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

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

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

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

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

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

Methods-Extension/Termination An extension/termination reaction was preformed for each labeled template 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 “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 The product was then loaded onto a gel and run in order to sequence the PCR products

Asymmetric PCR Asymmetric PCR is here one oligonucleotide primer is present in 100 fold greater concentration than the other 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 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 These were created using single stranded M13mp10 DNA that contained EcoRI sites This allowed one to not have to purify the product This allowed one to not have to purify the product

Results Taq DNA polymerase was found to be very fast 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 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 It was also found that many the initiated primers were completely extended before reinitiating to a new template

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

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

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 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 This caused base specific premature chain termination In order to overcome this high levels of dNTP’s and ddNtp’s were used In order to overcome this high levels of dNTP’s and ddNtp’s were used

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 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 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 By increasing one of the non-labeled dNTP’s the signals became clear

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

Conclusion Taq ideal for sequencing PCR products Taq ideal for sequencing PCR products Results appear superior than the previous ways 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 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 If all dNTP’s are present don’t observe mismatch pairing

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

More information 1. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. NatI. Acad.Sci. USA 74, Yanisch-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, Mills, D. R. & Kramer, F. R. (1979) Proc. Natl. Acad. Sci. USA 76,