by Subhankar Chakraborty

Created on: December 12, 2006   Last Updated: May 02, 2007

Subtractive hybridization

Subtractive hybridization (SH) is a technique which is used to determine the difference between two genomes, i.e. if two DNA samples are otherwise identical except for a region that has been deleted (as is common in several cancers) or added (as in case of infectious organisms whose genome might be added to that of an organisms), the difference can be detected using this technique.

SH is based on the principle of progressive differential enrichment of the DNA/mRNA sequence which is different in the sample DNA (also termed as the "tester") compared to the reference DNA (also termed the "driver"). Further, before the actual SH procedure is undertaken, the complexity of the two genomes has to be reduced. In case of the tester, this is attained by digesting the DNA with a restriction enzyme (like Sau 3A), while in case of the driver, the DNA is sheared into smaller fragments by sonication. In order to separate the tester DNA from the large excess of the driver in the later steps of the procedure, the tester DNA is labeled with biotin. This step of the process is described next.
The tester DNA is digested with a restriction enzyme that produces sticky ends (e.g. Sau3A). The ends of the DNA are then filled in with the Klenow fragment of E.coli DNA polymerase. In this step, dNTPs are added which provide the nucleotides for the filling-in-reaction. Of the four dNTPs, dTTP is modified such that it has biotin attached to it (this modified dTTP is called 12-SS-dUTP). Then, the tester fragments (now with filled in blunt ends) are ligated to an oligonucleotide (lets take in this case 5'-CTTACCATGGTAAG-3'). The oligonucleotide is so designed that if two oligonucleotides ligate to one another, it creates a HindIII restriction site. So, after the ligation reaction, the tester-oligonucleotide mixture is digested with HindIII to remove the self-ligated oligonucleotides. The tester DNA is now size fractionated on a Sepharose column to exclude low molecular weight contaminants. The next step is called Hybridization.
The oligonucleotide and biotin tagged tester DNA is now mixed with the sheared and size fractionated driver DNA which is in excess (tester: driver ratio of 1:200) and incubated in presence of sodium phosphate. During the process of hybridization, the DNA is first heat denatured and then allowed to reanneal until 90% of the driver DNA had reannealed. The mixture at the end of hybridization contains tester-ssdriver, tester-tester, driver-driver

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