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Old 09-14-2009, 08:39 AM   #1
nathan hunkapiller
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Location: San Francisco

Join Date: Sep 2009
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Default How do I sequence the area around a random trangene insert?

For my genotyping needs, I would like to design an assay to discriminate alleles of wild type mice from those that have one and also two copies of a randomly inserted transgene that I created. Problem is I don't know where the insert landed.

I know the sequence of the insert and was hoping to sequence outward from the flanking ends. Are there good methods for sequencing directly from genomic DNA without performing PCR? I obviously can't design primers outside of the insert.
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Old 09-14-2009, 01:10 PM   #2
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Location: SF Bay Area, CA, USA

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Hey Nathan,

Can't respond in detail now, but start your search for LAM-PCR. Pretty standard method that works well.

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Old 09-15-2009, 10:57 AM   #3
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Default Region-Specific Extraction

I worked with this group a while ago, and the technology was quite promising.

narrated ppt from European Federation of Immunohistocompatibility:
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Old 09-15-2009, 11:13 AM   #4
Location: Netherlands

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We have used so-called vectorette system in the past quite succesfully. Works well on bacterial as well as eukaryotic DNA.
Our protocol that time:

My guess is that this procedure required cloning and such at that time . Now I would choose to simply sequence the product and separate out the mixture of sequences

And some links with references:

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