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Dna Sequencing Fragments Terminator Bases Capillary Nucleotides Stop

This flashcard explains the principles of DNA sequencing, covering the capillary method which uses fluorescent terminator bases to halt DNA synthesis at varying lengths, and advanced sequencing techniques like high-throughput sequencing that utilizes a flow cell for massively parallel processing.

Cette fiche explique les principes du séquençage d'ADN, y compris la méthode capillaire utilisant des bases terminatrices fluorescentes pour arrêter la synthèse d'ADN à différentes longueurs, et les techniques de séquençage avancées comme le séquençage à haut débit qui utilise une cellule de flux pour un traitement massif et parallèle.

Front Principles of DNA sequencing
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  • Capillary method;
    • DNA for sequencing mixed with a primer, DNA polymerase, XS normal nucleotides and terminator bases (modified versions of A,C,G,T, that stop DNA synthesis when at location where actual base should be added, these have fluorescent tags (A-green, G-yellow, T-red, C-blue)), and placed in thermal cycler (at 96C, DNA strands seperate, 50C, primers anneal to DNA strand, 60C, DNA polymerase adds nucleotides to single-strand DNA template complementarily
    • each time terminator base randomly incorporated instead of nucleotide, DNA synthesis terminates, and forms fragments of diff lengths so after few cycles, all possible DNA chains formed, then fragments seperated via capillary sequencing (like gel electrophesis but in capillary tubes), fluorescent markers on terminator bases identify final base on each fragment and lasers detect diff colours (detects fastest fragments first, so allows sequence to be determined)
    • order of bases now show sequence of complimentary DNA strand, fed into computer which compares fragments and finds areas of overlap, and once genome assembled, can be used to find genes coding for specific characteristics
  • Advanced sequencing technique
    • high-throughput sequencing (next-generation sequencing/massivley parallel sequencing) - takes place on a flow cell (plastic slide, alternative to capillaries/gel), fragments of DNA attached to slide and replicated via PCR, still uses coloured terminator bases to stop reaction so image taken, so very efficent/fast, reduces costs

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