Synthesis of Gene Using PCR,Oligos,Sticky Ends,Khorana,DNA Fragments,Enzymatic Extention,Phosphorylation,Ligation,Cloning

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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 Using PCR
The methods for gene synthesis utilized earlier, whether manually or through automatic synthesizers, involved the following steps: (i) synthesis of oligos, (ii) annealing of oligos to give duplexes with single stranded cohesive or sticky ends and (iii) ligation of duplexes to obtain complete gene. This strategy was initially used by Khorana and was subsequently followed by many. Even, while using automatic synthesizers, only the oligos are synthesized on the synthesizer, but the production of gene from these oligos is achieved later following the usual procedure.

Crude synthetic oligonucleotides were sometimes also used for rapid generation of DNA fragments through PCR. In all these procedures oligos are first phosphorylated at the 5 ends, overlapping ends and finally by enzymatic extension at the 3 ends and finally nicks were joined with Dna ligase.

The full length double stranded DNA could be cloned in a plasmid/phage vector and multiplied in E. coli. Alternatively, it could be amplified by PCR, separated on gel, purified from gel and cloned. The above steps of phosphorylation, annealing, ligation and cloning could be dispensed with, in some of the recent methods of gene synthesis. For instance, overlapping oligonucleotides could be extended through several rounds of PCR. Full length double stranded DNA could then be amplified using short primers complementary to the 5 ends of two strands. In India, at NBRI, at Lucknow, Dr. Rakesh Tuli and his group standardized a method for construction of synthetic genes.

Synthesis of gene using polymerase chain reaction PCR

Gene Assembly Using 28 Oligos with 14-18 Base Long Overlaps and 6-16 Base Long Gaps

Gene Amplification Using Primers Complementary to 3' ends

In this method, overlapping oligos with gaps (6-16 bases long) between adjacent oligo were first synthesized on an automatic sequencer, the complementary oligos were annealed using 14-18 bases long overlaps and the assembled double stranded DNA was used for PCR to give complete DNA sequence by several cycles of PCR. Deep vent polymerase, instead of Taq polymerase was used to reduce error rate.

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