four questions about using BLAST search - (Jul/26/2006 )

Hi,

I am going to target mRNA with antisense oligonucleotide. I want to make sure that my oligo is not complementary to sequences other than my desired target.

My first question: Should I use BLAST for this purpose? If yes, can someone check if my way is right? Go to NCBI website, choose BLAST, under "Nucleotide" choose "nucleotide-nucleotide BLAST (blastn)", then input the sequence.

My second question: Should I input the original sequence of my antisense oligo, or its complementary (i.e. the mRNA sequence)?

My third question: which database should I use if I want to search within mRNA (including introns)?

My last question: if I want to limit to human genes, should I choose "homo sapiens"?

Thanks in advance for any help!

>>My first question: Should I use BLAST for this purpose? If yes, can someone check if my way is right? Go to NCBI website, choose BLAST, under "Nucleotide" choose "nucleotide-nucleotide BLAST (blastn)", then input the sequence.
You should use Search for short, nearly exact matches

>>My second question: Should I input the original sequence of my antisense oligo, or its complementary (i.e. the mRNA sequence)?
Input the target sequence (mRNA)

>>My third question: which database should I use if I want to search within mRNA (including introns)?
You can use refseq_rna, or nr. The reason for using nr is that targeting non-coding sequence or DNA may also have an effect.

>>My last question: if I want to limit to human genes, should I choose "homo sapiens"?
Yes.