Synthesis of Genes,Chemical Synthesis of tRNA Genes,H.G. Khorana,DNA Polymerase,Reverse Transcriptase,Electrophoresis,Digestion

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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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Synthesis of Genes
There are two approaches available for the synthesis of genes: (i) When the detailed structure of a gene is available, this gene can be synthesized by a purely chemical method as done by H.G. Khorana for the synthesis of gene for a tRNA (reported for the first time in 1970). (ii) If the detailed nucleotide sequence of the gene is not available, one may utilize 'RNA directed DNA polymerase' (reverse transcriptase) enzyme for the synthesis of the gene in question in the form of complementary DNA (cDNA) from the mRNA of the gene isolated in its pure form.

This cDNA gene, however would lack intron sequences and other gene sequences that are transcribed, but are removed during RNA processing. As discussed above therefore, cDNA is often used for isolation of a gene either from (i) DNA extracted from living cells utilizing restriction digestion, electrophoresis and the technique of Southern blot hybridization, or from (ii) a genomic library.

Chemical synthesis of tRNA genes
As outlined above, before one can start the chemical synthesis of a gene: the structure of gene should be known. The structure of a gene earlier could not be worked out by direct chemical analysis, since there were no means for isolating a gene (techniques for isolation of genes were developed later and were described earlier in this chapter). The structure of gene could, therefore, be inferred only from its product. For instance, if a gene is responsible for giving rise to a polypeptide chain and the structure of this chain is known, then from the genetic code dictionary, structure of the gene could be easily inferred. Such genes, were initially considered to be too long to be synthesized, because an average gene contains about 1,500 base pairs. On the other hand, since tRNA molecules are fairly small in size (about 80 nucleotides), a gene responsible for giving rise to a tRNA molecule was within the reach for synthesis.

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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