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Chemical synthesis of DNA By Prabhu Thirusangu

Chemical Synthesis of DNA by Prabhu Thirusangu

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Chemical synthesis of DNA By Prabhu Thirusangu

  1. 1. Introduction Synthesizing the Desired DNA/ Gene, has many applications in the Biochemical and Biotechnological experiments. I am here going to focus on the, “Advanced and Contemporary method of chemical synthesis of DNA” . In 1947, HAR GOBIND KHORANA, for the first time synthesized his gene of interest ( which is, alanine t-RNA of yeast)
  2. 2. The Evolution of chemical synthesis…  Many years ago, several laboratories, focussed on developing new procedures for polynucleotides synthesis, so that they would be readily accessible for biological and biochemical experiments.  In 1981, this goal has been achieved as methods were available that could be used for rapid preparation of deoxyoligonucleoties.  And those procedures were based on the earlier works of R.LETSINGER, which are generally known today as the “PHOSPHORAMIDITE METHOD”.
  3. 3. Significance of the, “Phosphoramidite” method… The Phosphoramidite method is so successful and it is currently widely used because of, I. READY ADAPTABILITY - to automated DNA Synthesis machines II. VERY HIGH YIELD and III. RELATIVELY HIGH PURITY - Of polynucleotides having 100 or more mononucleotides.
  4. 4. “Gene Machine” – The DNA synthesizer  An Automated machine which synthesizes the Desired Gene, chemically from the free nucleotides is known as the ‘Gene Machine’.  The Gene machine contains the, TEN CONTAINERS SYNTHESIZER COLOUMN VALVES SPECTROPHOTOMETER PROGRAMMED COMPUTER
  5. 5. The Gene Machine – Schematic form.
  6. 6. “Oligonucleotide synthesis” – In General  “Oligonucleotide synthesis”, is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure  Whereas enzymes synthesize DNA and RNA in a 5' to 3' direction, chemical oligonucleotide synthesis is carried out in the opposite, 3' to 5' direction.  In chemical synthesis, a DNA strand is synthesized from free nucleotides without the aid of TEMPLATE STRAND, and DNA POLYMERASE.
  7. 7. Deducing the Nucleotide sequence  Isolation and Purification of Target Protein from the source organism.  The Nucleotide Sequence is deduced from the Amino Acid sequence of Target Protein.  This is the “Nucleotide sequence of the Gene” for Protein.
  8. 8. Phosphoramidite Method  Before chemical synthesis, the deduced nucleotide sequence is divided into many small units of 60 – 80 base sizes.  The computer is programmed to make nucleotides sequence of one of the units.  The Reagents and Nucleotides are filled in the containers. To prevent the side reactions the amino group of Nitrogenous base, Demethoxytrityl (DMT), Methyl group, and Di-Iso propylamine group attaches to the Nucleotide. And this Chemically protected Nucleotide is called the PHOSPHORAMIDITE. Hence this method is called “Phosphoramidite Method!”
  9. 9. An Overview of Oligonucleotide Synthesis…
  10. 10. The stepwise process of Chemical synthesis…
  11. 11. Detritylation Process STEP 1 The First Nucleotide is linked with the Spacer Molecule, which when pumped into synthesizer column, binds with the glass beads, to form the ‘starting complex’, which acts as solid support for chemical synthesis. STEP 2 Unreacted Nucleotides are washed out by Acetonitrile and then Acetonitrile itself is washed out by Argon. As a result, ‘Starting complex’ alone exists in the column.
  12. 12. STEP 3 TCA is pumped into the column to release the DMT Group, from Nucleotide, which provides free 5’ OH – group for chain elongation. This is called Detritylation. STEP 4 TCA and DMT are washed out , and the amount of DMT released is estimated by Spectrophotometer
  13. 13. STEP 5 Activation and Coupling Reaction As programmed, the second nucleotide is pumped into synthesizer column, and simultaneously TETRAZOLE is pumped into it. The Tetrazole activates the Phosphoramidite to form a covalent bond between its 3’ P-group, and 5’ OH-group of previous nucleotide. This is called Activation and Coupling!
  14. 14. STEP 6 Capping Process… Unincorporated Phosphoramidite and Tetrazole are washed out by Acetonitrile and Argon. Acetic anhydride and Dimethyl amino pyridine which when pumped into the column add an acetyl group to the 5’ OH – group of previous nucleotide, which in turn prevents the further reactions. This is called Capping!
  15. 15. STEP 7 Oxidation… Iodine mixture is pumped into the synthesizer column for strengthening the phosphotriester bond between the two Nucleotides. This is the Oxidation Reaction!
  16. 16. Recovery of the Final Product  The steps 2 – 7 are repeated many times till the final nucleotide gets added to the growing DNA strand.  In every cycle, a new nucleotide is added to the DNA strand. At the end of the reaction, many single stranded DNAs with a unique sequences are found attached with the glass beads.  The DNA at the final nucleotide is removed by treating the column with TCA  The ssDNAs are eluted from the column using a buffer solution.  Methyl group at phophodiester bonds is removed from the DNA. Amino protecting groups are removed chemically.  The final DNA solution contains a mixture of single stranded DNAs. They differ in their length. The full length DNA strands are separated from others by electrophoresis.  In this way many different ssDNA units are made separately and joined together to make complete duplex DNA (Insert DNA).
  17. 17. Assembly of a Synthetic Gene All the ssDNA ‘s are mixed together and treated with T4 DNA Ligase. The Complementary strands hybridized with each other to form duplex DNA and the overlapping sequences bind the adjacent strands by hydrogen bonding. As a result Nicks are there in duplex DNA. The Nicks are sealed by T4 DNA Ligase. Final Product is the “Full Length Desired DNA” , which is amplified and used in Gene Manipulation.
  18. 18. The Structural View of Chemical Synthesis…
  19. 19. The Photograph of the Gene synthesizer…
  20. 20. The New 4 to 8 columns oligo synthesizer, the smallest one in the world, presented for the first time at ANALYTICA CHINA SHANGHAI in Sept. 2010! The smallest in the world…..
  21. 21. Conclusion Even though the other DNA Chemistries have also been explored and improved significantly during the past decade, like “Phosphate - Triester” approach, proposed by A. TODD,  THE OVERALL YIELD,  SPEED OF SYNTHESIS, AND  QUALITY OF DNA, From these procedures have not yet matched the “Phosphoramidite Methodology”
  22. 22. I Conclude hereby Stating that, the “Phosphoramidite Methodology”, Continues to be the most preferred procedures for the ‘Chemical Synthesis of DNA’. T. Prabhu 19th, November, 2010 Mysore
  23. 23. Webliography http://en.wikipedia.org/wiki/Oligonucleotide_synthesi http://bioenergy.asu.edu/photosyn/courses/bio_343/lecture/dnatech.html http://www.slideworld.com/.../Chemical-Synthesis-of-DNA-1-ppt-835455 http://med.stanford.edu/sgtc/resources/images/Chemical_DNA_Synthesis_Chart_3 -9-10_revised_PW.pdf http://www.nature.com/nnano/journal/v5/n11/full/nnano.2010.211.html http://www.ka-lab.de/

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