ow is an illustration explaining Sanger sequencing using fluorescently-labelled

Question

ow is an illustration explaining Sanger sequencing using fluorescently-labelled ddNTPs. Determine the sequence of the newly synthesized stands. [0.50] Newly synthesized strand: 5'- 3, . T C G G T C AA C TA C AA C C G G C TAAGG CTGGGACA-5' 5 -AG CCAGTTGATGTTGGCCAAGGATTCCGACCCTGT-3 | DNA Template R product) PC Template strand 3 -TCGGTCAACTACAACCGGTTCCTAAGG CTGGGACA-5 Primer DNA strands are synthesized in the sequencing reaction. All of the fragments end (terminate) with a fluorescent-tagged ddNTP DNA sequencing reaction 1) Taq polymerase 2) ONE primer 3) dNTP 4) labelled ddNTPs 5-AG CCAGTTG A-(Green) 5'-AGCCAGTTG-(Black) 5' -AGCCAGTTGATGTTGGC-(Blue) 5'-AGCCAGTTGATG T-(Red) 5' -AGCCAGTTGAT-(Red) 5' -AG CCAGTTGATGTT-(Red) 5 -AG CCAGTTGATGTT G-(Black) 5' -AGCCAGTTGATGTTGG-(Black) 5' -AGCCAGTTGATG-(Black) ddATP-(Green) ddCTP-(Blue) ddGTP-(Black) ddTTP-(Red) 5) DNA template (your PCR product) DNA fragments separate in the gel The "colors" (fluorescent-tags) of the DNA with the smaller fragments migrating bands in the gel can be read in a 5-to-3 faster through the gel compared larger fragments (bottom-to-top) direction. 5-AGCCAGTTGATGTTGGC(Blue) 5'-AGCCAGTTGATGTTGG (Black) 5-AG CCAGTTGATGTT G-(Black) 5'-AGCCAGTTGATGTT-(Red) 5, . A G C C A G T T G A T G T(Red) 5'-AGCCAGTTGA T G-(Black) 5 -AGCCAGTTG A T-(Red) 5-AGCCAGTTG A-(Green) 5-AGCCAGTTG-(Black) (Blue) (Black) (Black) Red) Red Black) Red) Green) (Black)

Explanation / Answer

The sanger sequencing uses dideoxynucleoside triphosphate (ddNTPs) analogs to interrupt DNA synthesis. When a ddNTP is inserted in place of dNTP, strand elongation stops as it lacks 3'-OH group needed for the next step. It results in the mixture of prematurely terminated labelled fragments, each ending with one of four ddNTPs. Each fragments are then allowed to separated through electrophoresis and then the sequence can be read directly from autoradiogram of the gel. Shorter fragments migrate faster, the fragments near the bottom of the gel represents the 5' end and the sequence is read in 5'---->3' direction, from bottom to top.

So the newly synthesized strand: 5'- AGCCAGTTGATGTTGGC -3'

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