back to Molecular Biology Homepage

back to Homework Page

NOTE: you may submit this assignment via e-mail (bkbaxter@lclark) if you prefer. I will reply to you as soon as I receive it. If the deadline is approaching and you have not received a reply, please assume that I did not receive it, and make other arrangements to get it to me on time!

The mechanism of ampicillin resistance is that the "amp/r" gene encodes an enzyme capable of degrading ampicillin. In the absence of this enzyme, ampicillin interferes will bacterial cell wall synthesis, effectively lysing the bacteria.

Given this information, why do you think the 30-60 min incubation is necessary? What is happening during this time that is important for ampicillin resistance?

2. (2 pts) To obtain oligonucleotide primers for use in PCR, as probes, or as sequencing primers, chemical synthesis is used. (This process is outlined in figure 7-9 in your textbook.) There are companies that specialize in doing this efficiently and cheaply, and most researchers (including us) simply order primers from these companies as we need them.

A limitation on the chemical synthesis of oliogonucleotides is "coupling efficiency," the efficiency with which new nucleotides are added to the growing chains. The company we're using claims a coupling efficiency of about 99% for every nucleotide after the first one (for which "coupling efficiency" is 100% by definition). In other words, 99% of the single nucleotides attached to the column will be successfully joined to the second nucleotide in the chain. 99% of these will be successfully joined to the third, and so on.

Given this efficiency, what is the percentage of oligonucleotides that will be full-length if the primer ordered is 20 nt long?

What is the percentage if the primer ordered is 100 nt long?

Comment on the usefulness of this procedure for accurately synthesizing long oligonucleotides.

3. (2 pts) The size of restriction enzyme recognition sites varies by enzyme, usually in the range of 4 - 8 basepairs. EcoRI, for example, has a 6-bp recognition site: GAATTC. AluI's recognition site is only four bp: AGCT.

You have a 5000 bp plasmid about which you have no sequence information. To begin to characterize this plasmid, you decide to do some restriction enzyme digests and agarose gels. From previous experience, you've learned that this is most successful if you have less than 8 or 10 bands per digest, and most are in the range of 800 - 7000 bp.

Based simply on probability, how often will an AluI site appear in DNA, on average? How many fragments would you get from an average 5000 bp fragment?

What about EcoRI? How often will it cut, on average, and how many fragments would you expect?

Which enzyme would be a better choice for your analysis?

4. (4 pts)

[disclaimer. The following is a fictional research situation, invented for the purposes of this question. Any resemblance to actual research questions is purely accidental, and probably minimal.]

You are an investigator studying changes in brain function. Specifically, you've long been interested in the biochemical changes that correlate with symptoms of Alzheimer's disease. This question, however, has been very difficult to pursue experimentally.

Recently there has been a breakthrough of sorts. A brain syndrome has been described in a simpler mammal, the furster, which seems to parallel Alzheimer's in humans. Some concerned furster owners have donated the brains of their recently deceased pets (both with and without the Alzheimer's-like syndrome) to your laboratory, and you have discovered to your surprise that you are able to grow cells from these brains in petri dishes. You have cultures of both normal and diseased cells.

Through biochemical analysis, you have identified a short protein which is consistently present in diseased cells and absent from normal ones. You've developed an assay that demonstrates that this new protein, which you've named plackogen, accumulates in plaque-like structures that resemble the plaques found in Alzheimer's tissue. However, preliminary sequencing of the protein has not revealed any homology to known proteins from any organism.

You are interested to know whether expression of plackogen is sufficient to trigger the formation of plaques in otherwise normal cells. To test this, you want to add plackogen protein to normal cells. However, you have been unable to get the normal cells to take up significant levels of plackogen protein when it is simply added to the medium. As an alternative, you propose to clone the gene for plackogen and transfer it to the normal cells. It isn't directly clonable by PCR, because you have been unable to obtain protein sequence from the ends of the protein.

a) What kind of library will you construct to clone the plackogen gene? Why?

b) In broad terms (at the level of detail that was used in class), how will you construct this library?

c) How will you screen the library to find the clone you want? Specifically, what will you use as a probe, and how will you design it? (You needn't describe the steps involved in screening, just the design of the probe.)

d) Once you have the gene, what changes will you need to make to your construct before you introduce it into cultured furster brain cells? (Assume that an efficient method of DNA delivery into cultured furster brain cells exists.)

e) Go back and think about your answer to d again. There are some fairly obvious changes to make, and some more subtle ones.

top of page

back to Homework Page

back to Molecular Biology Homepage