What is SELEX?

Constantin Polychronakos mc97 at musica.McGill.CA
Thu Aug 3 19:45:15 EST 1995


>   stephan.witte at uni-konstanz.de (Stephan Witte) writes:
>  Hi,
>  there is a method, called SELEX, for determing the target sequences of
>  RNA-binding proteins (?)
>  How does it work?
>  References?
>  
>  Stephan
>  
>                 
>  Stephan Witte
>  Inst. of Immunology
>  University of Constance
>  stephan.witte at uni-konstanz.de


	The method involves repeated rounds of selection and amplification from 
a library of degenerate RNAs that are retained in an affinity column made with the 
protein under study. A DNA oligo is synthesized that has defined sequences at each 
end, but contains 50-100 bases of random sequence in the middle. Ten nmols of this 
oligo contain 10 E15 molecules, each with an unique sequence (a small fracrtion of 
all possible sequences of that length). The sequence is converted to RNA by in vitro 
transcription, and applied to an affinity column. RNA molecules retained in the 
column are eluted, typically by heating to denature the RNA secondary structure, 
amplified by RT-PCR (or directly cloned in a cDNA library), in vitro transcribed, and 
the whole cycle is repeated, each time enriching the pool in RNA sequences that 
bind the protein. When most of the RNA binds to the column, individual clones are 
sequenced and the consensus sequence determined. It sound insane, but it works 
beautifully. See Tuerk and Gold, Science 249:505-510

Constantin Polychronakos
Department of Pediatrics
McGill University
Montreal, Canada





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