Isolation of Genes Coding for Specific Proteins,Immunizing,Antibody,RNA Polymerase Enzyme,Genomic DNA,Polysome

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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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Your Ad Here	Isolation of Genes Coding for Specific Proteins Isolation of genes for specific proteins became possible only after the discovery of reverse transcriptase enzymes in 1970. This enzyme can be easily used for the synthesis of copy DNA or complementary DNA (cDNA) from mRNA. This complementary DNA (cDNA) can then be used for the isolation of the corresponding gene form genomic DNA. It is, therefore, obvious that for the isolation of a specific gene, techniques should first be available for the isolation of specific mRNA. For this purpose, antibodies are produced against a specific protein for which the gene is to be isolated.
Therefore, isolation of a gene coding for a specific protein involves the following basic steps (i) purification of the protein product of the gene; (ii) production of antibodies against this protein product by immunizing animals like rabbit or mouse; (iii) precipitation of polysome engaged in synthesizing specific protein with the help of the antibody raised in step (ii); (iv) isolation and Purification of mRNA from the polysome fraction; Isolation of a gene for a specific protein (v) using mRNA for synthesizing cDNA with the help of reverse transcriptase; (vi) cloning of cDNA thus synthesized for preparation of a DNA library; (vii) immunological and electrophoretic analysis of the translation products of cDNA clones to identify specific cDNA clone meant for the specific protein in mind;
(vii) use of the specific cDNA probes selected in step; (vii) for the identification and isolation of the gene from genomic cDNA through screening a complete or partial genomic library (for preparation of cDNA and genomic DNA libraries, consult) In step (vi) above for preparation of cDNA library, cDNA may be cloned in expression vectors (vectors in which gene can be expressed due to the presence of promoter sequences for RNA polymerase enzyme) like l11 which can accept gene insertions into b galactosidase gene. The chimeric vector will then produce hybrid protein if correctly expressed. The hybrid protein can be identified using antibody as above. Plaques belonging to the chimeric vector carrying the desired gene can also be identified by the reaction of antibody attached to a radioactive protein	Your Ad Here
The above technique for isolation of gene has now gene successfully utilized both in plants and animals. However, the first genes isolated were those which existed in multiple copies and were expressed at high levels in specific tissues, e.g. ovalbumin gene in chicks, globin and immunoglobulin genes in mouse, genes for storage proteins in cereals and legumes, amylase genes in barley, actin genes in some legumes, etc

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Sequencing of a Gene or a DNA Fragment Maxam and Gilbert's Chemical Degradation Method Sanger's Dideoxynucleotide Synthetic Method Automatic DNA Sequencers Slab Gel Versus Capillary Sequencers Slab Gel Sequencing Systems Capillary Gel Sequencing Systems Direct DNA Sequencing Using PCR Ligation Mediated PCR LMPCR DNA Sequencing Through Transcription DNA Sequencing by Hybridization Using Microarrays on DNA Chips DNA Sequencing by DE-MALDI-TOF Mass Spectrometry Synthesis of Genes Synthesis of Gene for Yeast Alanyl tRNA Synthesis of Oligonucleotides Synthesis of Three Duplex Fragments Synthesis of Gene from Three Duplex Fragments Synthesis of Gene for a True Precursor tRNA Total Synthesis of a Human Leukocyte Interferon Gene Gene Synthesis Machines Protection Chemistry and Amidites Permanent Protecting Groups Temporary Protecting Groups Synthesis of Gene Using PCR Synthesis of Genes From mRNA