Spring 1998 Molecular Biology Exam #1 - Learning the Tools

Answer Key

There is no time limit on this test, though I have tried to design one that you should be able to complete within 2.5 hours, except for typing. You are not allowed to use your notes, or any books, nor are you allowed to discuss the test with anyone until Monday Feb. 2, 1998. EXAMS ARE DUE AT 8:30 ON MONDAY, FEBRUARY 2. You may use a calculator and/or ruler. The answers to the questions must be typed on a separate sheet of paper unless the question specifically says to write the answer in the space provided. If you do not write your answers on the appropriate pages, I may not find them unless you have indicated where the answers are.

Please do not write or type your name on any page other than this cover page. Staple all your pages (INCLUDING THE TEST PAGES) together when finished with the exam.

Name (please print here):

Write out the full pledge and sign:

How long did this exam take you to complete (excluding typing)?

6 pts.

1. Draw a nucleotide that makes DNA sequencing possible (not an ordinary nucleotide). To receive full credit, you must draw all of the atoms except for the base which you may label with a single letter. Also, label the 5' and 3' ends properly.

6 pts.

2. Draw the structure of a single amino acid. You do not need to include the side chain in your drawing but you do need to include all the other atoms. Also, indicated where the next amino acid would be added onto the one you have drawn.

The next amino acid would add where the -OH group is.

16 pts.

3. Tell me how you would do the following:

a. 250 mL of a solution that is 123 mM NaCl, 5% SDS (v/v if the stock solution of SDS is 20% w/v), 0.01 M Tris-HCl, pH 7.5. (FW NaCl = 58.44; FW Tris = 121; FW SDS = 288.4)

1.8 g NaCl
62.5 mL of hte 20% SDS stock
0.3 g Tris

Dissolve the solids in about 150 mL water, add SDS, pH to 7.5 with HCl, add water to 250 mL.

b. Determine the concentration of a stock DNA solution if 1 µL of the stock was added to 399 µL of water and this solution had an OD260 of 0.038. (1 OD260 unit has a concentration of 50 µg/mL dsDNA.)
760 µg/mL

c. Using the number you have calculated above, tell me how you would make a 10 µL solution that contains 100 ng of DNA.

Take 1 µL of the stock and add 9 µL of water. This has a concentration of 76 ng/µL. Take 1.32 µL of this new stock and add 8.68 µL of water. (You cannot pipet 0.132 µL of any liquid, it is too small.)

d. Make a solution that is 150 mM NaCl, 2 M KOH (stock solution is 5 M KOH), and 0.1 M SDS.
For 1 L:
8.77 g NaCl; 400 mL of 5M KOH; 28.8 g SDS
Dissolve the solids in about 500 mL of water, and add in the KOH. Then bring up to 1 L with water.

10 pts.

4. Write down the sequence of this DNA from 5' to 3' (cluster the letters into threes, please)

CTC CTA GGG GCC C ATG GCT CGA GCT TAA GCA TTA GTA CCA GTA TC

4 pts.

5. Find the ORF and indicate its location on your sequence for #4 above.
ORF = ATG......TAA

6 pts.

6. Translate this cDNA sequence using the table provided in this test. Translate as much as you can.
Met Ala Arg Ala stop

6 pts.

7. Go to the following URL: <bio.davidson.edu/Biology/Courses/Molbio/01seq/fluorseq.html>.

a. Answer the question at the top of the page.
The 3 ' UT region of the cDNA as you can see with the poly-T track which is the complementary strand to the poly-A tail of an mRNA/cDNA.

b. Is there any sequence shown that is from the plasmid? Explain your answer.
Yes, there must be some plasmid sequence. Since the poly-T tail is the terminus and since it is at the 5 ' end of the non-coding strand, it must be closer to the primer. Therefore, it is likely that the sequence upstream of the polyT tail is plasmid.

8 pts.

8. Below is a diagram that summarizes some data from a northern blot experiment. Your Favorite mRNA (YFmRNA) from the thymus has been probed at the same time as actin, which is expressed at constant levels at all times and in all cells. Interpret the data as fully as you can. The time indicates how long the cells had been incubated in a drug which alters calcium levels inside cells.

Three main points:

1) YFmRNA is a different size mRNA than actin mRNA (probably bigger with the top of the gel usually on top of a figure).
2) Unequal amounts of RNA was loaded in these lanes since the level of actin mRNA changes.
3) The drug does not seem to affect the expression of YFmRNA since its expression level is consistent with actin mRNA levels (as indicated by the thickness of the bands).

14 pts.

9. Provide the full names for these single letter codes for amino acids:

Y E L L W S M A R I N E Q T

tyrosine, glutamic acid, leucine, leucine, tryptophan, serine, methionine, alanine, arginine, isoleucine, asparagine, glutamine, threonine

8 pts.

10. Recently it has been learned what the molecular cause is for long term potentiation (LTP), which is the mechanism for memory (true story). It turns out that an ion channel called (NMDA) in the hippocampus is the primary cause for our ability to learn (for this question, let's assume there is only one kind of NMDA in the hippocampus). Explain to me how you would go about cloning the gene that encodes for this particular NMDA - you only need to list the major steps you would do. To receive full credit, you will need to demonstrate to me how you would know you had cloned the right gene.

There are several ways to do this, but since the GENE is asked for, I would start with a genomic library. Then I would use the cDNA from another NMDA (eitehr same species but different tissue, or same tissue but different species). I would use this as a probe for the genomic library. I would sequence the gene and compare it to other known NMDA gene sequences. To verify that I had the right one, I would do a northern blot using RNA isolated from the hippocampus. I should see one band of the appropriate molecular weight.

6 pts.

11. Aliens have in fact landed and are slowing killing all humans. The only way you can tell them from us (i.e. humans) is that their red blood cells glow green when exposed to UV light. Design a vector with an insert that would allow you to have every red blood cell in your body express the reporter gene GFP but no other cell would do this. (Let's assume that once you build this construct, getting it into all of your cells will be taken care of by someone else.) In your design, I want you to draw out every part of the DNA that would be important to the success of building and maintaining this vector. The future of all human beings depend upon your abilities. Good luck.

You would want a plasmid with an origin of replicaiton and drug resistance (e.g. ampicillin). Then you would want to have the cDNA/gene for GFP cloned downstream of a a RBC-specific promoter like the hemoglobin gene (alpha or beta) promoter. The promoter plus GFP should be cloned into the plasmid. This would allow you to maintain the construct.

10 pts.

12. Tell me the function of each of the following in a PCR reaction:

a. Taq
thermostable DNA plolymerase that polymerizes new strands of DNA

b. dNTPs
monomers used in DNA synthesis

c. DMSO
used to help with the specificity of annealing primers

d. 55° C
temperature where primers anneal to the target DNA

e. 95° C
temperature used to denature dsDNA so the polymerization can start over

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