Cycle sequencing protocol

Sequencing of parasite genes provides a powerful tool for the accurate identification of parasites and for systematic studies (Johnson and Baver-stock, 1989 Reddy, 1995 McManus and Bowles, 1996), and is based on either of the two original protocols (Sanger et al., 1977 Maxam and Gilbert, 1980). Cycle-sequencing (Murray, 1989), a PCR-based modification of the... 

Sears, L., Moran, L, Kissinger, C., Creasey, T O Keefe, P., Roskey, M., Sutherland, E., Slatko, B. (1992) CircumVent Thermal Cycle Sequencing and Alternative Manual and Automated DNA Sequencing Protocols Using the Highly Thermostable VentR (exo-) DNA Polymerase, BioTech-niques 13, 626-633. 

At the exact time set by the schedule, inject either the calibration mixture or sample into the chromatograph or make no iqjection (baseline blank). At the time of injection, start the chromatograph time cycle and the integrator/computer data acquisition. Follow the analysis sequence protocol for all subsequent repetitive analyses or calibrations. Since complete resolution of sample peaks is not expected, do not change the detector sensitivity setting during the analysis. 

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

Figure 6.6. Amplification of DNA by PCR. Target DNA sequence from a complex genome can be amplified by heat denaturation, providing appropriate conditions for the enzyme (Taq DNA polymerase) that allow it to cause exponential amplification of a particular DNA segment. Among components besides the enzyme that are essential for amplification process are oligonucleotide primers in opposite orientation to each other, shown by dotted arrows, deoxynucleotide triphosphates (dNTPs), Mg2+, and buffer. A 30-cycle amplification leads to a many-million-fold amplification of the discrete DNA segment, flanked by oligonucleotide primer sequences. (Reproduced from Short Protocols in Molecular Biology, 4th ed., F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., Wiley, New York, 1999, p. 15-1.)...

For amplification, follow the protocol of Saiki,24 except use approximately 3 ng template DNA, maintain the MgCl2 concentration at 0.2 mM, and amplify only through 10-15 cycles. With increased amplification, rather than obtaining a sharp band product when checked on an agarose gel, DNA sequences of a very broad range of sizes are produced (nonspecific amplification). 

Repeat for a total of 25 cycles. Note The thermal cycle of this protocol is optimized for traditional Ml3 forward and reverse sequencing primers. Other primer sequences may work most effectively at a different annealing temperature. Also, the extension temperature is lower than the temperature optimum of Taq polymerase, to allow the incorporation of the highly modified dye-labeled dideoxy terminators. 

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