Gene synthesis machines | Genetic Engineering and Biotechnology Isolation, Sequencing and Synthesis of Genes | Genetics, Biotechnology, Molecular Biology, Botany

⇒	Genetic Engineering and Biotechnology 3. Isolation, Sequencing and Synthesis of Genes
⇒	Isolation of genes
	⇒	Early attempts for isolation of ribosomal RNA genes in Xenopus
	⇒	Isolation of genes coding for known specific proteins
	⇒	Isolation of genes coding for an unknown product
⇒	Sequencing of genes or a DNA segment
	⇒	Maxam and Gilbert's chemical degradation method
⇒	Synthesis of genes
	⇒	Chemical synthesis of genes
	⇒	Synthesis of gene for yeast alanyl tRNA
	⇒	Synthesis of gene for a true precursor tRNA
	⇒	Gene synthesis machines

Gene synthesis machines
Methods for synthesis of DNA molecules of known base sequences have improved dramatically during 1980s and 1990s. Therefore, genes can now be synthesized rapidly and in high yields. For instance a gene for a tRNA took 20 man years effort during 1965-70 (described earlier in this section), but the same gene can now be synthesized within a matter of few days with the help of Gene Synthesis Machines. The key innovations which made the availability of gene synthesis machines possible, include the following : (i) development of silica based supports, which are insoluble and provide support for solid phase synthesis of DNA chains and (ii) development of stable deoxyribonucleoside phosphoramidites as synthons, which are remarkably stable towards oxidation and hydrolysis and thus are ideal for DNA synthesis.

Several versions of gene machines are now available. These machines, under the control of microprocessor, synthesize specific short sequences of single stranded DNA automatically. The desired sequence is entered on a keyboard and the microprocessor automatically opens the valves of the containers of successive nucleotides, reagents and solvents needed at each step, into a synthesizer column, which is packed with tiny silica beads. These beads provide support on which DNA molecules are assembled (Fig. 41.13).

Synthesis of gene from mRNA H. Temin and D. Baltimore, in 1970 discovered the presence of 'RNA directed DNA polymerase' enzyme which has the ability of synthesizing DNA on RNA template. This enabled molecular biologists to synthesize complementary DNA (cDNA) using mRNA as a template. If mRNA transcribed from a specific gene is made available in purified form, the complementary DNA (cDNA) synthesized with its help will represent the synthesized gene. To what extent this synthesized gene is.a faithful copy of native gene will depend on the fidelity of copying mRNA and also on the stability of cDNA thus synthesized. Moreover, since mRNA of a gene does not have the complete transcript of the gene in vivo, the synthesized gene will be smaller than the gene in vivo.

An outline of a gene synthesis machine, which synthesizes a gene automatically once the nucleotide sequences are entered in the microprocessor.

Fig. 41.13. An outline of a gene synthesis machine, which synthesizes a gene automatically once the nucleotide sequences are entered in the microprocessor.

By copying eukaryotic purified mRNA, several genes have been artificially synthesized. Most important of these genes are the genes for sea urchin histone proteins, ovalbumin gene in chicken and globin genes in mammals. The gene synthesized as cDNA from a β globin mRNA, has been inserted into a plasmid in order to study its behaviour. As earlier pointed out, this gene will lack any additional regulatory sequences absent in mRNA but present in a globin gene in vivo. This type of gene will also lack intron sequences found in eukaryotic split genes. These genes (cDNA), however, have already become a very important tool in molecular biology experiments.





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