hybridization - hybridization probes - types of hybridization and applications

1. NUCLEIC ACID HYBRIDIZATION
ThangaMallika.K
II M.Sc., Microbiology
18PY20

2. INTRODUCTION:
• A basic technique in molecular biology. in which single
stranded Nucleic acids are allowed to interact so that
complexes called HYBRIDS are formed by molecules with
similar complementary sequences.

4. NUCLEIC ACID HYBRIDIZATION:
• A technique which has the ability of indivudial single
stranded nucleic acid molecules to form double stranded
molecules.
• The principle of hybridization is the addition of a probe to a
complex mixture of target DNA. The mixture is incubated
under conditions that promote the formation of hydrogen
bonds between complementary strands.

6. ABOUT DNA:
• Watson and Crick model of DNA was followed and it possess
two strands which runs in antiparallel directions and those
strands are held by hydrogen bonds.
• According to Watson-Crick base pairing, Adenine pairs with
Thymine and Guanine pairs with Cytosine by hydrogen
bonding.
• The mechanism can be achieved by means of Denaturation
and Annealing.

7. • Since, Nucleic acid hybridization is a process used to identify
specific DNA sequences. This is performed by means of DNA
probes.
• Probes used in hybridization reactions are usually chemically
synthesized DNA or RNA that has been labelled with
fluorescent dye or radioactive isotope.
• Nucleic acid hybridization can be done in all combinations:
DNA-RNA, DNA-DNA or RNA-RNA, Insitu Hybridization
and FISH analysis.

8. FACTORS AFFECTING NUCLEIC ACID
HYBRIDIZATION:
 Strand length
 Base Composition
 Chemical Environment.

9. HYBRIDIZATION PROBES:
• It is a nucleic acid fragment that is complementary to another
nucleic acid sequence and thus, when labeled (with
radioisotope, fluorescent dye, etc.) can be used to identify
complementary segments.
• A probe actually hybridizes to single stranded nucleic acid
(DNA/RNA) molecules because of complementarity between
the probe and target.
• Nucleic acid probes can be synthesized in the laboratory, as
single and double stranded probes, but a working nucleic acid
should be a single stranded only to bind with complementary
target (sequence).

10. Probes are of three types:
o DNA probes: it is a short sequence of DNA labeled
isotopically or chemically that is used for the detection of a
complementary nucleotide sequences.
o RNA probes: it is a short sequence of RNA labeled
isotopically or chemically that is used for the detection of a
complementary nucleotide sequences. They are also known
as riboprobes or complementary probes and are often used in
insitu hybridization because of high sensitivity.

11. o Oligonucleotide probes: it is a short sequence of nucleotides
synthesized to match a region where a mutation is known to
occur and then used as a molecular probe to detect the
mutation.

12. LABELLING OF PROBES:
• Hybridization probes can be labelled by two methods:
1. Invivo Labelling
2. Invitro Labelling
• INVIVO LABELLING: By supplying labeled nucleotides to
the cultured cells.
• INVITRO LABELLING: An enzyme is used to incorporate a
labeled nucleotide in the probe.

13. TYPES OF HYBRIDIZATION:
• There are mainly three techniques of hybridization. They are
as follows:
1. Southern Hybridization
2. Northern Hybridization
3. Colony Hybridization

14. SOUTHERN HYBRIDIZATION:
• Southern blot is a techniques employed for detection of a
specific DNA sequence in DNA samples that are
complementary to a given RNA or DNA sequence.
• It was first given by E.M Southern, a British biologist. This
methods includes separation of restricted DNA fragments by
electrophorosis and then transferred to a nitrocellulose or a
nylon membrane, followed by detection of the fragment using
probe hybridization.

16. APPLICATIONS:
• Southern blots are used in gene discovery, mapping, evolution
and development studies.
• To identify specific DNA in the sample.
• To isolate desired DNA for construction of DNA.
• Used in phylogenic analysis.
• Used to make RFLP maps.

17. NORTHERN HYBRIDIZATION:
• Northern blotting was developed by James Alwine, George
stark and David Kemp (1977). In this technique, RNA is
being analysed instead of DNA.
• It is a technique by which RNA fragments are separated by
electrophorosis and immobilized on a membrane. The
identification of specific RNA is done by using nucleic acid
probes. It helps to study gene expression by detection of
RNA.

19. APPLICATIONS:
• To study the gene expression of various tissues, organs,
development stages, pathogen infections.
• mRNA splicing studies.
• Identification of transferred genes in transgenic individuals.

20. COLONY HYBRIDIZATION:
• It is a rapid method of isolating a colony containing a plasmid
harboring a particular sequence or a gene from a mixed
population.
• The colonies to be screened are first replica plated onto a
nitrocellulose filter disc that has been placed on the surface of
an agar plate prior to inoculation.

22. In situ Hybridization:
• It is a technique that employs a labeled complementary
nucleotide strand for localizing specific DNA or RNA
sequence targets within fixed tissues and cells. There are two
ways to detect DNA or RNA targets:
i) Chromogenic insitu hybridization
ii) Flourescence insitu hybridization.

23. CISH FISH

24. APPLICATIONS:
• Library screening
• Southern blot
• Northern blot
• ASOs ( Allele-specific oligonucleotides ) to detect mutations.