Summary
MBY364 Theme 5 summary
- Course
- MBY364 (MBY364)
- Institution
- University Of Pretoria (UP)
Full summary of theme 5 (screening and recombinant selection) made using lecture notes, the textbook and class notes.
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SCREENING AND RECOMBINANT SELECTIONBACKGROUND
- Isolating a recombinant from a population depends on the specific cloning
strategy
- An appropriate selection strategy should enable selection of a specific
recombinant clone from a large population of clones
- There are a number of techniques available to screen large numbers of
recombinants e.g. genetic selection, protein-dependent methods or nucleic
acid sequence-dependent methods
- Identification can be done by exploiting either the sequence of the clone or the
structure/function of its expressed product
GENETIC METHODS TO SELECT FOR THE PRESENCE OF A VECTOR
- Plasmids and cosmids:
- Antibiotic resistance - allows to select for transformed cells
- Replica plating and insertional inactivation of marker gene - selects for
recombinants
- Phage vectors:
- Plaque formation
- Size of cloned insert and Spi phenotype - selects for recombinants
- A limitation is that it only differentiates between transformed cells and
recombinant clones
IMMUNOCHEMICAL SCREENING
- Depends on the expression of a specific gene and the availability of a specific
antibody
- Is one of the most versatile expression cloning strategies - can be applied to
any protein for which an antibody is available
- No need for the protein to be functional
- Requires the use of an expression vector
- Molecular target is an epitope - short sequence of amino acids that folds into
a particular 3D conformation on the surface of the protein
- Epitopes fold independently of the rest of the protein - form even when the
polypeptide chain is incomplete
- Many epitopes can form under denaturing conditions when the overall
conformation of the protein is abnormal
- Polyclonal antibody recognises many different epitopes but a monoclonal
antibody recognises a single epitope
- Polyclonal antibodies are more sensitive - if the epitope is not
available, it can recognise another epitope
, - Monoclonal antibodies are more specific - there is a possibility for
polyclonal antibodies to cross-react with the host proteins
- Overall polyclonal antibodies are better for screening
- Generally indirect detection is preferred to direct detection methods
- Requirement of an antibody is a limitation
- Requires a linear epitope which is not dependent on the conformation
of the protein
- These detection methods use denaturing conditions
- Therefore a denatured protein may bind
- cDNA libraries are better when using immunochemical screening as they
consist of mRNA without introns
- Genomic libraries contain introns therefore they can’t express the protein -
only in the case of eukaryotes
- Libraries constructed from bacteria will not contain introns in the first place
- Direct detection:
- An antibody is radioactively labelled
- It binds to an antigen present in the wells
- The results are analysed through autoradiography
- Indirect detection:
- A primary antibody binds to the antigen present in the wells
- A secondary antibody with an enzyme conjugated to it binds to the Fc
receptor on the primary antibody
- Enzyme may be alkaline phosphatase or horseradish peroxidase (HP)
- This binding results in the release or breakdown of a colour solution
- The results are analysed through autoradiography
- Indirect detection depends on the activity of the enzyme for detection
- Screening using libraries constructed in plasmid vectors:
- Transformed cells plated out on petri dishes and allowed to form
colonies
- Replica plate obtained since cells will be killed during the procedure
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