Fundamentals of sequencing of difficult templates--an overview - PubMed (original) (raw)

Comparative Study

Fundamentals of sequencing of difficult templates--an overview

Jan Kieleczawa. J Biomol Tech. 2006 Jul.

Abstract

Despite enormous progress, the DNA sequencing of difficult regions, whether they are part of complex genomes or individual constructs, still presents a significant challenge and may require many trials, brute-force, or the intervention of a very experienced sequencer (and sometimes all of the above). Very early on, it was realized that sequencing of various types of difficult templates requires distinct treatments. To sequence through GC-rich regions, the addition of DMSO, NP-40/Tween-20 detergents, or the mix (4:1) of BD3.0:dGTP3.0 was sometimes helpful. To get through long poly-A/T tails, sometimes one would be successful using tailored poly- A/T (V/B) N primers or primers that spanned part of pre-tail and tail regions. A few years ago invitrogen introduced a set of sequencing additives that proved to be useful for many different types of difficult templates.

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Figures

FIGURE 1

Denaturation of pGem3zf under different conditions. Two hundred nanograms of pGem3zf was denatured using heat-denaturation conditions described in this paper and in several published papers. (1) control pGem3zf; (2) 2.5 min heat-denaturation (HD); (3) 7.5 min HD; (4) 15 min HD (DNA in lanes 1–4 was in 10 mM Tris-cl, 0.01 mM EDTA (pH 8.0) = TEsl. The DNA in lanes 5–14 was also in TEsl but had additional components): (5) in the presence of 2 mM MgCl2, 7.5 min HD; (6) in the presence of BigDye terminator v3.0/4X final dilution with MgCl2 at 2 mM, 7.5 min HD; (7) in the presence of 2 mM MgCl2 followed by 40 sequencing cycles [(10 sec/96°C)(5 sec/50°C)(2 min/60°C)]; (8) as in lane 6 but followed by cycling as in lane 7; (9) 7.5 min HD, add 2 mM mgcl2 followed by cycling as in lane 7; (10) 7.5 min HD, add dye-terminator as in lane 6, followed by cycling as in lane 7; (11) in the presence of 0.1 N NaOH incubated at room temperature for 5 min, neutralized with 0.1 N HCl; (12) in the presence of 0.2 N NaOH incubated at room temperature for 5 min, neutralized with 0.2 N HCl; (13) in the presence of 0.2 N NaOH incubated at 85°C for 5 min, neutralized with 0.2 N HCl; (14) in the presence of 0.1 N NaOH incubated for 3 min at 100°C, neutralized with 0.1 N HCl.

FIGURE 2

chromatograms of GC-rich template (about 67%) sequenced using a standard (A) and modified (B) protocol. The solid bar indicates identical sequence regions.

FIGURE 2

chromatograms of GC-rich template (about 67%) sequenced using a standard (A) and modified (B) protocol. The solid bar indicates identical sequence regions.

FIGURE 3

Chromatograms of GC-rich template (about 72%) sequenced using a standard (A) and modified (B) protocol. The solid bar indicates identical sequence regions

FIGURE 3

Chromatograms of GC-rich template (about 72%) sequenced using a standard (A) and modified (B) protocol. The solid bar indicates identical sequence regions

FIGURE 4

An example of a template with 59-bases GA non-repeat stretch sequenced using a standard (A) and modified (B) protocol. These views represent quality scores as displayed by Sequencher program. The light blue color indicates bases with higher Q quality and darker blue color indicates bases with lower Q values. The black bar shows the region with the same sequence.

FIGURE 4

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Fundamentals of sequencing of difficult templates--an overview - PubMed (original) (raw)