Subtractive Hybridization

BSBT 514Genetically Modified Organisms

Presented bySakshi Saxena

ASU2013010200124IBT VIth sem

Introduction

In higher eukaryotes, biological processes such as cellular growth and organogenesis are mediated by programs of differential gene expression.

To understand the molecular regulation of these processes, the relevant subsets of differentially expressed genes of interest must be identified, cloned, and studied in detail.

Subtractive hybridization is a technique for identifying and characterizing differences between two populations of nucleic acids.

It detects differences between the RNA in different cells, tissues, organisms, or sexes under normal conditions, or during different growth phases, after various treatments (ie, hormone application, heat shock) or in diseased (or mutant) versus healthy (or wild-type) cells.

Subtractive hybridization also detects DNA differences between different genomes or between cell types where deletions or certain types of genomic rearrangements have occurred.

Subtractive hybridization

Subtractive hybridization

Subtractive hybridization requires two populations of nucleic acids

Tester/Tracer

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.

Subtractive hybridization

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.

T D

TD DDTT DT

Subtractive hybridization

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.

T D

TD DDTT DT

Subtractive hybridization

Process

1. Choosing material for

isolating tester and

driver nucleic acids;

2. Producing tester and

driver3. Hybridizing

4.Removing driver-tester hybrids and

excess driver (subtraction)

5.Isolating of the complete sequence of

the remaining target nucleic

acid.

Subtractive hybridization1. PREPARATION OF DRIVER AND TESTER

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 not very complex and is easy to isolate.

Subtractive 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.

2. HYBRIDIZATION

Subtractive hybridization3. SUBTRACTION

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.

Subtractive hybridization4. ISOLATION OF TARGET SEQUENCES

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.

Subtractive hybridization5. ANALYSIS OF ISOLATED SEQUENCES

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

REFERENCES

1. Subtractive hybridization – genetic takeaways and the search for meaningRichard J Byers, Judith A Hoyland, Janet Dixon, and Anthony J FreemontInt J Exp Pathol. 2000 Dec; 81(6): 391–404.

2. [Subtractive hybridization. Theoretical analysis, and a principle of the trap].[Article in Russian]Sverdlov ED, Ermolaeva OD.Bioorg Khim. 1993 Nov

3. Subtractive HybridizationAccessed from https://www.thermofisher.com/in/en/home/brands/product-brand/dynal/streptavidin-coupled-dynabeads/subtractive-hybridization.html

4. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries LUDA DIATCHENKO, YUN-FAI CHRIS LAUt, AARON P. CAMPBELLt, ALEX CHENCHIK, FAUZIA MOQADAM, BETTY HUANG, SERGEY LUKYANOVt, KONSTANTIN LUKYANOVt, NADYA GURSKAYAt, EUGENE D. SVERDLOVt, AND PAUL D. SIEBERT. Proc. Natl. Acad. Sci. USA Vol. 93, pp. 6025-6030, June 1996 Biochemistry .