sequencing antibody mRNA

Mike Preston preston at warren.med.harvard.edu
Tue Nov 7 15:05:33 EST 1995


In article <Moulinou-0711951103190001 at mac-delcros.univ-rennes1.fr>,
Moulinou at Univ-Rennes1.fr (Jacques-Philippe Moulinoux) wrote:

> I am currently looking to get the sequence of the variable region of a
> monoclonal antibody that we obtained in my laboratory.
> Since I am new in the field I would like either to get detailed protocols
> on how to make it or to establish a collaboration with somebody who
> possesses the technology.
> The monoclonal antibody is an IgG1 that have been ellicited against the
> polyamine spermine.

I have used the PCR primers described by Coloma et al. to amplify the V
regions of a number of murine monoclonal antibodies.  For sequencing, I
clone the PCR products into a TA cloning vector (Invitrogen works for me,
no affil) and sequence using T7 and SP6 primers. I'm sure that there are
other primers that have been described that work as well or better but
these worked for me.  

1. look up a good protocol for isolating total RNA, then mRNA (or use a
kit if you have the money)

2. Reverse transcribe the mRNA to cDNA using random hexamer primers.

3. PCR using the conditions and primers described below.

Coloma, M. J., A. Hastings, L. A. Wims, and S. L. Morrison. 1992. Novel
vectors for the expression of antibody molecules using variable regions
generated by polymerase chain reaction. J. Immunol. Methods 152:89-104.

Mike

-- 
Michael Preston
Channing Laboratory
preston at warren.med.harvard.edu



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