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GENETICS: BI 381

MOLECULAR GENETICS: BI 381 Revised Fall 2011

Class home page: http://cstl-csm.semo.edu/gathman/bi381/

Instructor: Allen GathmanOffice: Polytechnic 301F

Phone: 651-2189 Email: agathman@semo.edu

Facebook: Im there.

Office hours: 11-12 M & F

Grading:Exams (3)300

Final Exam10090-100%A

Homework and quizzes20080-89%B

Scientific Papers10070-79%C

60-69%D

Writing with Sources > APA guide. Online sources are listed under "electronic media" in the list. Most web pages will be cited as Stand-Alone Documents without authors, like this:

GVU's 8th WWW user survey. (n.d.). Retrieved August 8, 2000, from http://www.cc.gatech.edu/gvu/ user_surveys/survey-1997-10/

Note that the URL given must be complete; if you paste it into a browser, it should take you to the exact page you were looking at. Don't try to type in the URL; highlight it and copy and paste it into your assignment.

You must cite both in the text, parenthetically (Author, year), and then in a list of sources cited, using full bibliographic entries. A web page without author or date is cited in the text with a shortened title of the page and "n.d." -- (GVU's, n.d.) -- and then cited in the list of sources as shown above.

Comparison of enzymology of DNA replication

in a prokaryote and a eukaryote

Function

E. coli

HeLa (human)/SV40

Helicase

dnaB

T antigen

Loading helicase/primase (assembly of primosome)

dnaC

T antigen

Single strand maintenance

SSB

RPA

Priming

dnaG

Pol /primase

Sliding clamp (holds polymerase to template)

subunit, Pol III

PCNA

Clamp loading

gd complex

RFC

Catalysis of DNA synthesis

Pol III core

Pol

Holoenzyme dimerization

?

RNA removal

Pol I

MF1

Ligation

ligase

ligase I

Adapted from Lewin, Genes VII.

One point of origin in proks (oriC), many in euks.

Rate: 1000 nuc/sec in proks, 100-200 nuc/sec in euks.

Mechanism of DNA Polymerase activity:

All DNA-dependent DNA polymerases operate in approximately the same way. The substrates are the 3'-hydroxyl end of the growing DNA strand, and a deoxyribonucleoside triphosphate or dNTP.

The oxygen in 3'-OH makes a nucleophilic attack on the alpha phosphate (the one closest to the sugar) of the dNTP. The oxygen has two orbitals full of electrons that aren't involved in bonding, and they are attracted to large nuclei with multiple protons, such as the phosphate nucleus. When these electrons move toward the phosphorus nucleus, there is the possibility of forming a covalent bond between the phosphorus and that oxygen. However, in order for this to happen, one of the oxygens already bound to the phosphorus must be displaced. Normally, this would be quite difficult, as the covalent bond between the oxygen and phosphorus is quite stable. In this case, though, one of those oxygens already bound to the phosphorus is also bound to another phosphate see the figure above. In fact, it's bound to a group of two phosphates, called pyrophosphate (and also a Mg2+ ion, which is a cofactor for the enzyme). This group of two phosphates is an example of what is known as a "good leaving group." If the pyrophosphate group leaves the dNTP, it will quickly react with water to form two inorganic phosphates (Pi). Since pyrophosphate is so unstable and reactive, it doesn't last long at all in water; this means that its concentration is always low. Think of the reactions like this:

dNTP -------> dNMP + pyrophosphate --------> dNMP + 2 Pi

Since the pyrophosphate concentration is always quite low, the reaction equilibrium is shifted forward; in other words, it's relatively likely that the pyrophosphate will come off the dNTP. That means that it's pretty easy to displace the oxygen in that pyrophosphate from the alpha phosphorus, so the nucleophilic attack succeeds. The result is that the dNMP (a deoxyribonucleoside monophosphate, or nucleotide) becomes covalently bound to the 3' carbon of the sugar at the end of the DNA strand, thus lengthening the strand by one nucleotide. Then the process repeats.

Why can't this happen at the other end of the strand? Assume that the active site of DNA polymerase is going to orient the incoming dNTP so that a 5'-3' bond would form (rather than joining the 5' carbon of the dNTP to the 5' end of the DNA strand, upside down). So instead, the dNTP would be coming down toward the 5' end of the existing strand with its 3' OH down, to make a nucleophilic attack on the 5' phosphate of the DNA strand. Draw this out and take a look at it.

In order for the nucleophilic attack to succeed, an oxygen has to leave the phosphate on the 5' end of the DNA strand, but there's nothing else attached to it. It's not a good leaving group at all, so this reaction isn't favored. Therefore, DNA always grows from the 5' to 3' direction, by adding nucleotides onto the 3' end of the existing strand. B. DNA Manipulations

Objectives:

Describe how a genomic or cDNA library may be created in a plasmid or phage vector.

Describe strategies for identifying and isolating a particular desired clone in a library.

Explain the technique of dideoxy (Sanger) DNA sequencing.

Explain how microarrays are used to compare gene expression under different conditions.

Explain techniques used in current genetics research articles.

On your own:

Explain what restriction enzymes (endonucleases) do, what types there are, and how they may be used in the lab.

Describe plasmid and phage vectors, and explain how they are used.

Explain the processes of Southern and Northern blotting and their applications.

Explain PCR (polymerase chain reaction) and its applications.

Problems:

1. If you digest the genomic DNA of a beaver (3 x 109 base pairs) to completion with HaeIII (a 4-cutter) about how many pieces of DNA will result? SHOW YOUR WORK.

2. You hit a beaver on I-55, and stop and throw it in your trunk. Back at home in your basement laboratory, you extract some RNA from its liver. How would you produce a beaver cDNA library using this RNA? Diagram a flow chart of the steps you would follow, using techniques and materials from the list on the next page.

3. You have made the beaver cDNA library mentioned above, and it contains 14,000 different cDNAs, one of which is the trypsin gene cDNA. How many colonies will you need to plate to be 95% sure of getting the cDNA for the trypsin gene? SHOW YOUR WORK.

4. You have the beaver cDNA library mentioned above. You want to find the beaver G6PD gene and get it in a form that you can use to produce lots of copies of it. Diagram a flow chart of the steps you would follow, using techniques and materials from the list on the next page.

5. You have a catfish, and you want to know if catfish have a G6PD gene. (But you dont care if you can clone the gene afterward). Diagram a flow chart of the steps you would follow, using techniques and materials from the list on the next page. You need not explain the techniques.

6. The E. coli genome is about 4.5 x 106 bp. If you had a mole of E. coli DNA, would you carry it around in a teaspoon, a lunchbox, a dump truck, or a river barge? SHOW YOUR WORK and explain.

7. You have a microgram of E. coli DNA in a test tube. How many copies of the E. coli genome is this? SHOW YOUR WORK.

8. For PCR, typically you want about a million copies of your template sequence. Youre trying to use PCR to amplify a sequence found once in the E. coli genome, and you have a solution of isolated E. coli DNA at a concentration of 500 ng per l.

a. What volume of this solution should you use in a PCR reaction?

b. The smallest volume you can reliably dispense is 0.1 l. What should you do? Explain and be specific.

10. a. Go to Entrez (http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi) and find the nucleotide and protein sequences for human hemoglobin beta (gi # 28302128). Paste them into a Word document. Convert to double spacing.

b. Find the start codon in the HBB gene and write the amino acid sequence in above the codons in the nucleotide sequence.

c. The sickle cell allele of this gene has a T instead of an A at position 70. Circle that nucleotide on the sequence.

d How will the protein sequence of the sickle-cell hemoglobin differ from that of the normal hemoglobin beta?

e) Why does this result in a disease condition? Explain.

f) The restriction enzyme MstII has the target site CCTNAGG. How can it be used to determine whether a human carries the sickle cell allele? Explain.

LIST OF TECHNIQUES AND ITEMS(Restriction endonucleases followed by target site, cleavage site marked with /)

molecular weight of DNA = 650 (that is, 650 g/mol per base pair)

Avogadros number

(6.022 x 1023) (no units)

acrylamide

anti-IIR antibodies

anti-IIIS antibodies

G6PD gene from ladybug

G6PD gene from chicken

G6PD gene from rat

G6PD gene from frog

G6PD gene from human

G6PD gene from shark

G6PD gene from dog

agarose

alkaline phosphatase

alkaline solution (NaOH)

ampicillin

anneal

autoradiography

bake

Bam HI (G/GATTC)

beer

boil

buy oligonucleotide

calcium chloride

call 911

call Dr. Gathman

call Dr. Lilly