screening methods of c DNA library.

3. CONTENT

8. SCREENING OF cdna library...
 The result of any cloning experiment that
begins with total DNA from specific source is a
library of clones.
 One of the key elements required to
Identify the gene during cloning is known
As Probe.
 A Probe is normally a cloned piece of
DNA that contains a portion of the sequence
For which you are searching.
 Typically , will make the probe radioactive
And add it to a solution.
 This is binding step is known as
Hybridisation.

11. Types of screening methods:
 By colony hybridisation,
 By plaque hybridisation,
 With labelled oligonucleotide probe,
 Immunological screening,
 Differential screening,
 Blue-white screening,
 By replica plating,
 Expression screening.

12. Colony hybridisation...
• Colony hybridisation is the “BLOT ANALYSIS
TECHNIQUE” where the bacterial cells are transferred
From the solid nutrient medium to the absorbent
Material.
• It can defines as a method for the isolation of
The specific DNA sequence or genes from the
Bacterial cells containing hybrid DNA, by the
Means of a nitrocellulose membrane filter.
• The transferring medium then goes through
Several chemicals and physical treatment.

13. Colony hybridisation involves the
following steps:
 First, inoculate the bacterial cell suspension o the
Solid agar medium to prepare the master plate.
 After inoculation, the number of bacterial colonies
Will develope with different plasmids which refer
As “master or reference plate”.
 Then transfer the bacterial cells from the master
Plate on to the membrane or filter by the means of
Nitrocellulose filter.
 Press the nitrocellulose paper over the surface of
The master plate.

14. Cont.....
 The compression of the filter membrane will form
Replicas or copies of the bacterial cella as that of master
plate.
 Then the treatment of filter medium with SDS to
lyse the bacterial cells.
 Treat the filter paper with alkali to separate the
DNA into single strands & fixation of dna onto the
filter medium.
 Addition of radioactive probe; it will code the
desired gene of sequence from the bacterial cells&then
washing and autoradiography.

16. Plaque hybridisation...
 Plaque hybridisation is a technique use in
molecular biology for the identification of recombinant
phages.
 The procedure can also be used for the detection
of differently represented repetitive dna.
 The technique [similar to colony hybridisation]
involves hybridising isolated phage DNA to a
label probe for the gene of study.
 The plaque hybridisation procedure has some
advantages over colony hybridisation due to the
smaller and well defined area of the filter to which
the DNA binds.

17. PLAQUE HYBRIDIZATION...

18. With labelled oligoucleotide probe:
 This type of screening based on a known
peptide sequence.
 The protein encoded by the gene of interest is
first digest with protease to breakdown as a amino
acids, and separate the each peptides.
 Then treat the peptides with the Edman’s
Reagent.
 The target sequence are complimentary
sequence of DNA which is used as a primer.

20. Immunological screening...
 Immunological screening of an expression c DNA
primary antibody and labelled secondary antibody;
note the label is often an enzyme label like alkaline
phosphate ,but it also be I125 .

22. Differential screening:
o Differential screening is probably the most
Direct approach for the identification of new genes
Whose expression is associated with a change in
Physiological conditions.
o In the recent years , other approaches have
Been developed, including the production of
Of substracted Cdna libraries , differential
Display and RNA fingerprinting.

24. Blue-white screening...
 It is mostly used in recombinant DNA
technology, sometimes this method is used in
screening of c DNA library.
 Foreign DNA is inserted into the plasmid,
where it inactivates the lac z gene.
 The recombinant plasmid is introduced
Into a bacterium which become amp-resistant
 All treated bacteria are spread on a nutrient
Agar plate containing ampicillin and beta-
galactosidase substrate and incubated.
 When colonies that appear must contain foreign
DNA .
 Blue colonies do not contain foreign DNA.

26. Replica-plating...
• Replica plating is a microbiological technique
In which one or more secondary Petri plates
Containing different solid selective growth media
Are inoculated with the same colonies of
Micro organisms from a primary plate,
Reproducing the original spatial pattern of
Colonies.
• The technique involves pressing a velveteen
Covered disk , and then imprinting secondary
Plates with cells in colonies removed from the
Original plate by the material.
• Replica plating is especially useful for negative
Selection.

27. • For example, if one want to select the colonies that
Were sensitive to AMPICILLIN , the plate could be
Replica plated on a secondary ampicillin agar plate.
• The purpose of replica plating is to be able to compare
The master plate and secondary plates, typically to
Screen for a desired phenotype.
• Common screenable phenotypes include
auxotrophy and antibiotic resistance.

29. Expression screening...
 Antibodies used to screen the expression library.
 The procedure has similarities to the plaque
hybridisation protocol.
plaque lift (taking by placing a
membrane on the dish of the plaque)
immersed in a solution of the
antibody.
detected by other antibodies.
repeat cycles of screening to
isolate pure plaques.