Chapter 16
Cloning Strategies: An Overview
FRIEDHELM HILDEBRANDT
lntroduction
When trying to clone a gene for a genetic disease, different approaches can
be followed, depending on the availability of prior information about the
function or nature of the gene or its product. Figure 1 gives an overview of
the different cloning strategies that are available. The relevant methods are
discussed in the subsequent chapters.
Post-purification cloning
If the relevant gene product is known, antihoclies against the protein can be
generated and used for cDNA library screening. Alternatively, protein purification can be attempted in order to obtain partial amino acid sequence.
cDNA sequence can be inferred from the amino acid sequence, and oligonucleotides can be synthesized for library screening or for use in a PCRbased cloning approach. Due to the degeneracy of the genetic code, the
cDNA sequence will not be unequivocal. This problern is discussed below.
Cloning by sequence similarity
In a situation where there is some prior sequence available of a related gene
in a different species, or of an isoform of a protein, this sequence information can be used to design oligonucleotides, that represent a "best guess"
with respect to the target cDNA sequence. When designing oligonucleotides
Friedhelm Hildebrandt, Universitäts-Kinderklinik, Mathildenstrasse 1, Freiburg, 79106,
Germany (phone +49-761-270-4301; fax +49-761-270-4533; e-mail hildebra@kkl200.ukl. uni-freiburg.de)
Cloning Strategies: An Overview
FRIEDHELM HILDEBRANDT
lntroduction
When trying to clone a gene for a genetic disease, different approaches can
be followed, depending on the availability of prior information about the
function or nature of the gene or its product. Figure 1 gives an overview of
the different cloning strategies that are available. The relevant methods are
discussed in the subsequent chapters.
Post-purification cloning
If the relevant gene product is known, antihoclies against the protein can be
generated and used for cDNA library screening. Alternatively, protein purification can be attempted in order to obtain partial amino acid sequence.
cDNA sequence can be inferred from the amino acid sequence, and oligonucleotides can be synthesized for library screening or for use in a PCRbased cloning approach. Due to the degeneracy of the genetic code, the
cDNA sequence will not be unequivocal. This problern is discussed below.
Cloning by sequence similarity
In a situation where there is some prior sequence available of a related gene
in a different species, or of an isoform of a protein, this sequence information can be used to design oligonucleotides, that represent a "best guess"
with respect to the target cDNA sequence. When designing oligonucleotides
Friedhelm Hildebrandt, Universitäts-Kinderklinik, Mathildenstrasse 1, Freiburg, 79106,
Germany (phone +49-761-270-4301; fax +49-761-270-4533; e-mail hildebra@kkl200.ukl. uni-freiburg.de)