module3–day3.doc

MODULE 3 – Day 3 Design siRNAs to knock down the expression of four genes that influence how cells respond upon exposure to DNA damaging agents; ATM, ATR, EXO1 and AAG Each group will design an siRNA sequence to knock down each of the four human genes listed below. i.e., every group will design siRNAs for all four sequences. Each gene is shown with its full name, common abbreviation, mRNA accession number, and the base pair region to be targeted. Ataxia telangiectasia mutated (ATM) NM_000051 bp 8700-8800 Ataxia telangiectasia and Rad3 related (ATR) NM_001184 bp 4450-4550 3-alkyladenine-DNA glycosylase (AAG, a.k.a. MPG) NM_002434 bp 700-800 Exonuclease 1 (EXO1) NM_003686 bp 1500-1600 The design of the siRNA will be based on Tuschl et al.’s criteria (http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html). These criteria have been incorporated into a search algorithm that was developed by a company called Ambion. Ambion’s website contains a search engine that will find multiple candidate sequences for each gene. You will identify candidate sequences that fall within the given base pair region for each gene. You will then perform BLAST searches to determine which candidate sequence has the highest selectivity for your gene of interest. For your interest the home page of Ambion is shown below. Finding the mRNA sequence of the gene target. Go to the NCBI homepage: /. Enter the accession number for your gene of interest. Make sure “Nucleotide” is selected from the left hand pull-down menu. Click on the Go button. Click on the link for your gene of interest. Display the mRNA sequence in FASTA format by selecting “FASTA” from the pull-down menu at the upper left next to the display button. Click on the Display button. Finding candidate siRNA sequences. Go to the Ambion siRNA design tool website: /techlib/misc/siRNA_finder.html. Copy and paste the mRNA sequence for your gene of interest into the mRNA sequence box. NOTE: Paste ONLY the FASTA sequence in –