This document discusses various methods for identifying clones, including hybridization probing and immunological screening. Hybridization probing techniques allow complementary nucleic acid strands to hybridize, identifying clones through colony and plaque hybridization with radioactive or non-radioactive labeling. Immunological screening identifies clones by detecting the translation product of the cloned gene using antibodies. Southern hybridization specifically identifies restriction fragments containing genes of interest. These clone identification methods are important techniques for analyzing cDNA libraries, identifying related genes through heterologous probing, and detecting recombinant protein expression in colonies.

1. PhD in Biotechnology/Microbiology/Biochemistry
UNIT- RDT
Topic: Methods for clone identification
By
Dr. Vijay Kumar
Assistant Professor
Department of Biosciences,
Swami Rama Himalayan University

2. Methods for clone identification
Hybridization probing
 Complementary nucleic acid strands hybridize to each other
 Colony and plaque hybridization probing
 Labeling with a radioactive marker
 Non-radioactive labeling
 Examples of the practical use of hybridization probing
 Abundancy probing to analyze a cDNA library
 Heterologous probing allows related genes to be identified
 Southern hybridization enables a specific restriction
fragment containing a gene to be identified
 Identification methods based on detection of the translation product of
the cloned gene
 immunological screening
OUTLINE OF THE TOPIC

3. Learning objectives
At the end of this learning session a student of M.Sc.
Microbiology/Biotechnology –Semester-II should be
able to:
 Learn about major methods used to identify
clones
 Hybridization methods
 Probe labelling methods
 Identification of clone by immunological
screening

4. Introduction
 Once a suitable library has been prepared, a number
of procedures can be employed to attempt
identification of the desired clone
 Few of these procedures are based on detection of
the translation product of the cloned gene
 It is usually easier to identify directly the correct
recombinant DNA molecule
 Can be achieved by the important technique of
hybridization and immunological screening

5. Complementary nucleic acid strands hybridize
to each other
Nucleic acid hybridization
An unstable hybrid
A stable hybrid Short non-complementary regions do not affect overall
stability
An unstable hybrid molecule formed between
two non-homologous DNA strands.
A stable hybrid formed between two
complementary strands
A DNA–RNA hybrid
A DNA–RNA hybrid, such as may be formed
between a gene and its transcript.

6. Colony and plaque hybridization probing
Nitrocellulose/nylon membrane Alkali +
protease
Bacteria attached to
membrane 80°C for 2 hours or
ultraviolet irradiation
DNA bound to the membrane
OR
Probe with
labelled DNA
Wash
Apply X-ray
film
The resulting
autoradiograph

7. Methods for labelling DNA
Labelling by nick translation Labelling by end-filling
Labelling by random priming
Labeling with a radioactive marker
DNA molecule is usually labeled by incorporating nucleotides that carry
a radioactive isotope of phosphorus, 32P

8. Non-radioactive labeling
Labelling with a biotinylated nucleotide
DNA probe Biotin–dUTP
Nick translation, end-
filling or random priming
Detect with avidin coupled to a fluorescent
marker

9. Labelling with horseradish peroxidase

10. mRNA can be cloned as complementary DNA
1. First strand synthesis
4. Ligation into a
vector
3. Second
strand synthesis
5. Transform 2. RNA
degradation

11. Probing within a library to identify an abundant
clone

12. Oligonucleotide probes for genes whose
translation products have been characterized
Bind first nucleotide to
the support

13. The use of a synthetic, end-labelled
oligonucleotide to identify a clone of the yeast
cytochrome c gene

14. Heterologous probing

15. Southern hybridization enables a specific restriction fragment
containing a gene to be identified
A long cloned DNA fragment may contain several genes in addition to the one in which we
are interested. B = BamHI restriction site
Southern hybridization.

16. Identification methods based on detection of the
translation product of the cloned gene
Antibodies. (a) Antibodies in
the bloodstream bind to
foreign molecules and help
degrade them.
(b) Purified antibodies can be
obtained from a small
volume of blood taken from a
rabbit injected with the
foreign protein.

17. Using a purified antibody to detect
protein in recombinant colonies.
Instead of labelled protein A, the
antibody itself can be labelled, or
alternatively a second labelled
antibody which binds specifically to
the primary antibody can be used.

18. Suggested readings
• Brown, T. A. (1990). Gene cloning (pp. 2-6).
Chapman and Hall.
• Old, R. W., & Primrose, S. B. (1981). Principles
of gene manipulation: an introduction to
genetic engineering (Vol. 2). Univ of California
Press.