpose of the ddNTPs in the sequencing reactions?
  • Be able to "read" a sequencing gel, or to draw the bands on a gel if given a sequence.
  • Know how primers should be oriented for PCR.
  • Know the temperature cycles in a PCR reaction, and what happens at each temperature.
  • Know how the repeated cycles lead to amplification of a DNA template.
  • Review applications of PCR –- obtaining more sample (DNA Fingerprinting, genetic testing, viral screening), creation of restriction enzyme sites for cloning.
  • Know the steps involved in Southern blotting
  • How is DNA transferred to the membrane?
  • Review applications of Southern blotting –- detect gene rearrangements/deletions; detect structurally related genes in same species/ detect homologous genes in other species; zoo blot; order genes along chromosomes;  identify specific DNA fragments.
  • Know the steps involved in Northern blotting.
  • Review applications of Northern blotting –- size determination; detect alternatively spliced mRNAs/alternate transcripts from a single gene; determine where a gene is expressed; determine the level of expression.
  • Be familiar with steps of ELISA and its applications
  • §Why is DNA fingerprinting done? (and why this over sequencing?)

    §How is DNA fingerprinting done (each method) – keep in mind it involves techniques we have discussed before –restriction enzyme digestion (not always), Southern analysis, PCR

    §What is the difference between the various techniques used for fingerprinting?

     

  • **If given a certain problem, be able to decide which technique (PCR, sequencing, Southern or Northern, DNA Fingerprinting) would be the most appropriate to address the problem.**

Transgenic Animals:

Terms to know:

  • Transgenic
  • Transgene
  • blastocyst
  • pluripotent
  • positive/negative selection
  • NeoR
  • gancyclovir

Concepts to understand:

  • Applications of trangenics – genes which enhance growth, study mammalian gene expression, animal model for human diseases, and production of pharmaceutically important proteins.
  • Making transgenic mice using retroviral vectors – be familiar with the steps in protocol.
  • Making transgenic mice by microinjection – be familiar with the steps in protocol.
  • Drawback of microinjection – random integration. What are the potential problems with this?
  • Be familiar with the ESC technique for making transgenic mice.
  • How do you select for cells with the transgene inserted at a specific location and against cells with the transgene integrated at a random site? (see figure 19.5, 19.6)
  • Specific gene disruption (making knockout mice). How is it done? Why is it done?
  • What are some applications of transgenics in cows -- production of pharmaceutically important proteins, change constituents of milk, improving livestock.

 

Laboratory:

Review labs: Solutions, LAL, Restriction enzyme digestion, construction and cloning of a DNA recombinant, PCR , Southern blotting, ELISA, Biolog, and Bioinformatics.

  • **Always worth reviewing your solution making. **
  • Know how to prepare and run an agarose gel, and how DNA is separated on the gel.
  • Know how to set up a restriction enzyme digestion.
  • How is Calcium chloride transformation done?
  • What was the recombinant plasmid selected during the cloning lab?
  • Know how to determine the orientation of an insert within a vector by restriction enzyme analysis.
  • Understand the principles and practice of PCR. What is included in a reaction, and how is the DNA amplified?
  • Understand the principles and practice of Southern analysis, and its use in DNA fingerprinting. Be able to identify a correct individual by looking at data.
  • Review basic procedure for ELISA
  • Understand the basic premise of using the Biolog system for bacterial identification
  • Review basic procedure for LAL test
  • What is bioinformatics?
  • What is GeneBank?
  • What do DNA searches allow you to do?