SUBTRACTIVE

HYBRIDIZATION
PREPARED BY – SOWNTHARYA V 20BT040
THIRD YEAR BIOTECHNOLOGY
MUTHAYAMMAL ENGINEERING COLLEGE
 BASIC PRINCIPLE

• IT IS A TECHNIQUE USED TO IDENTIFY THE AND CHARECTERZING DIFFERENCE

BETWEEN TWO POPULATIONS
• IT DETECTS DIFFERENCE BETWEEN RNA IN DIFFERENT CELLS,TISSUES OR
ORGANISM
• IT ALSO DETECT DIFFERENCE BETWEEN DIFFERENT GENOME
 BASIC PRINCIPLE

SUBTRACTIVE HYBRIDIZATION REQUIRES TWO POPULATIONS OF NUCLEIC ACIDS.

*1.TESTER/ TRACER

*2.DRIVER

*CONTAINS THE TARGET NUCLEIC ACID (THE DNA OR RNA DIFFERENCES THAT ONE WANTS TO IDENTIFY)

*IT LACKS THE TARGET SEQUENCES

*THE TWO POPULATIONS ARE HYBRIDIZED WITH A DRIVER TO TESTER RATIO OF AT LEAST 10:1. BECAUSE
OF THE LARGE EXCESS OF DRIVER MOLECULES, TESTER SEQUENCES ARE MORE LIKELY TO FORM
DRIVER-TESTER HYBRIDS THAN DOUBLE-STRANDED TESTER.
. ONLY THE SEQUENCES IN COMMON BETWEEN THE TESTER AND THE DRIVER
HYBRIDIZE.

HOWEVER, LEAVING THE REMAINING TESTER SEQUENCES EITHER SINGLE-

STRANDED OR FORMING TESTER-TESTER PAIRS.

THE DRIVER-TESTER, DOUBLE-STRANDED DRIVER AND ANY SINGLE-STRANDED

DRIVER MOLECULES ARE SUBSEQUENTLY REMOVED (THE “SUBTRACTIVE” STEP),

• LEAVING ONLY TESTER MOLECULES ENRICHED FOR SEQUENCES NOT FOUND IN
THE DRIVER.
 PROCESS INVOLVED IN SUBTRACTIVE
HYBRIDIZATION

• CHOOSING MATERIAL FOR ISOLATING TESTER AND DRIVER NUCLEIC ACIDS

• PRODUCING TESTER AND DRIVER
• HYBRIDIZING
• REMOVING DRIVER-TESTER HYBRIDS AND EXCESS DRIVER (SUBTRACTING)
• ISOLATING OF THE COMPLETE SEQUENCE OF THE REMAINING TARGET NUCLEIC
ACID
 PREPARATION OF TESTER AND DRIVER

*IN PRINCIPLE, BOTH TESTER AND DRIVER SAMPLES CAN BE EITHER DNA OR RNA, BUT IT IS OFTEN MOST
PRACTICAL FOR THE TESTER TO BE DNA (BECAUSE THE TESTER IS PRESENT IN A LOW CONCENTRATION,
AND DNA IS MORE STABLE THAN RNA), AND FOR THE DRIVER TO BE RNA (AFTER HYBRIDIZATION,
EXCESS DRIVER RNA CAN BE ELIMINATED ENZYMATICALLY OR BY ALKALI DEGRADATION).

*RNA FROM THE TESTER SOURCE IS REVERSE TRANSCRIBED INTO COMPLEMENTARY DNA (CDNA) AND
HYBRIDIZED TO POLY A+ DRIVER RNA.

*THE TESTER-DRIVER HYBRIDS ARE REMOVED, EXCESS FRESH DRIVER IS ADDED, AND THE
HYBRIDIZATION IS REPEATED ONCE.
*THE REMAINING “TARGET” CDNA IS EITHER CLONED OR USED TO MAKE A PROBE. THIS BASIC
PROCEDURE IS USEFUL IF THE STARTING MATERIAL IS NOTCOMPLEXED IS EASY TO ISOLATE.
 HYBRIDIZATION

* DURING SUBTRACTIVE HYBRIDIZATION, THE HYBRIDIZATION STEP IS DRIVEN BY THE EXCESS

DRIVER SEQUENCES, SO TESTER SEQUENCES THAT HAVE COMPLEMENTARY SEQUENCES IN THE
DRIVER POPULATION RAPIDLY FORM DRIVER-TESTER HYBRIDS, WHEREAS SEQUENCES UNIQUE TO
THE TESTER POPULATION REMAIN SINGLE-STRANDED OR FORM TESTER-TESTER PAIRS MORE
SLOWLY.
*THE RATIO OF DRIVER TO TESTER, THE OVERALL CONCENTRATION OF DRIVER, THE
TEMPERATURE, AND THE LENGTH OF HYBRIDIZATION SHOULD BE CHOSEN BASED ON THE
COMPLEXITY OF THE DRIVER AND TESTER, THE ABUNDANCE CLASS OF THE TARGET NUCLEIC
ACIDS, AND THE LENGTH OF THE DRIVER AND TESTER SEQUENCES USED.
 SUBSTRACTION

THE PURPOSE OF THE SUBTRACTION STEP IS TO REMOVE DRIVER- TESTER HYBRIDS

FORMED DURING THE HYBRIDIZATION STEP. LEAVING BEHIND TESTER ENRICHED FOR
THE TARGET SEQUENCES.

HYDROXYAPATITE CHROMATOGRAPHY IS USED TO BIND DOUBLE- STRANDED DRIVER

AND DRIVER-TESTER HYBRIDS, LEAVING SINGLE- STRANDED NUCLEIC ACIDS BEHIND.
• THIS IS A GOOD CHOICE IF THE DRIVER IS RNA BECAUSE SINGLE- STRANDED RNA
CAN BE REMOVED CHEMICALLY OR ENZYMATICALLY, LEAVING ONLY SINGLE-
STRANDED CDNA TESTER AFTER THE SUBTRACTION.
 ISOLATION OF TARGETED SEQUENCE

AFTER ONE OR MORE HYBRIDIZATION AND SUBTRACTION STEPS, THE RESULTING

TESTER NUCLEIC ACIDS SHOULD BE GREATLY ENRICHED FOR TARGET SEQUENCES.

THE REMAINING TESTER SEQUENCES ARE ISOLATED AND ANALYZED IN A VARIETY

OF WAYS. TESTER CAN BE MADE INTO AN ENRICHED LIBRARY AND PROBED WITH
DRIVER AND TESTER SEQUENCES TO LOOK
• FOR TESTER-SPECIFIC CLONES, OR THE TESTER IS LABELED AND USED TO PROBE
TESTER AND DRIVER LIBRARIES AND TO ISOLATE FULL-LENGTH CLONES.
 ANALYSES OF ISOLATED SEQUENCE
AND
ALTERNATIVE METHODS
• FURTHER ANALYSIS OF ISOLATED TESTER SEQUENCES CAN BE DONE BY NORTHERN BLOTTING, IN
SITU HYBRIDIZATION OR PCR METHODS TO DETERMINE WHETHER THE SEQUENCES ARE TRULY
TESTER-SPECIFIC
• ALTERNATIVE METHODS:
• POSITIVE SELECTION
• REPRESENTATIONAL DIFFERENCE ANALYSIS (RDA)
• SUPPRESSION SUBTRACTIVE HYBRIDIZATION
• SERIAL ANALYSIS OF GENE EXPRESSION (SAGE)
• MICROARRAYS
•

THANK YOU
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