Subtractive hybridization (SSH) is a technique for identifying differentially expressed genes between two populations of nucleic acids. It involves two hybridizations - in the first, an excess of driver cDNA is added to normalize the tester cDNA. In the second hybridization, the normalized cDNA is mixed with more driver cDNA to selectively amplify sequences present in the tester but not driver (E-hybrids). Only the E-hybrids can be exponentially amplified due to their different adapter sequences, allowing isolation of differentially expressed genes between populations. SSH is commonly used for plant and cancer research and to study various diseases.

1. SUBSTRACTIVE
HYBRIDIZATION (SSH
ANALYSIS)
Submitted By,
LOGESWARAN KA
P20BIT1013
I-MSC. BIOTECHNOLOGY
PERIYAR UNIVERSITY.

2. CONTENTS
INTRODUCTION
PRINCIPLE
WORKFLOW OF PCR SUPPRESSION EFFECT
HYBRIDIZATIONS (FIRST & SECOND)
CONCLUSIVE EVENTS
PROS AND CONS
CURRENT RESEARCH
BIBLIOGRAPHY

3. INTRODUCTION
PCR Based cDNA Substraction Procedure.
Pioneers – Luda Diatchenko, Sergey Lukyanov And His
Colleagues In 1996.
Very Effective In Tissue Specific cDNA synthesis And Functional
Sequences Identification.
Mainly Based On PCR Suppression Effect (Panhandle Like Loop
Structure Formation) Caused By Long ITRs (Inverted Terminal
Repeats).
Undesired DNA Fragments Can Be Removed From The Mixture
Of Target DNA Samples.
One Round Of SSH Analysis Results In 1000 Fold Differentially
Expressed cDNA Synthesis.
Removed cDNA Mixture Can Be Used As Hybridization Probe
For Screening rDNA Libraries.

4. PRINCIPLE
Driver And Tester cDNA Samples Was Prepared.
Tester Contains Target cDNA And Driver Contains No Or Low
Amount Of Target cDNA.
Both Were Exposed To 4-Base Blunt End Cutting Restriction
Enzymes And Digested.
The Resulting Tester Sample Divided Into 2 Portions (A&B)
And Ligated With 2 Different Adapters (A&B) At 5’End And 2
Populations Of Tester Samples Were Prepared.
The Adapters Designed Without PO4-Groups For Covalent
Binding Of Longer Strand Of Both Adapters (A&B) To 5’End Of
cDNA.
SSH Analysis Contains 2 Hybridizations.
First Hybridization.
Second Hybridization.

5. Workflow Of PCR Suppression Effect

6. HYBRIDIZATIONS
FIRST HYBRIDIZATION
Excess Driver Sample Added
To Both Tester Samples A&B.
Heat Denatured And Allowed
To Anneal.
Tester A Becomes Normalised
Due To Homohybrid cDNA
Production.
So, It Is Enriched With cDNA
For Differential Expression .
Normalisation Means
Concentrations Of High and
Low Abundance Of cDNA In A
Samples Becomes Roughly
Equal.
Tester B Is Faster For More
Abundant Compounds Due
To Second Order Kinetics.
SECOND HYBRIDIZATION
Mixing Of 2 samples From
First hybridization.
New Hybrids B, C, D, And E
Were Produced By
Reassociation.
Another Portion Of Driver Is
Added And It Enriches E-
Hybrids For Differential
Expression.
E-Hybrids Has Different
Adapter Sequences At Their
5’End.
One Is From Tester Sample A
And Another One Is From
Tester Sample B.
These 2 Adapter sequences
Ensures Proper Amplification
Of E-Hybrids In PCR.

7. CONCLUSIVE EVENTS
Exponential Growth Only Takes Place In E-Hybrids. E-Hybrids
Has Different Adapter sequences At Their 5’End.
B-Hybrids Cannot Be Served As Template For Exponential
Growth. Because, They Have Long ITRs At The End And Results
In PCR Suppression Effect (Formation Of Panhandle Like
Structure) Due To Intramolecular Annealing.
A-Hybrids And D-Hybrids Do Not Have Primer Binding Sites.
C-Hybrids Can Be Amplified At Linear Rate.
So, Only The E-Hybrids With Different Adapter Sequences At
The End Can Be Amplified Exponentially.
In This Method, Undesirable Sequences Can Be Removed And
Selective Amplification Of cDNA Can Be Achieved.

8. PROS AND CONS
PROS
Characterization Of Two
Populations Of Nucleic
Acids.
Substraction Of Plant And
Bacterial Genomes.
Generation And Cloning Of
Tissue Specific cDNA.
Isolation Of Differentially
Expressed Genes.
cDNA And Genomic DNA
Library Construction.
Detection Of DNA And RNA
Differences Between
Different Genomes, Cells
And Organisms.
CONS
During RNA Comparison,
Few Micrograms Of
Poly(A)+ RNA From 2 Cell
Populations Is Needed. It
Is Difficult To Obtain.
Appearance Of False
Positive DNA During DNA
Studies Is A Major
Drawback. Mirror
Orientation Selection
(MOS) Is The Solution.

9. CURRENT RESEARCH
Plant Genome Research To Fight Extreme Environmental
Conditions.
Different Cancers Treatments.
Different Animal Diseases Treatments.
Different Human Diseases Treatments.
Different Bacterial Diseases Treatments.
Different Fungal Diseases Treatments.
Different Viral Diseases Treatments.

10. BIBLIOGRAPHY
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC39182/
(Pioneers Research Article)
https://pubmed.ncbi.nlm.nih.gov/14970460/
https://www.sciencedirect.com/topics/medicine-and-
dentistry/suppression-subtractive-hybridization
http://evrogen.com/technologies/SSH.shtml
http://what-when-how.com/molecular-biology/subtractive-
hybridization-molecular-biology/
https://www.ahajournals.org/doi/full/10.1161/01.STR.32.4.10
20 (Case Study - Using SSH Analysis For Differential Gene
Expression In Strokes)

11. THANK YOU