Sequencing – classical method

Česká verzia

Authors: Monika Šedivcová ¹; Petr Martínek ¹; Jan Stehlík ¹; Petr Grossmann ¹; Jana Kašpírková ^1,2; Tomáš Vaneček ^1,2
Authors place of work: Bioptická laboratoř, s. r. o., Plzeň ¹; Šiklův ústav patologie LF UK a FN, Plzeň ²
Published in the journal: Čes.-slov. Patol., 49, 2013, No. 3, p. 122-128
Category: Přehledový článek

Summary

In this article the basic methods of reading nucleotide sequences in DNA molecules are summarized. Sanger sequencing is described most thoroughly as it is the most frequent routine method currently being utilized. The article describes in detail the principle of sequence determination through the production of fragments with a known end base using chain termination synthesis of DNA and ways of separation and detection of the fragments. Some alternative methods of sequencing are mentioned in short. Basic approaches of analyzing sequence data are explained as well as different outcomes, obstacles and challenges.

Key words:
sequencing – Sanger – synthesis – ddNTP – fluorescence – capillary electrophoresis – mutation – electropherogram

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