[Automated-sequencing] Re: Direct Genomic DNA Sequencing of mammalian cells

Phillip San Miguel via autoseq%40net.bio.net (by pmiguel At purdue.edu)
Thu Nov 9 12:11:58 EST 2006


Hi Alejandro,
I think this is not possible to accomplish directly. With DNA preps, the 
molar concentration of the sequence the primer anneals to will be 
inversely related to the genome size. That is, if the genome you 
sequence from is 1 megabase, then you need 650 ug of DNA to give you a 
picomole of it. The ABI 3730XL is pretty sensitive, but it is touch to 
sequence directly from E. coli genomic DNA (~4 megabase genome). A 
mammalian genome will be almost 1000 times the size of of the E. coli 
genome.

Indirect methods should be possible, however. Even where you don't know 
the sequence of all the DNA you intend to sequence, TAIL PCR can be used 
(Genomics. 1995 Feb 10;25(3):674-81.)

Where you know the (approximate) sequence, you might be able to use even 
simpler methods. Maybe you could put 2 primers in your sequencing mix, 
but put one in at 1/100th or 1/1000th the concentration of the other. If 
the primers flank the area you want to sequence, like normal PCR 
primers, you would get PCR amplification occurring until the sequencing 
reaction ran out of the low concentration primer.  That might bring the 
PCR product up to a concentration that would produce usable sequence.

That is pretty simple, so there are probably published protocols. Or you 
could probably whip one up yourself in a week or so.
Phillip


Alejandro Gonzalez wrote:
> Hi everybody, I'm working at automated sequencing using MegaBACE 1000 DNA
> sequencer at my job and my question is, does anyone know of a reliable
> method of sequencing that can be applied to genomic DNA directly? but avoid
> pre-Pcr amplification step. Just directly sequencing of genomic DNA of the
> mammalian organism.
> 
> Any help would be appreciated,
> 
> Sincerelly,
> 
> Alejandro D González
> Gema Biotech sa
> agonzalez At gemabiotech.com
> 
> ----- Original Message -----
> From: "David Reynolds" <dreynold At aecom.yu.edu>
> To: <autoseq At oat.bio.indiana.edu>
> Sent: Tuesday, October 03, 2006 2:12 PM
> Subject: [Automated-sequencing] Re: Autoseq Digest, Vol 16, Issue 1
> 
> 
>> Hi Ian,
>>
>>
>> This list should be able to help you.
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>> At 01:01 PM 10/3/2006, you wrote:
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>>> Message: 1
>>> Date: Tue, 3 Oct 2006 13:24:20 +0100
>>> From: "IanMc" <zebedeeboy At hotmail.com>
>>> Subject: [Automated-sequencing] Laboratory Robotics
>>> To: bionet-genome-autosequencing At magpie.bio.indiana.edu
>>> Message-ID: <eftkpi$jj6$1 At gemini.csx.cam.ac.uk>
>>>
>>> Hi does anyone know of a lab robotics interest group or newsgroup. We
> have
>>> recently acquired an MWG RoboSeq and I would like to talk with other
> users
>>> about hardware and software topics.
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>>> TIA
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>>> Ian Mc
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