DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments

Abstract

The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

Fluorescent Oligonucleotides - Versatile Tools as Probes and Primers for DNA and RNA Analysis

“…60,[82][83][84][85][86][87] Those fluorescent analogues are used in enzymatic DNA sequencing. Automated techniques refer to a variation of traditional Sanger sequencing, 88 like in the ABI approach where four differently labeled dye terminators are utilized. 89 The fluorescent dyes are introduced via alkine linkers attached to the C-5 base position in the pyrimidine series and the C-7 position in the deaza purine series (Figure 3).…”

Strategies for introducing non-radioactive labels during the automated sequence analysis of nucleic acids

“…In addition, the set of dye-terminators could not be directly transferred to other polymerase systems but required an optimisation. Thus, for Taq DNA polymerase, essential for performing the cycle sequencing protocol, a set of rhodamine-based terminators was developed (Lee et al, 1992) which produced acceptable sequences, when used in conjunction with 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates). The intrinsic peak height variation due to sequence dependent differential incorporation of the terminators by the Taq DNA polymerase was recognised and in an effort to utilise the processivity of T7 DNA polymerase to attain more even signal patterns a new set of labelled terminators was designed (Lee et al, 1992).…”

“…Thus, for Taq DNA polymerase, essential for performing the cycle sequencing protocol, a set of rhodamine-based terminators was developed (Lee et al, 1992) which produced acceptable sequences, when used in conjunction with 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates). The intrinsic peak height variation due to sequence dependent differential incorporation of the terminators by the Taq DNA polymerase was recognised and in an effort to utilise the processivity of T7 DNA polymerase to attain more even signal patterns a new set of labelled terminators was designed (Lee et al, 1992). Nevertheless, in order to maintain the convenience of the cycle sequencing system, a detailed analysis of the peak variation with respect to the sequence context provided a collection of empirical rules for the interpretation of Taq DNA polymerasegenerated patterns (Parker 1995).…”

AmpliTaq ® DNA Polymerase, FS Dye-Terminator Sequencing: Analysis of Peak Height Patterns

“…Direct sequencing of DNA amplified by the polymerase chain reaction (PCR) is being used increasingly in large-scale sequencing projects and in scanning specific regions for polymorphisms and mutations (1)(2)(3)8). Among the available sequencing chemistries, cycle-sequencing with thermostable DNA polymerases and dye-labeled dideoxy chain terminators is the most versatile because it eliminates the need for specially modified sequencing primers, since the PCR primers can also be used as sequencing primers (6,8).…”