Recombinant DNA
Prof Peter Chantler
Molecular Biology of the Cell
Molecular Cloning
e. Creation of an expression library
If nucleotide sequences are not available as probes for library screening (eg. sequence is not known) antibodies could be used for screening, if available. However, to do this one must create an expression library ie. a library that not only contains the DNA fragments of interest but one that can actually manufacture the protein coded by the fragment so that it may be detected by the antibody. This requires that the cDNA fragment within the vector be inserted downstream of a bacterial promoter which will cause the inserted fragment to be expressed.
In this example (see animation below), the phage vector, lgt11, has an EcoRI cloning site located within the coding region of the E. coli lacZ gene. If the insert is in the correct reading frame it will be translated to form a fusion protein made up partly from b-galactosidase and partly from the protein product of the cDNA insert, which may be detected by the antibody, using replica plates. Once the desired plaques are detected, one may return to the master plate and rescue the original material.
Course
1 Molecular Cloning
a Choice
b Insertion
c DNA Library
d Screening
e Exp Library
2 DNA Sequencing
3 PCR
4 VNTRs
5 RAPD
Contacts
Prof Peter Chantler
RVC eMedia Unit
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