Synthesis of Gene for Yeast Alanyl tRNA,R.W. Holley,H.G. Khorana,Thymine DNA,Long Chain,Synthesizing DNA

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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 Gene for Yeast Alanyl tRNA
As readers may know, structures of large number of tRNAs are now known. The first tRNA whose structure could be known was yeast alanyl tRNA. R.W. Holley (who died in early 1993) and his coworkers (in 1965) gave the detailed structure of yeast alanyl tRNA. This information was immediately used by H.G. Khorana and his coworkers to deduce the structure of the gene for yeast alanyl tRNA. The structure of this gene would obviously be such that one of the two strands of DNA (gene) would be complementary to base sequence of yeast alanyl tRNA. The other strand would then automatically have the same sequence as in tRNA except that in place of uracil of tRNA there would be thymine in DNA.

Synthesis of such a long chain (77 base pairs) of double stranded DNA was rather difficult in 1965 (not in 1990s and thereafter) but Khorana and his coworkers had extensive experience of synthesizing DNA of known base sequences. It was decided that such a long chain could not be synthesized by adding a single base each time. Therefore, it was decided that small oligodeoxyribonucleotides ranging in length from 5 to 20 nucleotides should first be synthesized. These segments would be single stranded and would cover the whole length of both the strands of DNA. These would then be joined to form double stranded DNA, 77 nucleotide pairs long. In actual synthesis of yeast alanyl tRNA gene, the following steps were involved.

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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