The document discusses nucleic acid hybridization, a key technique in molecular genetics for detecting DNA or RNA sequences through the formation of double-stranded molecules. It outlines the stages of hybridization, including probe synthesis, target DNA processing, denaturation, and methods of transferring target DNA to solid carriers. Various applications of hybridization such as Southern, Northern, and Western blotting are explained, along with the visualization techniques used for identifying hybridized sequences.

1. By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. SYNOPSIS:-
INTRODUCTION
HYBRIDIZATION STAGES
PROBE SYNTHESIS
PROBE MARKING
TARGET DNA PROCESSING
TARGET DNA DENATURATION
TARGET DNA TRANSFER TO SOLID CARRIER
VISUALIZATION
CONCLUSIONS
REFERENCES

3. INTRODUCTION
Nucleic acid hybridization is a fundamental tool in molecular
genetics which takes advantage of the ability of individual
single-stranded nucleic acid molecules to form double
stranded molecules (that is, to hybridize to each other)
The nucleic acid hybridization is the process wherein two DNA
or RNA single chains from different biological sources, make
the double catenary configuration, based on nucleotide
complementary and of sequence homology of the two sources,
resulting DNA-DNA, RNA-RNA or DNA-RNA hybrids.
Hybridization can detect identical or similar sequences.

4. Nucleic acid probe:
A short known sequence of single stranded DNA or RNA designed
to hybridize with the target nucleic acid.
 Type of nucleic acid probe

5. 1 probe synthesis
DNA and RNA probes could be labeled in vitro by one of two
methods:
1) Strand synthesis-
• By using DNA or RNA as a template to generate a labeled DNA
strand.
• DNA or RNA polymerase are used and one of the four dNTPs in
the reaction usually has a labeled group e.g. 32P.
• DNA could be labeled by nick-translation, random primed
labeling.
2) End-labeling:
• Used in labeling single strand probes by adding one (kinase
end-labeling) or very few (fill-in end-labeling) labeled groups
at the 5’ end.
Hybridization stages

6. Strand synthesis-
Randomized primers

7. fill-in End-labeling

8. 2.Probe marking
Labeling can be done using radioactive or non-radioactive labels.
 Radioactive
The common radioactive isotopes used for labeling include
P 32, S 35.
 Once hybridized, the labeled probes can be detected by X-ray
autoradiography.
 Disadvantages include:
 Higher expense
 Difficulty in handling
 Health hazard
 Non-Radioactive
 Non-radioactive marking uses repoter molecules that are
directly or indirectly detectable.
 The non-radioactive labels include biotin, acridinium esters.

10. 3 Target DNA processing
 Target DNA processing the source DNA isolation and
purification and its digestion with a suitable restrictase so as
the target oligonucleotidic sequence is obtained as a linear,
double-standed molecule.
 Seprated obtained by the restriction reaction are seprated by
electrophoresis in the agarose gel.

11. 4 Target DNA denaturation
 Denaturation or melting: Separation of the two DNA strands
from each other. Melting could be done by either:
Heat (90-100 °c) thermal denaturation
Alkaline pH
Low salt concentration

12. 5 Target DNA transfer to solid carrier
Nitrocellulose paper, Nylon membrane are used.
 Target DNA transfer can be carried out in three ways:-
 capillary blotting
 electroblotting
 vacuum blotting

13. Electroblotting
apparatus

14. blotting
When hybridization takes place on solid carrier is named blotting
and is divided in 3 categories:-
1 )Southern blotting where by DNA molecules are identified using
DNA or RNA probes.
 Southern blotting are 4 steps:
 DNA extraction
 Electrophoresis to separate
 Transfer to membrane
 Use labeled probes, which will hybridize to specific sequence,
to identify sequence of interest.
2) Northern blotting where by RNA molecules are identified using
RNA or DNA probes.
3) Western blotting where by protein sequences are identified
using specific antibodies.

15. Southern blotting

16. Northern blotting

17. Western blotting

18. 6. Visualization
 Hybridization is carried out by incubating the labelled probe,
under appropriate conditions, with the membrane in which the
nucleic acids extracted from the sample have been spotted.
 After several washes to remove unbound probe, the membrane
is exposed to X-ray film (if the probe is labelled with an isotope)
or processed for colorimetric visualization of results if non-
radioactive radiation is used.
RNA hybridized

19. CONCLUSIONS
 Nucleic acid hybridization is widely used for scientific
applications but essentially restricted to specialized
laboratories.
 The commonly used recombinant plasmids as the
preparation of the DNA probe is very simple and it can
easily be labeled directly, Nucleic Acids and used for
hybridization.
 Microarrays can be used in many ways to analyze gene
expression in various cell types, in response to various
stimuli.

20. REFERENCES
1. TEXT BOOK OF BIOCHEMISTRY
LEHNINGER NELSON & COX (iv EDITION)
2 PDF FILE:-
I. wolf49.pdf fail
II. MBPS.Ch14(1).pdf