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Sequence, question!


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8 replies to this topic

#1 Evgeny Denisov

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Posted 18 March 2010 - 03:19 AM

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!

Attached Thumbnails

  • post_9409_1268888463.jpg


#2 Clare

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Posted 18 March 2010 - 04:06 AM

Hello :lol:

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 :D Perhaps someone else has better input! :lol:

Clare

PS: If you repeat the sequencing do you get the same results?


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!


Edited by Clare, 18 March 2010 - 04:06 AM.


#3 Evgeny Denisov

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Posted 18 March 2010 - 04:56 AM

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-))

#4 Clare

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Posted 18 March 2010 - 05:28 AM

No probs :D

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

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-))



#5 Evgeny Denisov

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Posted 18 March 2010 - 07:35 AM

-))))

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.

#6 Clare

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Posted 18 March 2010 - 07:47 AM

Is this intron area highly conserved?
C

-))))

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.



#7 Evgeny Denisov

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Posted 18 March 2010 - 08:30 AM

To tell the truth I don't know-))

Probably, I'll sequence this sample once again. May be with reverse primer.

#8 perneseblue

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Posted 18 March 2010 - 08:20 PM

Probably, I'll sequence this sample once again. May be with reverse primer.


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.
May your PCR products be long, your protocols short and your boss on holiday

#9 hanming86

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Posted 20 March 2010 - 09:19 PM

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.
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