BIOT251 Review sheet Exam 3
Exam date: Wednesday April 30, 2008 12:15
Exam format: exam will include short answer (few word to few sentence), problems, and drawing of certain structures or diagrams. I will ask you to APPLY the things we went over in class to new situations.
Study tips: review your notes, read the material I gave you to read, look over the questions at the ends of the Chapters, and think, think, think!
Don't hesitate to email with questions. You can also call me--email for phone #--I don't want to post here.
Recombinant DNA Techniques - PCR, Southern, Northern Sequencing, DNA Fingerprinting, ELISA:
Terms to know:
Primer
Probe
Thermostable polymerase
Nitrocellulose/nylon membrane
Dideoxy nucleotide triphosphate (ddNTP)
Primer
Probe
Nitrocellulose/nylon membrane
SAAP
biotinylated DNA
RFLP analysis
VNTRs
STRs
mtDNA
monoclonal antibody
polyclonal antibody
epitope
Concepts to understand:
 | What is necessary for a PCR reaction? (What must you include in the reaction) |
| Know how primers should be oriented for PCR. |
| Know how to calculate the annealing tempertature of a primer. |
| 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 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. |
| What is necessary for a sequencing reaction? How does the reaction work? What is the purpose of the ddNTPs in the sequencing reactions? |
| Basic steps involved in a sequencing reaction. |
| Principles of sequencing reaction - how do different bands originate? What is included in the reaction? |
| Be able to "read" a sequencing gel, or to draw the bands on a gel if given a sequence. |
| What is primer walking, and why is it used? |
| 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? Why would mtDNA sometimes be used? |
| Know the steps involved in an ELISA, and what it is used for. |
| What is the difference between polyclonal and monoclonal antibodies? How are monoclonal antibodies made? |
| **If given a certain problem, be able to decide which technique (PCR, sequencing, Southern or Northern, ELISA, or DNA Fingerprinting) would be the most appropriate to address the problem.** |
Laboratory:
Review the PCR, VNTR and DNA fingerprinting
labs. Be sure to review the handouts and your lab reports. Some of things that
you should review are listed below:
| **Always worth reviewing your solution making. ** |
| Understand the principles and practice of PCR. What is included in a reaction, and how is the DNA amplified? |
| What specifically did we amplify in the PCR-based fingerprinting experiment? |
| What is the difference between the two types of fingerprinting methods that we used in class? |
| 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. |
| How were the DNA bands visualized on the membrane in the DNA fingerprinting
experiment? How was the technique different than what would be done normally? |