Download DNA Chip Analysis and Bioinformatics

DNA Chip Analysis and Bioinformatics
All underlined tasks require you to record information. A human genome microarray was used to screen for
genes that were expressed in target samples from smokers (S), non-smokers (NS) and former smokers (having
quit for at least 2 years) (FS)
The following DNA probe sequences gave an expression signal from the target samples that are noted after each
sequence. The target samples were prepared using human lung tissue obtained from biopsy.
You will use the National Center for Biotechnology Information (NCBI) resources to identify the sequences
GENE 1 - CTCATCAGTAATGGTCAGAGCAT (Smoker (S))
GENE 2 – TATGTCTCCCTAAGGGCCCAACA (Non Smoker (NS))
GENE 3 – CTGGGGTTAGTGCTGGCGATCCT
(NS, Former Smoker (FS))
GENE 4 – CCAGGAGCCCCAGTTACCGGGAG (S)
GENE 5 – GCGGCGGCAGCCCTGGGGGTCA (S)
GENE 6 – CATGATAAGGCTCTTACCCCCTT (S, FS)
GENE 7 – GTCATGGCGACTGTCCAGCTTTG (NS, FS)
GENE 8 – ATGGGGTCCGCAGCGTTGGAGAT (S, FS)
GENE 9 – GTCACTGGAAGTTGTTATTGTGGT (NS)
Control 1– CCATGGATGATGATATCGCCGC (S, NS, FS)
Control 2 –TGAACGCATTCATCGTGTGGTCT (no signal)
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Choose at least one sequence to analyze. Copy the sequence.
Go to the National Center for Biotechnology Information (NCBI) site http://www.ncbi.nlm.nih.gov/
From the menu on the right click on “BLAST” (Basic Local Alignment Search Tool).
Choose the BASIC BLAST program "Nucleotide Blast".
Paste the probe DNA sequence into the query box, scroll down and select “show results in a new
window” and click “ BLAST”. Leave all other parameters as they are.
6. Wait until the page loads (this could take a minute or so - be patient).
7. Scroll down to “Sequences producing significant alignments” and note approx. how many “ hits” your
search produced (less than 10, more than 10, more than 100, etc.). (Each line is a "Hit' that matches your
sequence to some sequence in the database of the human genome.)
8. Note the E-values (expect value). This number relates to the likelihood that your probe DNA sequence
really matches whatever gene is listed. For example, small numbers are better than large ones e.g. 6e-4
is a lot smaller than 9.2, and is a more likely match. An E-value of 0.0 means it matches exactly.)
9. For the first few hits from Homo sapiens, and NOT a "Predicted" gene, note the E-value.
10. On the left, click on the accession for the hit that looks promising (usually the first or second hit).
11. Scroll down and familiarize yourself with the information shown. Look at the “DEFINITION” . This
will give the gene name and the gene symbol in parentheses (e.g. CYP1A1)” Scroll down to see how
many introns and exons are in the gene. Note how many nucleotides are in this gene. Click on “CDS”
to view the sequence of the coding region for that gene.
12. Look at the resources on the right of the screen to help you determine why the gene might be expressed
in smokers, non-smokers or former smokers. Try the following links Record your findings.
A. Gene (look under “related information”)–Scroll down and Note the gene’s ontology
(explaineded below) Look for the graphic representation (in green) of the gene. You can mouse
over sections under the green graphic for links to HPNC and HPRD. Try these links.
B. Articles - Is this gene is unique to lung tissue or are there published articles about this gene in
other tissues of the body? Summarize your findings.
C. GEO Profiles (under “related information”)- Each Profile is presented as a chart that displays the
expression level of one gene across samples from a microarray experiment. Experimental
context is provided in the bars along the bottom of the charts making it possible to see at a glance
whether a gene is differentially expressed across different experimental conditions. Profiles have
various types of links Look at the types of experiments, and also for obviously different
expression patterns in the graphic display. In what other experimental conditions is this gene
differentially expressed? Record your findings.
D. Homologs (under “related information”)- – are there homologous genes in other organisms? If
so, what organisms? Go to Homologene and click on the blue graphic to see the structure of the
superfamily of proteins this gene codes for.
18. Copy the sequence for the negative control and BLAST it. Use the resources and skills you used above
to determine whether this is a good control for this microarray experiment. Explain your reasoning and
provide supporting evidence.
19. Go back to the NCBI home page, on the left click on “Genes and Expression” and click on the “Tools”
tab at the top right. Click on “Digital Differential Display” and select “Homo sapiens” species from the
pull-down menu. Click continue.
20. To select at least two tissue pools of ESTs to compare, scroll down to Lung and check one or more
boxes of lung tumor libraries (click on the link next to each box to determine its source). Scroll up and
type ‘lung tumor’ to name pool A and click “continue”. For pool B, scroll down and click on boxes for
tissues you identified from articles you researched. Click continue. What genes are differentially
expressed in lung tumors vs. other tissues, based on these results? Record your findings.
Gene Ontology (GO) describes three aspects of a gene
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Molecular function - Describes the function or activities of a gene product
Biological process-Biological process that involves one or more gene products in an
overall, ordered assembly of molecular functions.
Cellular compartment or Localization-the location in the cell (or outside the cell)
where the gene product exists.
“Molecular Class” is a fourth descriptor that many scientists find helpful.
NOTE: Gene Ontology includes the extracellular environment of cells; a gene product may be a
component of one or more parts of a cell