1 / 7

DNA Sequencing Scenario

DNA Sequencing Scenario. DNA replication and chain termination. ddTTP No OH group = chain termination at T residue. dATP. O H. Ö H. H. 5 ´. T. P. P. P. P. P. P. P. P. P. P. C. T. A. G. A. A. G. T. C. T. A. T. C. P. P. P. P. P. P. P. P. 3 ´.

vevina
Download Presentation

DNA Sequencing Scenario

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. DNA Sequencing Scenario

  2. DNA replication and chain termination ddTTP No OH group = chain termination at T residue dATP OH ÖH H 5´ T P P P P P P P P P P C T A G A A G T C T A T C P P P P P P P P 3´ Adapted from Lehninger Principles of Biochemistry 4th Edition Primer strand The 3´ hydroxyl (OH) group interacts with the phosphate group on the incoming nuclueotide to form a phosphodiester bond Template strand

  3. Nucleosides & Nucleotides Taken from: http://en.wikipedia.org/wiki/Nucleotide Nucleoside = a heterocyclic base (A, G, C, U or T) and a five carbon sugar (pentose) either ribose for RNA or deoxyribose for DNA Nucleotide = a heterocyclic base and a pentose sugar with one or more phosphate groups (mono-, di- or tri-phosphate). Nucleotides are the monomers of nucleic acids, with three or more bonding together in order to form a nucleic acid.

  4. Base O H H H H O- O- O- O- O- O- O- O- O- OH OH P P P -O O P O P O CH2 -O -O O O P P O O P P O O CH2 CH2 O O O O O O O O O Base O H H H H OH H Base O H H H H H H Nucleotide structure continued Nucleoside tri-phosphate (NTP) With a ribose sugar (OH at C2 & C3) used to synthesise RNA. Deoxynucleoside tri-phosphate (dNTP) With a deoxyribose sugar(OH at C3) used to synthesise DNA. Dideoxynucleoside tri-phosphate (ddNTP) With a dideoxyribose sugar(no OH groups) used for the Sanger method (dideoxy) sequencing.

  5. phosphodiester bond http://en.wikipedia.org/wiki/Phosphodiester_bond Nucleotides are joined together by phosphodiester bonds Phosphodiester bonds require a hydroxyl group (OH) to be present on the 3´ carbon to react with a phosphate on in 5´ carbon of the sugar residue of the next nucleotide. If no 3´ hydroxyl group is available no further nucleotides can join the DNA strand and the chain terminates.This is how dideoxynucleotides prevent chain elongation – they lack a 3´ OH group on the sugar.

  6. Components of the reaction mix • DNA template • DNA primer • DNA polymerase -Taq polymerase • Mixture of deoxy (dNTPs) & fluorescently labelled dideoxy nucleotides (ddNTPs) Computer analysis – base calling Outline of Fluorescent Dideoxy DNA Sequencing 20 – 35 cycles Apply to a capillary gel & subject to electrophoresis The fragments are detected after excitation of the labelled ddNTPs by a laser Fluorescent peaks and bases are visualised and analysed

  7. T TC PRIMER TCAGAT PRIMER PRIMER PRIMER PRIMER PRIMER PRIMER PRIMER TCA TCAG TCAGATAG TCAGA TCAGATA The sequencing reaction continued Components in the reaction mix Taq polymerase OH 5´ PRIMER + dATP, dCTP, dGTP, dTTP, + ddATP, ddCTP, ddGTP, ddTTP AGTCTATATC 3´ TEMPLATE Termination products obtained Remember that the sequence obtained with be complementary to the template sequence! Therefore our new 5´  3´ complementary sequence to our original template sequence reads TCAGATAG.

More Related