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
**************************************************************
Bradley Turner
Beth Israel Hospital
Harvard Medical School 617-667-1215 phone
Division of Gastroenterology 617-667-2767 fax
Room Dana 536 tur...@sprcore.bih.harvard.edu
330 Brookline Avenue bstu...@mbcrr.harvard.edu
Boston, MA 02215 btu...@bih.harvard.edu
**************************************************************
brett
>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).
Maybe you could comment on the cost effectiveness of this method. As you
describe it, you'd be adding an aditional $20 in nucleotides per PCR just to
clone the thing. I have never had too much difficulty in standard RE cloning of
PCRs, so I'm not convinced of the utility of LIC.
Brett Lindenbach
Program in Immunology
Washington University - St Louis
br...@borcim.wustl.edu
"I own my own pet virus. I get to pet and name her." - Cobain
Bradley Turner
Dear Brett,
I am primarily using LIC in 3' & 5' RACE-PCR reactions, in which
I have no idea of the restriction sites that may be present in my
PCR products. Thus, LIC allows me to clone my RACE (rapid amplification
of cdna ends) products without subjecting them restriction digestion,
and possibly cleaving internal sites.
It also has the advantage of allowing me to clone my PCR products
directionally, which saves a little time in sequencing, expression
and probe production.
I am also using LA-PCR methods which may remove the terminal A's
of PCR products thereby reducing ligation efficiency into traditional
TA cloning vectors.
I haven't really compared the cost of adding LIC sites (11-12 nt)
to my PCR primers to that of adding restriction sites to the ends of
primers (4-8 nt *PLUS* a few extra bases to insure that the
restriction enzymes will be able to cut near the ends of the PCR
product and so that TAQ or ligase won't remove the added restriction
sites) but it seems that the cost shouldnn't be that different.
Thus for my applications, the possible added slight cost is offset
by the other potential advantages of the LIC technique.
CAVEAT:
So far I've only used LIC with positive controls and not with my
samples yet, so I would welcome any other helpful comments or
advice regarding the LIC technique or PCR cloning in general.
Thanks,
Brad
**************************************************************
Bradley Turner
Beth Israel Hospital
Harvard Medical School 617-667-1215 phone
Division of Gastroenterology 617-667-2767 fax
Room Dana 536 tur...@sprcore.bih.harvard.edu
330 Brookline Avenue bstu...@mbcrr.harvard.edu
Boston, MA 02215 btu...@bih.harvard.edu
**************************************************************
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To: met...@net.bio.net
From: br...@borcim.wustl.edu (brett)
Subject: Re: Seamless Cloning->LIC (ligation independent cloning)
Date: 16 Aug 1996 14:05:40 -0700
Message-ID: <1996081621...@wustl.edu>
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