Seamless Cloning->LIC (ligation independent cloning)

[Bradley Turner]

Dear Jeanne,


You might also want to consider LIC (ligation-independent-cloning),which
is described in the references below, as allowing directional cloning
of PCR products without using ligations, restriction enzymes or T/A
vectors.

Basically, one adds an extra 11-12 bases to the 5' end of your
PCR primers. After PCR, the PCR product ends are digested in the 
presence of T4DNApol with dTTP or dATP which exposes the 11-12
LIC site at the ends of your PCR product. This is *annealed*
with the LIC vector at RT for about 30min and then transformed
into e coli (NO ligations, NO restriction digests).

References:

Rapid, reliable ligation-independent cloning of PCR products using
modified plasmid vectors
RS Haun, IM Serventi, J Moss
Biotechniques 13(4)515-8, 1992

Ligation-independent cloning of glutathione S-transferase
fusion genes for expression in Escherichia coli
RS Haun, J Moss
Gene 112:37-43, 1992

Ligation-independent cloning of PCR products (LIC-PCR)
C Aslanidis, P deJong
Nucleic Acids Research 18(20)6069-74, 1990

Minimal length requirement of the single-stranded tail for
ligation-independent cloning (LIC) of PCR products
C aslanidis, PJ deJong, G Schmitz
PCR Methods and Applications 4:172-7, 1994

Novel methods for cloning and engineering genes using the polymerase
chain reaction
A Rashtchian
Current Opinion in Biotechnology 6:30-36, 1995

Ligation-independent cloning of PCR products with primers containing
nonbase residues
S Kaluz, APF Flint
Nucleic Acids Research 22(22)4845, 1994

Also, Pharmingen (800-848-6227) and Novagen (800-526-7319) 
and Clontech (PCR-Direct, 800-662-2566) all sell
LIC vectors and kits [no affiliation with these companies].
Also the LIC-vectors described in the articles by Haun and Moss 
are available from ATCC (at minimal cost) as follows:

pBluescript II KS(+)/LIC, ATCC #87047
pGEM-7Zf(+)/LIC-F,        ATCC #87048
pGEM-7Zf(+)/LIC-R,        ATCC #87049

I hope you find this helpful,
Brad Turner

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		    Bradley Turner
	         Beth Israel Hospital

Harvard Medical School		617-667-1215 phone
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