Sequence, question! - (Mar/18/2010 )
Hi
I don't know how i should analyze this sequence file. See attached!
How can you interpret these N? It's single mutations or any errors.
Please, help!
P.S. The purpose of this analysis is the search of mutations!
Hello 
What exactly are you sequencing??
If it were a clone or something I would say your N is a definite A. BUT if you are looking at a particular species for mutations, is it possible they are heterozygous for A and T? That's just my guess 
Perhaps someone else has better input!
Clare
PS: If you repeat the sequencing do you get the same results?
Evgeny Denisov on Mar 18 2010, 11:19 AM said:
I don't know how i should analyze this sequence file. See attached!
How can you interpret these N? It's single mutations or any errors.
Please, help!
P.S. The purpose of this analysis is the search of mutations!
Thanks for reply!
I sequence of TP53 gene (exons 4-8) from tumor samples. These N are found only in this sample!
Although I didn't perform repeat of sequence-))
No probs
What are the Ns in your normal control samples? Are these Ns throughout the whole seq or just in the little area that you showed us? Do they result in an amino acid change?
Clare
Evgeny Denisov on Mar 18 2010, 12:56 PM said:
I sequence of TP53 gene (exons 4-8) from tumor samples. These N are found only in this sample!
Although I didn't perform repeat of sequence-))
-))))
The normal nucleotides (control) are A. These N-s are found only in this little area... It's so strange!-)
Really, these Ns are affected introns.
Is this intron area highly conserved?
C
Evgeny Denisov on Mar 18 2010, 03:35 PM said:
The normal nucleotides (control) are A. These N-s are found only in this little area... It's so strange!-)
Really, these Ns are affected introns.
To tell the truth I don't know-))
Probably, I'll sequence this sample once again. May be with reverse primer.
Evgeny Denisov on Mar 18 2010, 08:30 AM said:
I agree with Evgeny, Sequence again with the reverse primer. And see how that pans out. If the result is repeatable, clone your PCR product into a plasmid and sequence again. This allow sequencing of individual PCR products. You should then have two types of plasmids.
Considering it's from cancel cell then .. i wouldn't think of it as weird. when u work with mammalian cell .. i think they have differing number of chromosome. allele? it could have just been that it's a combined pcr product from different allele or so.
go with the AT cloning business adn get the individual plasmid as suggested above.