Lecture 12-080228
Download file We do not have lecture notes 10 and 11 because they correspond to the two days in which we had paper presentations
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Download file We do not have lecture notes 10 and 11 because they correspond to the two days in which we had paper presentations
2/4- Genomics Lecture covering intro, extraction and isolation, and PCR: On the slide detailing centrifugation, what are the final units of the equation RCF = (w^2*r)/g? We were just trying to understand what this equation says.
Answer: dimensionless. W^2*r has acceleration units (e.g. cm/s^2). The acceleration of gravity is the same. This equation tells us the relative acceleration relative to that of gravity on earth.
2/5- Genomics Lecture covering PCR and restriction fragment analysis: For the equation N'=N(o) * (1+E)^n -2n, we hypothesized that the '2n' term is derived from the amplicons created of incorrect length. Is this accurate?
For the slide on Typical PCR Conditions, there is a photo of an agarose gel slide. Why is showing us the different lengths of reaction primers significant?
Answer: If I understand correctly your question, each lane on the left side of the 100 bp ladder represents a reaction done with different sets of primers for the same template. For each reaction, the amplicon has a different length; the position in the gel has to be different.
Quantitative PCR: Real time PCR- we were having a difficult time piecing together these slides. Here is what we took from them: At different concentrations of DNA, it will take a different number of PCR cycles to reach the threshold fluorescence for each due to the various concentrations. We then were confused on how to determine an unknown concentration of DNA from this. We theorize that by comparing the number of cycles it takes an unknown amount of DNA to reach fluorescence can then be compared to the number of cycles it took a known amount of DNA and the unknown solution can be determined.
Answer: You are basically right. Paraphrasing, first you make a calibration curve. That is use different amounts of DNA (not concentrations) and determine the number of cycles required to reach the fluorescence threshold. This is a plot of number of cycles versus the log(No), where No is the amount of DNA. Second, you analyze your unknown and determine the number of cycles to reach the threshold. Third, use the calibration curve to determine the initial amount of DNA in the sample. Make sure that you understand how to derive the equation from the basic PCR expression.
2/7- Genomics Lecture covering PCR, Restriction Fragment analysis, and Separation of DNA by electrophoresis: We began questioning the binding affinities for SNP molecular beacons. A SNP can be detected from the wild type DNA by using a fluorescent molecular beacon complementary to the SNP and not to the wild type. However, how accurate will the final fluorescence be? There has to be some binding to the wild type strand since all of the other bases are complementary except the SNP.
Answer: Good point. This is a possibility. The trick is in designing the beacon and selecting the temperatures in the various stages of thermal cycling. The best thing to do is to have controls to confirm for false positives.
2/12- Genomics Lecture covering sequencing: We were very confused by the CE detection system diagram. What should we be taking from this photo?
Answer: There are two excitation lasers and four filters that select the fluorescence of each one of the fluorescently-labeled primers. Each laser is synchronized with two corresponding filters. This gives the ability to measure, in the same run, the DNA fragments resulting from the four chain-termination reactions.
2/14- Genomics Lecture covering arrays:
What is the significance of the short vs. long DNA probe discussion?
Answer: Both long and short have been used by different companies. The short ones have fewer mismatches, hybridize faster, do not have secondary structure. On the other hand, the short sequences may appear in the genome several times and one needs to have several partial sequences to make sure that you got the correct gene. These arrays are less sensitive than those that use long probes. Also, it is hard to control the hybridization efficiency of the long probes.
NEW QUESTIONS ADDED ON MARCH 1ST, 2008
For Problem 2 of the open book portion. I don't understand at all what Part A is asking. Could someone please explain the answer to me. I'm really confused by the question.
Answer, Question 2, exam example: You are given a DNA template and you need to PCR part of that sequence. The task is to find two primers that you can use to amplify part of the given DNA sequence. You have a lot of flexibility in what part of the sequence to amplify. The only condition is that you mark it in the sequence so that one can confirm that your primer selection is correct.
For Part C in that same problem do we use the same formula we used in Part B and just plug in the number of cycles to get the correct number of amplicons. This is what I did for Part B and C is this right?
Part B
Nm = No (1+x)^n
10000 = 10 (1+1)^n
1000 = 2^n
log(1000) = n [log (2)]
n= log (1000) / log (2) = 9.96 so about 10 cycles
Part C
Nm = No (1+x)^n
Nm = 10 (2)^10 = 10,240
Answer, Question 2, exam exmple: Part C requires to know the number of cycles (n). The number of amplicons with the incorrect length is 2n. That is, 2(10) = 20 amplicons do not have the correct length. 10220 amplicons have the correct length.
Also, in Question 12 this is the FISH method right? And the cross linked strands are found in the most dense bright part of the picture, the long DNA fragments are found on the outskirts of the dense region, and the short DNA fragments are found in the tail. Correct?
Answer, Question 12, exam example: This is not the fish method. This is a comet assay that determines the degree of fragmentation of DNA in single cells. This questions resulted from a paper presented in class in the previous year. This material is not covered in this exam.
And finally, for Question 11 on the open book. The primer sequence in Part A should be AGTCCCTGAC GAAGTTCCTA. For Part B the answers should be as follows.
Answer, Question 11A, exam example: The Question 11 was part of the closed book. Because extension by the polymerase happens at the 3' end of the primer. For the sense strand, the sequence of the primer would start...gctggtaagt.
Answer, Question 11B, exam example: The first three fragments should be defined by the first time that ddT is incorporated as the polymerase extends the sequence that originated with the primer.
For Part C, the electrogram should only have peaks where the T residues are correct. The rest should be a baseline or region where no DNA fragment is detected. This is the part I'm unsure about because then the electrogram I drew only has four peaks where the T residues are. The rest is a flat line.
Answer Question 12, part c, exam example: This is correct. The electropherogram will have some flat regions, in which no peaks are detected.
Answer: Yes
Milan Mrksich's Seminar
Feb 19, 2008, 9:45 AM
Smith 117/119
THIS IS THE LINK FOR MY SUMMARY! IT IS NOW IN PDF! THE FIGURES STILL AREN'T THAT GREAT - SO REFER TO THE PAPER! PLEASE LET ME KNOW IF YOU CAN'T GET THIS!
IF YOU CANNOT DOWNLOAD THIS SUMMARY, PLEASE LET ME KNOW AND I WILL PRINT OUT COPIES. THIS INCLUDES DOWNLOADING A DOCUMENT FULL OF RANDOM SYMBOLS.
Please click here for the genomics paper summary.
Hi Everyone -
I noticed that the cut and paste job that I did to the original did not work. This is the actual title of the paper, so use this when you are using Google Scholar
Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified by an ENA residue.
Sorry about this! Everything else about the link is the same!
Courtney
Please see the attached file.
Please see the attached pdf for my summary.
Reference: Alonso, A. et al. “Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies,” Electrophoresis, 2006, 29, 5101-5109.
Please click see attached PDF for Exercise 8.
see attached PDF
Here is the paper I will present for class. The citation is as follows:
Fan H., Quake S. Anal. Chem. 2007, 79, 7576-7579
Exams in Chem 8157 usually have two parts. In the open book part you may use anything you want; you are not allowed to consult with other people. In the closed book part you rely on your memory, your understanding of the material, and in its application to solve problems.
Questions are also based on exercises, assigned readings (posted, copies of articles, and textbook), and the material introduced via the paper presentations.
Nancy Allbritton's Seminar
Feb 19, 2008, 9:45 AM
Smith 117/119
Link to seminar announcement and speaker's web site
Instructions for blogging about this seminar
Please add your comments within 48 hours after the seminar is finished.
"Multichannel PCR-CE microdevice for genetic analysis"
Liu, CN; Toriello, NM; Mathies, RA, Anal Chem, 2006, 78, 5474-5479.
http://pubs.acs.org.floyd.lib.umn.edu/cgi-bin/abstract.cgi/ancham/2006/78/i15/abs/ac060335k.html
Steinberg, K.M., Okou, D.T., Zwick, M.E., Applying Rapid Genome Sequencing Technologies to Characterize Pathogen Genomes, 80, 2008, 520-528.
Margulies, M., et. al; Nature, 437, 2005, 486-490.
The Steinberg paper discusses newer methods of rapid DNA sequencing including the topic pyrosequencing. It is more of an introduction/review than a research discussion. The Margulies paper discusses using pyrosequencing in research. I will be using both for my presentation.
"Improvement of single nucleotide polymorphism genotyping by allele-speciWc PCR using primers modiWed with an ENA residue," M. Koizumia, K. Moritaa, M. Takagi, Y. Hiroaki, A. Kasuya. Analytical Biochemistry 340 (2005) 287–294.
Let me know if you have trouble getting this paper - I will print copies of mine. I was not able to access this paper at home, so I cannot get you the direct link now. But, as long as you are on campus, you should be able to google scholar it and go directly to it!
"Single-Nucleotide Polymorphism Genotyping by Nanoparticle-Enhanced Surface Plasmon Resonance Imaging Measurements of Surface Ligation Reactions"
Yuan Li, Alastair W. Wark, Hye Jin Lee, and Robert M. Corn, Anal Chem, 2006, 78, 3158-3164.
(http://pubs.acs.org/cgi-bin/article.cgi/ancham/2006/78/i09/pdf/ac0600151.pdf)
The instructions for this exercise are in the last slide of the Lecture notes-6. You also need to access the web site given in the slide on Agarose Gel Electrophoresis (Lecture Notes - 6).
Due Feb 14.
"Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies"
Alonso, A. et al. Electrophoresis 2006, 27, 5101-5109.
Click here for full text.
"Application of cadmium sulfide nanoparticles as oligonucleotide labels for the electrochemical detection of NOS terminator gene sequences"
W. Sun, J. Zhong, B. Zhang, K. Jiao. Anal Bioanal Chem (2007) 389: 2179-2184.
http://www.springerlink.com/content/u314835lw176g437/
If you were unable to post your comments or did not submit comments for posting in the blog, please let me know ASAP. There are some issues with the notifications and I need to know who is being affected.
Thanks
Edgar