uses dideoxynucleoside triphosphates, generating uniform band intensities in sequencing experiments (with dGTP). These properties make the enzyme ideal for generating high-quality DNA sequences using cycle-sequencing methods. It is stable at 90°C for at least 1 hour and retains 50% of its activity when incubated at 95°C for 60 minutes. The Thermo Sequenase polymerase in this kit combines the advantages of both Sequenase DNA polymerase and Taq DNA polymerase. It produces bands (with Mg2+) that are nearly as uniform as those produced with Sequenase DNA polymerase with Mn2+ (10), yet is thermostable like Taq DNA polymerase.
Cycle sequencing is the name given to the process of using repeated cycles of thermal denaturation, primer annealing, and polymerization to produce greater amounts of product in a DNA sequencing reaction. This amplification process employs a single primer so the amount of product DNA increases linearly with the number of cycles. (This distinguishes it from PCR* which uses 2 primers so that the amount of product can increase exponentially with the number of cycles.)
The earliest examples of cycle sequencing used 32P-labeled primers and a non- thermostable polymerase which was added after each denaturation cycle (11,12). Later improvements included the use of thermostable Taq polymerase
(13,14) and the use of alpha-labeled dNTPs in place of the labeled primer using mixtures of nucleotides similar to those used originally by Sanger (15,16). The labeled-primer methods make efficient use of 32P giving a sequence with as little as 4∝Ci of [γ-32P]ATP (14). The methods using internally-labeled products were less efficient, requiring either 10∝Ci of [α-33P]dATP or 20∝Ci of [α-35S]dATP for a sequence. This is a consequence of the relatively low specific radioactivity and the small number of labeled bases in short product molecules. This kit makes very efficient use of [α-33P]ddNTP, requiring less than 1∝Ci of
33P per sequence. Cycle sequencing is necessary with this kit when using less than 0.2-0.5pmol of template DNA. Non-cycle (or very few cycle) protocols may be used with more than ~0.5pmol of template.
*See license information on back cover.