title: Fundamentals of sequencing of difficult templates--an overview.
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.
authors.affiliation: Wyeth Research, Biological Technologies Department, Cambridge, MA 02140, USA. jkieleczawa@wyeth.com
authors.forename: Jan
authors.initials: J
authors.lastname: Kieleczawa
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
chemical_list: D011933:Reagent Kits, Diagnostic; D004247:DNA