BioSci 145B week4 papers page 1 © copyright Bruce Blumberg All rights reserved Classic paper –Goal was to automate DNA sequencing to facilitate sequencing of human genome –Describes new form of sequencing using two-step PCR to generate long, even reads First step amplifies template DNA by PCR Second step does asymmetric amplification (linear) to perform actual sequencing reaction Proof-of-principle for automated sequencing

BioSci 145B week4 papers page 2 © copyright Bruce Blumberg All rights reserved Innis et al., Figure 1 In this figure the authors are optimizing extension temperature and time –70 C and 2 minutes appear optimal

BioSci 145B week4 papers page 3 © copyright Bruce Blumberg All rights reserved Innis et al., Figure 2 Testing effects of limiting one dNTP in the reaction –WHY? –Because reaction with ddNTP will have limiting amounts of corresponding dNTP e.g. ddGTP and dGTP –B) Note that there are regions where polymerization terminates when one dNTP is limiting –C) Some of these are not recoverable by adding large amount of dNTPs later

BioSci 145B week4 papers page 4 © copyright Bruce Blumberg All rights reserved Innis et al., Figure 3 (A)Shows effects of incubation temp – 70 is best (B) shows that must preheat reaction to get good extension (Not true really) (C) shows that very long sequences can be read by extended electrophoresis

BioSci 145B week4 papers page 5 © copyright Bruce Blumberg All rights reserved Innis et al., Figure 4 Testing ability of Taq polymerase to read through regions of strong secondary structure Left panel shows inability of reactions containing dITP to be extended well –Also not really true

BioSci 145B week4 papers page 6 © copyright Bruce Blumberg All rights reserved Innis et al., Figure 4 Left side shows classic sequencing artifact – compression due to secondary structure Right side - Nucleotide analog 7- deaza-dGTP resolves the sequence Lot of noise about such things in 1980s and we used to use such analogs to resolve sequences Compression is a gel running artifact that can be totally eliminated by heating the gel when running –Never see this in capillary electrophoresis since the capillaries are run at high temperature

BioSci 145B week4 papers page 7 © copyright Bruce Blumberg All rights reserved Innis et al., Figure 5 A is ss M13 template sequenced in traditional way B is cycle sequenced product Claim that cycle sequencing gives much longer reads and this appears to be the case BUT –Difference in the 2 is an artifact of how the reactions were run –A could have been as long as B with different ratios of ddNTPs/dNTPs

BioSci 145B week4 papers page 8 © copyright Bruce Blumberg All rights reserved Innis et al. Conclusions drawn –Taq polymerase is good for sequencing Long reads (processive) Even banding patterns (no 3’ exonuclease activity) Incorporates nucleotide analogs to improve sequencing gels –Developed new sequencing method Optimized temperature and time for Very suitable for automated sequencing Value of the method –This is how all sequencing is done today (with labeled ddNTPs) –Advances in template prep (TempliPhi) and –Terminator chemistry (Big Dyes) –Have led to rapid completion of genome projects ahead of schedule and under budget