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This protein sequence is highlyconserved across several species, a trait that is often a sign of physiological importance. Scroll through the protein alignments. Notice that alignmentbetween amino acids is illustrated differently than alignment between nucleotides. An identical match is shown by listing the one letter amino acidcode in the middle row, between the two aligned sequences. A mismatch is indicated by a blank, but similarity is indicated by the "+" sign, meaning theamino acids are not identical, but they have some of the same chemical or structural properties. Gaps are indicated by hyphens in the sequence that contains the gaps. Gaps are penalized in an alignment, and cause the normalized score to be lower.

BLAT , stands for Blast-like alignment tool (2), and is used forsequence comparisons against an entire genome. Review the difference between BLAT and BLAST at the BLAT FAQ site . Execute aBLAT search, using the sequence above, from the UCSC Genome Bioinformatics Site . Click on the link in this web page entitled "BLAT".Search the human genome, leaving the settings at the default values. The search results will first appear in summary form. Identify the sequence our query most closely matches by the highest score.

What human chromosome (number) contains the sequence our query most closely aligns?

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What percent identity exists between our sequence and the aligned subsequence(s)?

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Click on the link entitled "details". First, the query sequence is presented,with matching bases capitalized in blue font. Bases in cyan mark the beginning and end of aligned subsequences, indicating a gap in either the reference or the querysequence. Second, the genomic sequence from the selected chromosome is shown, and blue, capitalizedbases illustrate the matching region(s). Notice that the query cDNA sequence is missing a largeregion of DNA that is present in the chromosome. The pairwise alignment is shown below the genomic sequence. Return to the original results summary andclick on the link entitled "browser". The missing region is illustrated in graphical form here, where the chromosome band is shown in grey, extendingacross the graph and the query sequence, labeled at the left as "YourSeq", is in black, below the STS Markers.In fact, the examples in this module use an EST, or an expressed-sequence tag, for the query sequence. ESTs are mRNA-derivedrepresentations of the genes expressed in a given tissue and/or at a given developmental stage.

Knowing that our sequence is an EST, how could this explain the region of DNA that appears in the genomic sequence, but not in our query sequence?

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If the answer to this question is not intuitive, read the EST section of the NCBI primer. Scroll down to the section entitled "Genes and Gene Prediction Tracks" and change the display options under Ensembl to "pack" and the setting under GenScan to "pack". Next, scroll to the bottom of the page and hit the refresh button. (For a description of the different display options for annotation tracks, read the User Guide . The GenScan predicted genes for this area of the chromosome are shown in brown, while the Ensembl gene predictions are in maroon.

How does our sequence line up with the predicted genes?

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Now, design an independent search in order to answer the following question. What is the GI number and definition for the EST that aligns most closely with our query sequence, according to BLAST?

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This has been an introduction to BLAST sequence alignment, designed to give an idea of some of the different ways to optimize searches and problems that require the useof sequence alignment tools. Subsequent modules in this course will build on this foundation, exploring advanced techniques and additional alignment tools.

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Source:  OpenStax, Bios 533 bioinformatics. OpenStax CNX. Sep 24, 2008 Download for free at http://cnx.org/content/col10152/1.16
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