2. CONTENTS
Introduction
Methods of clone identification
I. Nucleic acid hybridization
II. Screening by PCR
III. Blue white screening
IV. Immunological screening
V. Insertional inactivation
VI. Screening by functional complementation
3. INTRODUCTION
Clone: A genetically identical copy of a cell or an organism
Clone identification or screening : It is an experimental
technique in which a number of procedures are employed
for the identification of a desired clone
4. METHODS OF CLONE IDENTIFICATION
Nucleic acid hybridization: this method can be used to identify a particular recombinant clone if a DNA or
RNA probe complementary to the desired gene is available
Any two single stranded nucleic acid molecules have potential to form base pairs with one another with most
pairs of molecules the resulting hybrid structures are unstable as only a small number of individual inter strand
bonds are formed
6. COLONY
HYBRIDIZATION
It allows the screening of
colonies plated at high density
using radioactive DNA probes
Discovered by David S.
Hogness and Michael
grunstein
7. PLAQUE
HYBRIDIZATION
It is also known as plaque lift
It was developed by Benton
and davis in 1997 and
employs a filter lift applied to
phage plaques
This method is applied to
isolation of recombination
plaques by nucleic acid
hybridization and probably is
the most widely applied
method for library screening
8. SCREENING BY PCR
PCR screening is employed for the identification of rare dna
sequences in complex mixtures of molecular clones by increasing the
abundance of particular sequence
It is possible to identify any clone by PCR only if there is available
information about its sequence to design suitable primers
9. BLUE WHITE SCREENING
It is a negative selection system using bacterial lactose metabolism as an indicator of successful cloning
Across the vector’s cloning site lies a DNA sequence encoding a peptide which can be visually detected as blue
colonies
When DNA of interest is inserted in plasmid the gene is disrupted and bacterial colony becomes white in
colour
10.
11. IMMUNOLOGICAL SCREENING
THIS INVOLVES USE OF ANTIBODIES THAT SPECIFICALLY RECOGNIZE
ANTIGENIC DETERMINANTS ON THE POLYPEPTIDE
IT DOES NOT RELY ON ANY PARTICULAR FUNCTION OF EXPRESSED FOREIGN
PROTEIN BUT RECOGNIZE AN ANTIBODY SPECIFIC TO PROTEIN
INSTEAD OF RADIOLABELING OF DNA MOLECULES ANTIBODIES ARE USED TO
IDENTIFY THE COLONIES DEVELOPED THAT SYNTHESIZE ANTIGENS ENCODED
BY FOREIGN DNA PRESENT IN PLASMIDS OF BACTERIAL CLONES
12.
13. INSERTIONAL INACTIVATION
IT IS THE INACTIVATION OF A GENE UPON INSERTION OF ANOTHER GENE
IN ITS PLACE OR WITHIN ITS CLONING SEQUENCE
THIS HELPS IN SELECTION OF RECOMBINANT COLONIES IN RDNA
TECHNOLOGY
14.
15.
16. SCREENING BY FUNCTIONAL
COMPLEMENTATION
IT IS A PROCESS OF COMPENSATING A MISSING FUNCTION IN A MUTANT
CELL BY A PARTICULAR DNA SEQUENCE FOR RESTORING THE WILD TYPE
PHENOTYPE
IF MUTANT CELLS ARE NON VIABLE ,THE CELLS CARRYING THE CLONE OF
INTEREST CAN BE SELECTED AND ISOLATED
IT IS VERY POWERFUL METHOD OF EXPRESSION CLONING AND ALSO USEFUL
FOR IDENTIFICATION OF GENE FROM AN ORGANISM HAVING SAME RULE AS
THAT FOR DEFECTIVE GENE IN ANOTHER ORGANISM
17.
18. REFERENCES
Chapter 8 of Gene cloning and data analysis by T.A.brown
https://www.thermofisher.com/Screening methods every scientist should know
https://www.Slideshare.net/Selection and screening of recombinant clones
https://www.Slideshre.net/Methods of screening
NPTEL-biotechnology-genetic engineering and applications –module 4
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20. TITLE LOREM IPSUM
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