PCR ?

Bradley Turner bsturner at mbcrr.harvard.edu
Sat Jul 8 18:44:33 EST 2000


Hi David,

Sounds like a job for "transposons".  They have been used for quite 
a while to integrate 'randomly' into prokaryotic genomes
to cause mutations that can later be mapped by sequencing
out from the transposon ends.  There are now even commercially
available kits for doing this, and even constructing your
own transposons with your selectable marker of choice.

Try the following:

Epicentre Technologies
1202 Ann Street
Madison, WI 53713 USA
608-258-3080 phone
608-258-3088 fax
<http://www.epicentre.com>

EZ::TN pMOD Transposon construction vector
EZ::TN Insertion kit (for creating bacterial gene knockouts)

Some background reading on the above would be (based on Tn5)

Tn5 in vitro transposition
Journal of biological Chemistry 273(13)7367-7374, 1998
IY Goryshin, WS Reznikoff

Tn5tac1, a derivative of transposon Tn5 that generates
conditional mutations
Proc Natl Acad Sci USA 85, 6468-72, 1988
W-Y Chow, DE Berg

Tn5 lacZ Translation fusion element: Isolation and
analysis of transposition mutants
Meth Enzymol 217(22),312-22 1993
WS Reznikoff,R Jilk, MP Krebs, JC Makris, PL Nordmann
M Weinreich, T Wiegand

Transposon Tn5 mutagenesis to map genes
Meth Enzymol 154(11)175-196, 1987
FJ deBruijn

-----------------------------------------------

New England BioLabs produces a transposon kit GPS
(Genome Priming System) based on Tn7

New England BioLabs
32 Tozer Road
Beverly, MA 01915-5599 USA
978-927-5054 phone
978-921-1350 fax
<http://www.neb.com>

Transposon Tn7
Curr Topics Microbiol Immunol 204, 27-48, 1996
NL Craig
---------------------------------------------
Primer Island Transposition Kit based on 
yeast Ty1 system

PE Applied Biosystems
850 Lincoln Centre Drive
Foster City, CA 94404 USA
415-570-6667 phone
415-572-2743 fax

Efficient integration of artificial transposons into plasmid
targets in vitro: a useful tool for DNA mapping, sequencing
and genetic analysis
Nucleic Acids Research 22(18)3765-72, 1994
SE Devine, JD Boeke
--------------------------------------------
The Template Generation System based on
the Mu transposon from 

Finnzymes OY
PO Box 148
FIN-02201 ESPOO
+358-9-584 121 phone
+358-9-5841 2200 fax 
<http://www.finnzymes.fi>

An efficient and accurate integration of mini-Mu transposons
in vitro: a general methodology for functional genetic
analysis and molecular biology applications
--------------------------------------------

A good general reference might be:

Transposable element tools for microbial genetics
CM Berg, DE Berg, Chapter 140 pp. 2588-2612 in
Escherichia coli and Salmonella cellular and
molecular biology vol.2 2nd edition
Ed. FC Neidhardt et al. ASM press Washington, DC 199?

Systematic identification of essential genes by in vitro
mariner mutagenesis
Proc Natl Acad Sci USA 95, 8927-32, 1998
BJ Akerley, EJ Rubin, A Camilli, DJ Lampe, HM Robertson,
JJ Mekalanos

----------------------------------------------

I'm not as familiar with the literature and techniques
for eukaryotes but you might start with:

Resident aliens: the Tc1/mariner superfamily of 
transposable elements
Trends in Genetics 15(8)326-32, 1999
RHA Plasterk, Z Izsvak, Z Ivics

or medline search or basic biochem/molec biol text
on anything about 'mobile DNA elements' 

Hope this helps,
Brad Turner [no affiliations with above companies]


****************************************************************
                    Bradley Turner
                Beth Israel Deaconess
                    Medical Center

Harvard Medical School          617-667-1215 phone
Division of Gastroenterology    617-667-2767 fax
Room Dana 605                   bsturner at biosun.harvard.edu
330 Brookline Avenue            bturner at caregroup.harvard.edu
Boston, MA 02215                bsturner at mbcrr.harvard.edu
****************************************************************


==============================================================


On 8 Jul 2000, David Keszenman-Pereyra wrote:

> I want to integrate a selectable cassette randomly into the genome. 
> Then I want to sequence the flanking regions of the integration site.
> Is there any PCR method to do it quickly ????? (single primer 
> amplification ?????)
> 
> 
> David Keszenman-Pereyra
> University of Sheffield
> Department of Molecular Biology & BIotechnology
> Firth Court, Western Bank, Sheffield S10 2TN
> UK 
> 
> 
> 
> 
> ---
> 
> 
> 


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