LAB - Bioinformatics

DNA sequence alignment viewed with the multiple alignment editor Se-Al. On the far right there are insertions and/or deletions in several sequences (click pic for full-size image)

We had laptops for each student in the lab and had a small and crude (quick and dirty?) overview of some of the few tools available in bioinformatics.

The main focus of our exercise was the use of the algorithm known as BLAST (Basic Local Alignment Search Tool), available at the NCBI webpage. After an overview of some of the main features of the site (which is updated VERY frequently), we "BLASTed" (used BLAST) a few unknown protein (using 'protein BLAST') and nucleotide (using 'nucleotide BLAST') sequences. We found out what the sequences were and to what organisms they belonged. This is one of many possible uses of the BLAST algorithm.

During this lab students were also introduced to the fasta file format and its use for sequence alignment. After finding out what our problem nucleic acid sequences were (by BLASTing)and to what organisms they belonged we made a fasta file with all of them and aligned them using an on-line version of ClustalW available on the European Bioinformatics Institute website.

The following learning outcomes should have been met:

• Introduction to the concept and field of bioinformatics
• Introduction to the main sequence data repository in the Americas and one of the main in the world: NCBI
• Introduction to the main database in the NCBI website: GenBank
• Basic understanding of the fasta file format
• Basic use of the BLAST algorithm
• Introduction to the concept of sequence alignment

Lab report

The lab report can be typed and it won't follow the format of the previous reports. Please include:

• An brief introduction explaining what bioinformatics is
• A summary of the exercises performed in the lab (methods)
  
• Findings about the problem sequences. What organisms did they belong to? What proteins and genes were they? - Include the three first hits for each search if available. No need to include the actual sequences
• Research question: What are the potential uses of a sequence alignment?

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LAB - Silver staining a polyacrylamide gel

Download the Bio-Rad Silver Stain Plus protocol in pdf format by clicking here.

pGLO plasmid restriction digest, PAGE, and visualization through silver staining.
The staining procedure didn't work as expected (due to longer than needed staining and shorter than required drying...), so no picture can be taken. Here are drawings of the band patterns:

• How many bands did you expect based on the number of restriction sites for BamH I? (check your plasmid map)
• What may explain the band pattern you got?

Speculate about these questions in your discussion.

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Exam 02

In the exam 80% of the questions were multiple choice, with the remaining 20% being short essay questions. I decided to include essay questions becuase people had not been doing very well in quizzes, but this section was actually the strongest for most people.

Exam stats:

• Mean score: 79 (86 in exam 1)
• Standard deviation: 10.9 (8.8 in exam 1)
• Mode: 82 (90 in exam 1)

Scores were down in average and the standard deviation increased compared to exam 1. Most likely because of the material being newer for most students, and a little more complex. Any other hypothesis?

Score frequency distribution

Toughest questions (statistically):

12. Mutation rate can be measured experimentally...
b. comparing the genomes of an ancestor and its descendants

Comment: A bacterial genome can be sequenced, then culturing the strain for a number of generations, and then sequencing the genome of a descendant. By comparing both genome sequences and knowing the number of generations past, the mutation rate can be measured.


13. Mutation rate can be masured by observation...
c. comparing the genomes of multiple species

Comment: When generation times are too long to actually allow many generations to go by (e.g. in mammals) an experimental measurement of mutation rate is not possible. In such cases it can be estimated by comparing the genomes of multiple related species and measuring generic distances.


20. A primer strand...
b. is the strand of DNA being extended

Comment: Primer strand is the technical term for a "daughter" strand, whereas template strand is the name for the "parent" strand. Most people circled the answer for 'what is a primer'. A primer is a strand of oligonucleotides, but it's not called 'primer strand'. Just 'primer'.


25. Single strand DNA-binding proteins...
e. straighten the template strand during relpication

Comment: They prevent the single tremplate strand of forming loops that can interfere with the reading of the DNA plymerase.

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DNA-Binding proteins and genetic switches

We covered pages 416-432 of the book, dealing with the properties that gene regulatory proteins have, mainly the fact that they can read the information in the DNA double helix without mamking direct contact with the bases, but by 'reading' chemical signals in the phosphodiester backbone unique to each nucleotide.

We also talked about the main structural motifs that are common to gene regulatory proteins (helix-turn-helix, zinc finger beta-sheet, leucine zipper, and helix-loop-helix).

We also covered pages 432-439, explaining how simple genetic switches work. They have been studied in bacteria and have the particularity that are composed by a single gene regulatory protein. We introduced the concepts of operon, operator, activator, and repressor.

Next class: Complex genetic switches (in eukaryotes), and post-trancriptional control

Quiz #13 Q&As:

1. In a multicellular organism what process ensures that the only proteins and RNAs to be produced in a particular cell are those that that particular cell (and, therefore, tissues and organs) requires?
Control of gene expression

2. In a multicellular organism most cells have all the information required to build a whole organism. Why does a cell “know” that only a fraction of the information must be used, and not all of it?
Control of gene expression

3. What is transcriptional control?
A mechanism through which gene regulatory proteins activate or repress trasncription of a gene

4. Mention two kinds of control of gene expression (other than transcriptional)
RNA processing, RNA transport and control, translational mRNA degradation, RNA editing, RNA differential splicing...

5. What is the difference between a simple genetic “switch” and a complex one?
In a simple one only one protein turns de gene on or off. In a complex one many proteins are involved, usually hundreds.

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