Molecular Analysis: Genomic

(Hybridization, Southern Blot,

Northern Blot, In situ Hybridization)
 Molecular diagnostics is a collection of
techniques used to analyze biological markers
in the genome and proteome, and how their
cells express their genes as proteins, applying
molecular biology to medical testing
 Hybridization is the
process of combining
two complementary
single-stranded (DNA
– DNA or DNA - RNA
or RNA – RNA)
molecules and
allowing them to
form a single double-
stranded molecule
through base pairing.

Nucleic acid hybridization provides a means for detecting specific

DNA or RNA sequences that are complementary to any isolated
nucleic acid and labeled by being the presence of radioactive
nucleotides or nonradioactive
Principle: single stranded DNA molecule recognize and specifically
bind to a complementary DNA strand in a mixture of other DNA
strand in membrane through capillary transfer

 Basic Procedure:
Single stranded target DNA is bound to
a membrane support

DNA probe labeled with detector

substance is added

DNA probe pairs with the

complementary target DNA

Sequence of nucleotide in the target

DNA can be identified
 Probes
• Probe is a short length of single
stranded DNA / RNA that has a
complementary base sequence to the
gene extract
• The probe is labelled
• Radioactive labeling or isotopic
labeling
• Nonradioactive labeling or
nonisotopic labeling
• When the probe is mixed with DNA
fragments is forms hydrogen bonds
with stretches of DNA complementary
to its own base sequence (annealing)
 Sources of Probes

• Nuclear DNA:
•Genomic libraries
•cDNA
• Cytoplasmic DNA
• The species specificity of many single-locus probes
Types of Probes
Probes
Synthesis
 Non-radiolabeled labeling and detection
• The use of nonradioactive labels has several advantages:
• safety
• higher stability of a probe
• efficiency of the labeling reaction
• less time taken to detect signal
• detection in situ

• Major types
• Direct nonisotopic labeling (ex. nt labeled with a fluorophore)
• Indirect nonisotopic labeling (ex. biotin.-streptavidin system)
 Non-radiolabeled Probe

This detector system is based on the enzymatic conversion of a

chromogenic substrate or chemiluminiscent susbtrate.
How does the probe work?

A specific sequence can be

detected in total cell DNA
A

by hybridization with a
radiolabeled or non-
radiolabeled DNA probe.

The radioactive probe

hybridizes to
complementary sequences
in cell DNA, which can then
be detected as radioactive
double-stranded molecules.
 Using Probes

Probes can be used to locate specific sequences

- Identify the same gene on a variety of different
genomes
- Locate a specific desired gene
- Identify the presence or absence of an allele for
genetic disease
 Steps for Hybridization

Denaturation

Renaturation

Hybridization
Steps for Hybridization
 Steps for Hybridization:
DNA Purification
• Isolate the DNA in question from the rest of the cellular
material in the nucleus.
• Incubate specimen with detergent to promote cell lysis.
• Lysis frees cellular proteins and DNA.
• Proteins are enzymatically degraded by incubation with
proteinase.
• Organic or non-inorganic extraction removes proteins.
Steps for Hybridization:
DNA Fragmentation

• Cut the DNA into different sized pieces.

• Use restriction endonucleases (RE)
• Nucleases hydrolyze the bonds that connect bases within the
strand, resulting in cleavage of the strand.
• They cleave the double stranded nucleic acid only at specific
points In vivo, they are involved in DNA metabolism and repair
or in bacterial host defense.
Steps for Hybridization:
Gel Electrophoresis

Sorts the DNA pieces by size

• Gels are solid with microscopic pores
• Agarose or polyacrimide
• Gel is soaked in a buffer which controls the
size of the pores
• Standards should also be run
 Steps for Hybridization: Blotting
Blotting refers to process of immobilization of sample nucleic
acid in solid support. The blotted are then used as target in the
hybridization experiment for specific detection.

• Transfer the DNA from the gel to a

solid support.
• The blot is usually done on a sheet of
nitrocellulose paper or nylon.
• DNA is then neutralized with NaCl to
prevent re-hybridization before
adding the probe.
• Transferred by either electrophoresis
or capillary blotting.
Hybridization Technique:
Southern Blot
 SOUTHERN BLOTTING
• The technique was developed by E.M. Southern in 1975.
• The Southern blot is used to detect the presence of a
particular piece of DNA in a sample.
• The DNA detected can be a single gene, or it can be part
of a larger piece of DNA such as a viral genome.
 SOUTHERN BLOTTING

• The key to this method is hybridization.

• Hybridization-process of forming a double-stranded DNA
molecule between a single-stranded DNA probe and a single-
stranded target patient DNA.
• Southern Blotting could be used to locate a particular gene
within an entire genome
Principles of Southern blot
 Southern Applications
• Detection of DNA rearrangements and deletions found
in several diseases

• Identification of structural genes (related in the same

species (paralogs) or in different species (orthologs))

• Construction of restriction maps

Hybridization Technique:
Northern Blot
 Nothern Blot
• A northern blot is a laboratory method used to detect
specific RNA molecules among a mixture of RNA.

• Northern blotting can be used to analyze a sample of

RNA from a particular tissue or cell type in order to
measure the RNA expression of particular genes.

• This method was named for its similarity to the

technique known as a Southern blot.
Principles Tehnique of
 Main Component Of Nothern Blot

GEL - RNA PROBE

RNA samples are most often
separated on agarose gels A probe is a fragment used for
containing formaldehyde as a nucleic acid hybridization
denaturing agent for RNA to
limit the secondary structure.
In Situ Hybridization
 In Situ Hybridzation (ISH)

• ISH was invented by Joseph G Gall.

• In situ hybridization indicates the localization of gene
expression in their cellular environment
 In Situ Hybridization

ISH is a type of hybridation that uses a labeled

complementary DNA or RNA strand. A labeled RNA or DNA
probe can be used to hybridize to a known target mRNA
or DNA sequence within a sample. This labeled RNA or
DNA probe can then be detected by using an antibody to
detect the label on the probe. The probes can therefore be
used to detect expression of a gene of interest and the
location of the mRNA.
 Aplication Of ISH

• Prenatal test during pregnancy to identify choromosomal

abnormalities such as Down Syndrome
• Treatment Pharmacogenomics – to predict how quickly
metabolize particular drugs.
• Pathogenomics – to identify infectious diseases
 Type Of ISH

a. FISH  Flourescence In Situ Hybridization

Enable to gain genetic information in the context of tissue
morphology

a. CISH  Chromogenic In Situ Hybridization

Enable you to assay multiple targers simultaneously and
visualize co-locazation within a single specimen.
Step of ISH
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