small fragment library construction

Chris Boyd chrisb at hgu.mrc.ac.uk
Tue Sep 28 02:45:32 EST 1999


Frank O. Fackelmayer (Frank.Fackelmayer at uni-konstanz.de) wrote:
: Hi all,
: I´m collecting ideas on how to make a library of short DNA fragments in
: an expression vector. I have a 2.7kb cDNA fragment that I want to split
: into many overlapping fragments in the 30-80 base pair size range. For
: cloning, it would be best to have a restriction site on both sides of
: the fragment, although in principle I could also use blunt end cloning
: if no other possibility should work. I would prefer to have a PCR-based
: method rather than an enzymatic digestion with e.g. DNase or micrococcal
: nuclease. Digesting with a combination of restriction enzymes (or CviJI)
: is not an option because of a quite long, highly GC-rich sequence that
: is either not cut at all (by RE having A or T in their recognition
: sequence) or chopped up to too small fragments (with GC-enzymes).

Dear Frank,

It is my impression that conventional cloning of very small fragments
is much more problematic than cloning moderately sized fragments,
contrary to what theory would predict. I think this is because getting
the vector:fragment ratio and DNA concentrations just right is more
critical than normal. As a way round this, have you thought about in
vivo cloning (IVC)? Make your small fragments by whatever method, then
ligate on catch linkers L1 and L2 with 30 bp or so homology to
appropriate regions of the vector as shown and PCR up the fragments.
Then co-electroporate a suitable recombinogenic strain (e.g. JC8679)
with a mixture of PCR products and linearized vector:


    L1            insert               L2
    ---------##################----------
        X                          X
    ---------                  ----------
    |                                   |
    |                                   |
    |___________________________________|
            linearized vector

Homologous recombination in the host will generate the desired
constructs. Because your inserts are so small, the process could be
quite efficient. With a suitable design of primers, you could even try
IVC and conventional cloning with the same PCR fragment pool.

We have used methods similar to this to excellent effect in many
cloning experiments.

Some references:

1. Jones, D. H. (1994) `PCR mutagenesis and recombination in vivo.'
   PCR Methods and Applications, 3, S141-S148.
2. Bubeck, P., Winkler, M. and Bautsch, W. (1993) `Rapid cloning by
   homologous recombination in vivo.' Nucleic Acids Res., 21, 3601-3602.
3. Ali, S. A. and Steinkasserer, A. (1995) `PCR-ligation-PCR mutagenesis
   - a protocol for creating gene fusions and mutations.'  BioTechniques,
   18, 746.
4. Boyd, A. C. and Porteous, D. J. (1997) `PCR-generated crossover
   linkers for site-directed mutagenesis.' BioTechniques, 23, 827-830.


Best wishes,
-- 
Chris Boyd                      | from (but not \  MRC Human Genetics Unit
Christopher.Boyd at hgu.mrc.ac.uk  | much longer)  /      Crewe Rd, Edinburgh
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd          EH4 2XU, SCOTLAND



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