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Writtin by: Michael A. Innis, Kenneth B. Mayambo, David H. Gelfand, and Mary Ann D. Brow

DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Writtin by: Michael A. Innis, Kenneth B. Mayambo, David H. Gelfand, and Mary Ann D. Brow. DNA sequencing. At the time Sagner dideoxynucleotide method used to sequence DNA

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Writtin by: Michael A. Innis, Kenneth B. Mayambo, David H. Gelfand, and Mary Ann D. Brow

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  1. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA Writtin by: Michael A. Innis, Kenneth B. Mayambo, David H. Gelfand, and Mary Ann D. Brow

  2. DNA sequencing • At the time Sagner dideoxynucleotide method used to sequence DNA • Basic overview: • Hybridizing oligonucleotide primer to a double stranded template • extend primer with DNA pol using 4 different labels • Resolving the 4 sets in a gel • Making an autoradiographic image of gel to infer DNA sequence

  3. Introduction • Had been refined over the years. However, still lacked the ability to perform sequencing on a large scale • Needed to improve the speed and automation

  4. Solution! • PCR, Polymerase Chain Reaction, was though to be the key to these problems • Can amplify genomic sequences more than 10 million fold accurately • That piece of can can be cloned into a vector or purified for sequencing

  5. Taq DNA polymerase • Found in Thermus aquaticus • Ideal for using in DNA sequencing • Thermostable, can use at ideal temperature to increase specificity • This increases chance of finding rare targets • Don’t need to replenish enzymes after each PCR cycle

  6. Methods- Sequencing • Using taq DNA polymerase and an altered PCR procedure DNA was sequenced • Annealing and labeling reactions using an annealing mixture of oligionucleotide primers and taq sequencing buffer was used on each set of sequencing reactions

  7. Methods-Labeling • A labeling mix was added to the annealing reaction mixture • The labeling mixture contained dGTP, dCTP, D ttp, AND dATP

  8. Methods-Extension/Termination • An extension/termination reaction was preformed for each labeled template • “G Mix” “A Mix” “C Mix” and “T Mix” were used to terminate extension. Each mix contained dNTP and ddNTP along with magnesium • The product was then loaded onto a gel and run in order to sequence the PCR products

  9. Asymmetric PCR • Asymmetric PCR is here one oligonucleotide primer is present in 100 fold greater concentration than the other • One of the two PCR primers is depleted during early thermal cycles generating single-stranded products with the remaining primers • These were created using single stranded M13mp10 DNA that contained EcoRI sites • This allowed one to not have to purify the product

  10. Results • Taq DNA polymerase was found to be very fast • A major benefit was that it could be run at a variety of temperatures and still be effective unlike others where the polymerase falls off due to the heat • It was also found that many the initiated primers were completely extended before reinitiating to a new template

  11. Factors affecting sequencing • KCL was found to inhibit the extension due to a slight enzyme inhibition • Geletin used in PCR as a stabilizer created distortions during electrophoresis. • Nonionic detergents stimulated Taq DNA pol and reduced the background

  12. G+C Rich DNA, eliminating band compression • Usually dITP or base analog cGTP to resolve the compression found in rich G+C DNA • Replaces DGTP • When used with Taq cGTP effective • dITP requires four times as much in order to achieve the same results

  13. Magnesium • It was found that Taq DNA pol was extremely sensitive to Magnesium ion concentration. It caused a lot of misincorperation of dDTP’s and ddNTP’s since their stock contains MgCl2 • This caused base specific premature chain termination • In order to overcome this high levels of dNTP’s and ddNtp’s were used

  14. Two step labeling and extension protocol • Because of the need for high d and ddNTP’s a two step protocol for labeling and extension was used similar to that created by Tabor and Richardson • The first step exposed the DNA to low concentrations of all 4 dNTP’s at low temperature. Only one of which is labeled • By increasing one of the non-labeled dNTP’s the signals became clear

  15. Sequencing and PCR • Using asymmetric PCR templates purification of the product wasn’t needed • When compared to using a regular template the results showed that there was very little difference between the two

  16. Conclusion • Taq ideal for sequencing PCR products • Results appear superior than the previous ways • Taq can work over a large range of temperatures, allowing one to straighten DNA which usually hinders transcription • If all dNTP’s are present don’t observe mismatch pairing

  17. Modern Times • Now day’s Taq DNA polymerase has been found to have problems • Discrimination of ddNTP’s is found leading to uneven incorperation • A strong bias for ddGTP isfound

  18. More information • 1. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. NatI. Acad.Sci. USA 74, 5463-5467. • 2. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, 103-119. • 3. Mills, D. R. & Kramer, F. R. (1979) Proc. Natl. Acad. Sci. USA 76,2232-223

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