BLAST_exercise_in_class

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York University *

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4290

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Biology

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Feb 20, 2024

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docx

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7

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BIOL 4290 BLAST in-class assignment 2023-10-02 BIOL 4290: In-class group assignment: using Nucleotide BLAST to determine the size of the PAP PCR fragment BLAST is a bioinformatics tool that find regions of similarity between biological sequences. When biological sequences are determined, they are loaded on servers (websites) and stored there for others to access. A very popular site is the NCBI site (below). You will use this site to examine the PAP gene, to determine where the PCR primers should bind, and where restriction enzyme sites would be. 1. Call up the NCBI site: https://blast.ncbi.nlm.nih.gov/Blast.cgi 2. You will see two BLAST choices: Nucleotide BLAST and Protein BLAST. Pick the nucleotide option! After you click on the BLAST icon, you will see the search page; Page 1 of 7
BIOL 4290 BLAST in-class assignment 2023-10-02 In order to search for a particular DNA sequence you need to know some information. 3. Copy and paste the PAP sequence (see below) into the query box and press “BLAST”. At the bottom of the page, you will see this block: You don’t really need to change settings from the default settings but if you want you can play with the settings to learn more. If you were unsure of your sequence or you wanted to search for similar but not identical sequences (for instance if you wanted to find the same gene in other species), you could change the setting from Highly Similar (see above) to somewhat similar etc. Once you insert your sequence (again below) in the Query box and hit the BLAST icon, it will take a few minutes to get results. As an example, I searched for a different sequence (yeast dUTPase) and got this result: Page 2 of 7
BIOL 4290 BLAST in-class assignment 2023-10-02 You’ll note that several options pop up. Many people can upload the sequence for the gene – often having slight differences in sequence leading to multiple entrys. Also the gene may have introns (genomic source sequencing) and so if the sequencing template was from a cDNA template, an entry deduced from that source will lack introns and even though the sequence represents the same gene, they will have different sequences. Also, there are several different isoforms of genes - again leading to multiple entries. You want to find one with the highest percentage similarity (can you see that in the picture above?). The gene is the DUT1 gene from Saccharomyces cerevisiae and has an accession number of X7463.1. This number is essentially a catalogue number. Q – What do the prefixes on the Accession numbers refer too? (ie NM, CP, X, etc) It refers to which data base that collected this data Click the Accession number to get the information for this gene. Here is the DUT information; Page 3 of 7
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