DNA Sequencing Through Transcription,Thermo Sequenase,Dideoxynucleotide Method Chain Termination,Transcriptional Sequencing

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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

Home >> Biotechnology and Genomics >> Isolation, Sequencing and Synthesis of Genes >> DNA Sequencing Through Transcription

Your Ad Here	DNA Sequencing Through Transcription Recently, DNA sequencing was successfully achieved using RNA polymerase enzyme for transcription, instead of using 'sequenase' or 'thermo-sequenase' used in Sanger's dideoxynucleotide method of chain termination. This method of sequencing described as 'transcriptional sequencing' makes use of 3' dNTPs for terminating transcription in the same manner as ddNTPs are used for terminating DNA synthesis in Sanger's method (whenever 3'dNTP is available at 3' position, no OH or PO4 group will be available for the formation of phosphodiester bond).
The 3'dNTP can be labeled with fluorescent (rhodamine) dyes, so that four dNTPs with for colour dyes (TMR-3'dUTP, ROX-3'dCTP, R6G-3'dATP, RII0-3'dGTP) can be used along with sufficient quantities of all -the four NTPs (ribonucleoside triphosphates). This will allow RNA synthesis to proceed normally with the help of RNAP, till occasionally a 3'dNTP (dye labelled) is incorporated. This will yield RNA chains of different lengths, each having at its terminus, a specific dNTP, which will be identified by its colour on an automatic sequencer. The method is believed to be superior, firstly, because incorporation of NTP during transcription does not require a primer, thus making it faster (240 bases/see) than DNA replication by Taq polymerase (60 bases/see), secondly, because a large amount of product can be obtained from small template (600 molecules of RNA from one DNA molecule), and thirdly, because direct PCR sequencing can be done, without the elimination of primers and, 2' -dNTPs.
The template in this method will have to be prepared by PCR using primers, to which phage promoter sequences are appended, which facilitate binding of RNAP for transcription. The residual primers and 2' dNTPs need not be removed, because they will not interfere with RNA synthesis during transcription.	Your Ad Here

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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

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