YL5

ASMPH YL5 Biochemistry Page of N. Rojas

Notes and comments for my biochemistry classes at the ASMPH.

2009.07.16. Exercise for presentation on Wednesday, July 22, 10:00 am

Using your assigned groups (new, diffierent from last SGD), analyze the molecular biology of the influenza virus.

• To get up to speed on sequence analysis, I suggest that you do th virtual lab on DNA fingerprinting at http://www.hhmi.org/biointeractive/vlabs/.
• Some sites that might be useful would be NCBI, CDC, WHO, and more.

2009.07.03. Welcome to Batch 3, the Class of 2014! Check out my page at http://ch151admu.wordpress.com for useful links and downloads to biochemistry materials.

2008.10.24. Make-up ELISA laboratory for excused absence during the hema-immuno module

• Go to http://www.hhmi.org/biointeractive/vlabs/immunology/index.html and perform the virtual lab.
• Answer elisa-virtual-lab and submit to me via the ASMPH office on or before November 3, 2008.

2008.07.19. More on Human Genome Lab: Sequence 4

For those who are working on sequence 4: A number of you told me you had problems tracking down your sequence, and even when I tried to help troubleshoot, it wasn’t working out. I tried running the full sequence today, and here’s what I suggest:

1. Take the sequencer data from lanes 1-2 to just before the polyA run and use that as your sequence (~266 nt). It seems that a shorter run does not work. I’ve attached the
   fasta file (text file) for your convenience, since I am sure you all have learned already
   how to read the sequencer output.
2. Use BLASTn on ALL REFSEQ mRNA, not just on the human, and see what comes up. You
   might smile about the identity of your patient, but we’re not quite there yet.
3. Take the gi# or NM# of the result and BLASTn this against the human genomic +
   transcript database and you should see the proper human gene.
4. Proceed with the rest of your analysis. Please use BLASTn, it seems to work better.

Apologies, and I will send feedback to our sequence source about our difficulties.

Have a good weekend!

2008.07.15. Human Genome Dry Lab Assignment

• Assignment per usual working group is due Monday, July 21, 5:00 pm to my email address!!!
• Scenario: You are trying to track down a gene associated with cancer, and have taken samples from two patient volunteers for DNA sequencing. The sequencer output just arrived on your desk.
• Read and encode about 50-70 nucleotides of your sequence as a text file. Sequence 1 has already been partially read for you– all you need to do is continue reading the sequencer output some more. (For more about this format, check out Wikipedia’s entry on the FASTA format.)
• Go to the BLAST site at http://blast.ncbi.nlm.nih.gov/Blast.cgi and go to nucleotide BLAST. Enter your sequences one at a time to find what is the most likely gene coded for by your DNA sequences. In the options: choose as database the mRNA Reference Sequence Database, organism = human, and  MegaBLAST under Program Selection, since you are looking for a fairly exact match. Look for the closest match, with the most documentation/links. Note down the accession/GI numbers.
• What is the most likely gene encoded for by each of your DNA sequences?
• From which part of the mRNA RefSeq file is your raw sequencer output? upstream end? downstream end? middle? Is your raw sequence that of the sense strand (same as the mRNA file) or the antisense or noncoding strand?
• From the mRNA RefSeq, you can link to the file for the protein product. Note the gi or accession numbers down and explore the links.
• Link to OMIM or enter your gene name in the OMIM database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM). On what chromosome is this gene found? What is its function?
• Look at the information from your two sequences:
  • Are your two genes functionally related? If yes, how so?
  • Using the full-length RefSeq data files (you can use the accession numbers), align the two sequences using pairwise BLAST (bottom of the page of http://blast.ncbi.nlm.nih.gov/Blast.cgihttp://blast.ncbi.nlm.nih.gov/Blast.cgi). Use BLASTn, MegaBLAST, and default for all other options.
  • Are the two DNA sequences related in sequence? Which portions of the genes are similar?
• Are the protein products related in sequence? Do a protein pairwise alignment of the protein RefSeq to find out (same page as the pairwise BLAST for nucleic acids, but choose program = BLASTp; Matrix= BLOSUM 62, default for all other options. Note any segments or domains that are similar.
• How are the two sequences related to the cell cycle?
• Discuss any sources of error or ambuiguity in this experiment, and how does it impact your conclusions?
• Given the scenario, what would be the likely next steps you would pursue after this particular experiment?
• Submissions:
• Data and results:
  • Table with Sequence No. for your group, Gene Name, mRNA RefSeq GI number, Protein RefSeq number, OMIM entry number. If you cannot narrow down to one, pick the best and below it note “runner up” entries.
  • Additional attachments: the Notepad/Text Files of your DNA sequencer output in FASTA format.
• Discussion: use the above questions to make a brief, coherent discussion on the scenario and your recommended next steps.

2008.07.04. Some notes:

• Summary on proteins
  • Functions in general
  • Protein structure
    • Primary structure: amino acids, peptides, and sequence
    • 3-D structure: secondary, tertiary, and quaternary; prosthetic groups
    • What holds structure together:
      • Primary structure: peptide bonds
      • 3-D structure: interactions and disulfide bonds
      • Native vs. denatured structure and what denatures a protein
  • Protein techniques
    • Size
      • Native: gel filtration or size exclusion
      • Denatured: SDS-PAGE
      • Exact: mass spectrometry
    • Charge
      • Native: Ion-exchange chromatography
      • Denatured: Isoelectric focusing
    • Presence and amount of protein
      • Ninhydrin
      • Coomassie blue staining of gels
      • Bradford protein assay
    • Specific binding
      • Affinity chromatography
    • Activity (to be discussed later under enzymes)
    • 3-D structure (no need for details)
      • NMR (a relative of MRI)
      • X-ray crystallography
• References:
  • Boyer Chapters 3 and 4, excluding antibodies, antibody-based techniques, and hemoglobin
  • http://ch151admu.wordpress.com/downloads-and-links-2/ for links to molecular visualization resources for Monday, July 7.

2008.06.17. Welcome to this page. You might want to visit http://ch151admu.wordpress.com to check out the site for basic biochemistry (Ch 151) that we offer at the Loyola campus. It was good to see you at yesterday’s school opening, and I look forward to meeting  the class of 2013 when the Cell Module starts on July 3.