Chain termination sequencing
This kit is designed to eliminate sequencing artifacts such as stops (or BAFLs— bands across four lanes) and background bands. BAFLs can result from the enzyme pausing at regions of secondary structures in GC-rich templates, producing prematurely aborted primer extension products of the same length in all four termination reactions. Background bands can be caused by primer extensions aborting prematurely at random positions, such as when a template is rich in a certain base and the complementary nucleotide in the reaction becomes depleted.
Traditional chain termination sequencing methods (1) involve the synthesis of a DNA strand by a DNA polymerase in vitro using a single-stranded DNA template. Synthesis is initiated at the site where a primer anneals to the template. Elongation of the 3' end of the annealed primer is catalyzed by a DNA polymerase in the presence of 2'-deoxynucleoside-5'-triphosphates (dNTPs), and is terminated by the incorporation of a 2',3'-dideoxynucleoside-5'- triphosphate nucleotide analog (ddNTP) that will not support continued DNA elongation (hence the name ‘chain termination’). Four separate reactions, each with a different ddNTP, (ddG, ddA, ddT, or ddC), give complete sequence information. A radiolabeled dNTP (2,3) or primer is normally included in the synthesis, so the labeled chains of various lengths can be visualized after separation by high-resolution gel electrophoresis (4,5). In this kit, a radioactive label is incorporated into the sequencing reaction products at the 3' end by the use of an [α-33P]ddNTP, thus ensuring that only properly terminated DNA strands are labeled and are visible in the sequence. This results in a cleaner, more reliable and easier to read sequence with fewer background bands and virtually no BAFLs.
The accuracy and readability of the sequence obtained depends strongly on the properties of the polymerase used for chain termination. Some polymerases, such as Sequenase™ Version 2.0 DNA polymerase, generate much more uniform, readable bands than others like Klenow and Taq DNA polymerase (6,7,8). Thermostable polymerases, such as Taq polymerase, can be used for multiple rounds (cycles) of DNA synthesis, generating stronger signals. Tabor and Richardson (9) have discovered that DNA polymerases can be modified to accept dideoxynucleotides as readily as the normal deoxynucleotide substrates. Using this technology, a new DNA polymerase for DNA sequencing was developed. This enzyme, called Thermo Sequenase DNA polymerase, is thermostable and possesses many of the excellent DNA sequencing qualities of Sequenase DNA polymerase. The properties of this DNA polymerase include activity at high temperature and absence of associated exonuclease activity. Like Sequenase DNA polymerase, derived from T7 bacteriophage, it readily