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Molecular biology Subtractive hybridization

techniques:

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 fillingin-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 hybrids as well as single stranded tester and driver DNA. The ssDNA is separated from the ds DNA by passing the mixture over hydroxyapatite (which retains the dsDNA). The resultant ssDNA (assumed to be enriched in unhybridized tester DNA) is then eluted and mixed again with large excess of the driver DNA and the process repeated at least three times. The process described above thus consists of two steps. In the first step, sequences that are identical in the tester and driver DNAs including those with partial identity will anneal to each other during the hybridization step. This will remove such similar sequences from the reaction and is called SUBTRACTION. The other part of the reaction process HYBRIDIZATION is what gives this method its name.

After repeated cycles of subtractive hybridization, the mixture is enriched for single stranded tester DNA. But it still has a large excess of the driver DNA that needs to be removed. This is achieved by biotin/avidin affinity chromatography to separate the biotin labeled tester from the unlabelled driver. The tester DNA is released from the avidin by treatment with dithiothreitol. Now, we have single stranded tester DNA that has the oligonucleotide ligated to it at its 5' end. The amount of this tester is very small. So we employ PCR to amplify the tester DNA. The primer designed is such that it contains the Sau3A (enzyme used earlier to shear the tester DNA into smaller fragments) restriction site at the 3' end, while the 5' end is identical to the oligonucleotide ligated to the 5'end of the tester (in this case the primer would be 5'CTTACCATGGTAAGGATC-3'). After amplification of the tester DNA, it is digested with Sau3A once more in order to cleave off the oligonucleotide attached to the 5'end of the DNA. Thereafter, the products are run on a Sepharose column to remove low molecular weight contaminants and ligated to a suitable vector (in this case BamHI cleaved and phosphatase treated pUC118 vector). Competent E.coli cells are then transformed and selected for ampicillin resistance. Subsequently the cloned fragment can be sequenced.1

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