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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Genes : Nature, Concept and Synthesis

 
     
 

Gene Machine
Recently, fully automated commercial instrument called automated polynucleotide synthesizer or gene machine is available in market which synthesizes predetermined polynucleotide sequence. Therefore, the genes can be synthesized rapidly and in high amount. For example, a gene for tRNA can be synthesized within a few days through gene machine. It automatically synthesizes the short segments of single stranded DNA under the control of microprocessor. The working principle of a gene machine includes (i) development of insoluble silica based support in the form of beads which provides support for solid phase synthesis of DNA chain, and (ii) development of stable deoxyribonucleoside phosphoramidites as synthons which are stable to oxidation and hydrolysis, and ideal for DNA synthesis.


A gene machine and its working mechanism (diagrammatic)

Fig. 2.14. A gene machine and its working mechanism (diagrammatic)

 

Content

Chemical nature of DNA

 

Chemical composition

 

Nucleotides, nucleosides

 

Polynucleotides

 

Chargaff's rule of equivalence

Physical nature of DNA

 

Watson and Cricks model of DNA

 

Circular and superhelical DNA

 

Organization of DNA in eukaryotes

Structure of RNA

Gene concept

Units of a gene

 

Cistron

 

Recon

 

Mutan

Split genes (introns)

 

RNA splicing

 

Ribozyme

 

Evolution of split genes

Overlapping gene

Gene organization

Gene expression

Gene regulation

 

Transcription

 

 

The lac operon (structural gene, operator gene, promoter gene and repressor gene)

Artificial synthesis of genes

 

Synthesis of a gene for yeast alanine tRNA

 

Synthesis of a gene for bacterial tyrosine tRNA

 

Synthesis of a human leukocyte interferon gene

Gene synthesis by using mRNA

Gene machine

The PCR

 

Amplification of DNA (melting of target DNA, annealing of primers, primer extension)

 

Application of PCR technology

   

The mechanism of a gene machine is shown in Fig. 2.14. Four separate reservoirs containing nucleotides (A,T,C and G) are connected with a tube to a cylinder (synthesizer column) packed with small silica beads. These beads provide support for assembly of DNA molecules. Reservoirs for reagent and solvent are also attached. The whole procedure of adding or removing the chemicals from the reagent reservoir in time is controlled by microcomputer control system i.e. microprocessor.

If one desires to synthesize a short polynucleotide with a sequence of nucleotides T,G,C, the cylinder is first filled with beads with a single 'T' attached. Thereafter, it is flooded with 'G' from the reservoir. The right hand side of each G is blocked by using chemicals from the reservoir so that its attachment with any other Gs can be prevented. The remaining Gs which could not join with Ts are flushed from the cylinder. The other chemicals are passed from the reagent and solvent reservoirs so that these can remove the blocks from G which is attached with the T. In the same way this cycle is repeated by flooding with C from reservoir into the cylinder. Finally the sequence T.G.C is synthesized on the silica beads which is removed chemically later on.

The desired sequence is entered on a key board and the microprocessor automatically opens the valve of nucleotide reservoir, and chemical and solvent reservoir. In the gene machine the nucleotides are added into a polynucleotide chain at the rate of two nucleotides per hour. By feeding the instructions of human insulin gene in gene machine, human insulin has been synthesized.

 
     
 
 
     



     
 
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