Subtractive Hybridization for Gene Isolation,DNA,Avidin Coated Beads,Biotin,Southern Blots,Isolated Gene,Hybridize

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Isolation, Sequencing and Synthesis of Genes Isolation, Sequencing and Synthesis of Genes - Introduction Isolation of Genes Isolation of Genes Coding for Specific Proteins Isolation of Genes with Known or Unknown Products having Tissue Specific Expression Isolation of Genes with Known or Unknown Products Using DNA or RNA Probes Use of Heterologus Probes Use of Synthetic Probes Isolation of Genes Coding Use of Transposable Elements Transposon Tagging T-DNA-Insertion Mutagenesis for Isolation of Plant Genes Promoter Enhancer and Gene Trap for Isolation of Genes Differential Screeing and Differential Display Technique for Isolation of Genes Subtractive Hybridizatioon for Gene Isolation Map Based Cloning for Gene Isolation Isolation of Novel Genes by Asis Data at JNU New Delhi

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Subtractive Hybridization for Gene Isolation
Subtractive hybridization may help in isolation of a gene for which deletion mutants are available. In this technique, the genomic DNA of wild type may be digested and hybridized by an excess of sheared and biotin labelled DNA from the deletion mutant. The DND that dons not hybridize, can be separated by passing the DNA thought a column with avidin coated beads so that biotin labelled and hybridized DNA will be bound on the beads and the unlabelled unbound DNA will pass out form the column.

Different step involved in isolation of a gene using the technique of subtractive hybridization

Different Steps Involved in Isolation of a Gene Using the technique of Subtractive Hybridization

Avidin coated beads so that biotin labelled and hybridized DNA will be bound on the beads and the unlabelled unbound DNA will pass out form the column. This process may be repeated through several cycles. The eluted DNA is amplified using PCR (adapters of known sequence may be used for designing primers) and then cloned. The clones are then used on the Southern blots of the mutant and wild type to select clones that hybridize only to the wild type and not to mutant type. Further confirmation may be needed to identify the isolated gene.

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