Re: [DIYbio] Genomic integration of DNA

keywords:
"homologous recombination"
insertion
vector
homology

http://en.wikipedia.org/wiki/Homologous_recombination

On Tue, May 1, 2012 at 2:35 PM, Mega <masterstorm123@gmail.com> wrote:
> Hey guys,
>
> I've learned very much of the function of DNA and plasmids here...
>
> What I yet don't know:
>
> What are the methods to get DNA into the genome?

It depends on your understanding of "genome"... it isn't the same as
chromosome. The genome of an organism contains plasmids, larger
elements like chromosomes, and potentially viruses and their
associated DNA or RNA molecules (these could be contamination or for
cloning).

For integrating with chromosomes, all I know is homologous
recombination, and synthetic assembly like they've shown at JCVI.
Traditional cloning (restriction enzymes (REs) and ligase) might work,
but I bet the efficiency would be pretty low... here's how JCVI got
their 1MBp synthetic chromosome into a DNA-free cell:

"Genome Transplantation in Bacteria: Changing One Species to Another"
http://www.scribd.com/doc/92047145/Science-2007-Lartigue-632-8

and a presentation on that paper:
http://openwetware.org/images/0/0e/Derek_Ju_Presentation_4-8-10.pdf


>
> Could you give me some keywords so that I can do a google research on that?
>
>
>
> (I know there are e.g. Agrobacterium transformation and bacteriophage, but
> what about gene gun etc. ? )

Electroporation is my favorite way, its very versatile... there are
phages, PEG buffers (which can cause cell fusion too)... Agrobacterium
tumerfaciens first needs to be transformed with an engineered Ti
plasmid, then can be used to transform plants, which it does by
injecting the Ti plasmid into plant cells (usually flowers are dipped
in Agro solution... sonoporation, gene gun

>
>
> Am I right, that you can insert DNA of interest into a short gene sequence
> of the host, and then put it into the host?
> So it will make homologuos recombination with the strand and the insert is
> in the genome??
>

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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