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    DNA Sanger Sequencing Worksheet Exercise: TH TH

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    Joshua Prince
    This document describes a DNA sequencing worksheet exercise that simulates the Sanger sequencing method. Students are provided a short DNA template sequence and primer. They are asked to write out the different DNA fragments that could be synthesized during the Sanger reaction using the provided primer and reaction chamber contents. Finally, students are tasked with indicating where fragments would appear on an electrophoresis gel, completing a sequence read-out graph from laser detection data, and answering questions about the biochemical steps involved.

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    DNA Sanger Sequencing Worksheet Exercise: TH TH

    Uploaded by

    Joshua Prince
    720 views3 pages
    This document describes a DNA sequencing worksheet exercise that simulates the Sanger sequencing method. Students are provided a short DNA template sequence and primer. They are asked to write out the different DNA fragments that could be synthesized during the Sanger reaction using the provided primer and reaction chamber contents. Finally, students are tasked with indicating where fragments would appear on an electrophoresis gel, completing a sequence read-out graph from laser detection data, and answering questions about the biochemical steps involved.

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    T3- Sanger Worksheet

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    This document describes a DNA sequencing worksheet exercise that simulates the Sanger sequencing method. Students are provided a short DNA template sequence and primer. They are asked to write out the different DNA fragments that could be synthesized during the Sanger reaction using the provided primer and reaction chamber contents. Finally, students are tasked with indicating where fragments would appear on an electrophoresis gel, completing a sequence read-out graph from laser detection data, and answering questions about the biochemical steps involved.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    720 views3 pages

    DNA Sanger Sequencing Worksheet Exercise: TH TH

    Uploaded by

    Joshua Prince
    This document describes a DNA sequencing worksheet exercise that simulates the Sanger sequencing method. Students are provided a short DNA template sequence and primer. They are asked to write out the different DNA fragments that could be synthesized during the Sanger reaction using the provided primer and reaction chamber contents. Finally, students are tasked with indicating where fragments would appear on an electrophoresis gel, completing a sequence read-out graph from laser detection data, and answering questions about the biochemical steps involved.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 3

    DNA

     Sanger  Sequencing  Worksheet  Exercise  


    LB145  S13  
     
    >>This  replaces  the  Sanger  Sequencing  Summary  Homework  on  page  79  of  the  lab  manual<<  
    >>DUE  on  April  15th/16th<<  
    >>Worth  5  points<<  
     
    Let’s  suppose  you  have  isolated  and  amplified  a  short  fragment  of  DNA  using  Polymerase  
    Chain  Reactions.  After  completing  this,  you  would  like  to  take  a  closer  look  at  the  sequence  of  
    nucleotides  in  the  DNA  fragment.  You  use  a  method  called  dideoxy  Sanger  sequencing.  
     
    In  this  worksheet  you  will  mimic  the  Sanger  sequencing  method  to  see  how  DNA  is  sequenced.  
     
     
    [Let’s  Look  Into  The  Future]   Here  is  what  a  DNA  sequencing  lab  will  tell  you…  
     
    This  fragment  of  DNA  that  you  have  amplified  has  been  sequenced  as  follows:  
     
    3’ – CGAATATGCGAGTCTGGCAACTCC – 5’ (TEMPLATE STRAND)
    ||||||||||||||||||||||||
    5’ – GCTTATACGCTCAGACCGTTGAGG – 3’

     
    While  I’ve  already  spoiled  the  mystery  by  showing  you  the  sequence  of  the  DNA  fragment  
    above,  the  important  thing  I  want  you  to  learn  is  HOW  DNA  sequencing  is  accomplished  using  
    the  Sanger  Method.  

    Primer:  
    You  have  used  a  primer  that  has  a  short  nucleotide  sequence  of:  5’  –  GCTTA  –  3’  
     
    Your  Task:  
    -­‐ Given  this  primer,  your  task  is  to  write  out  the  set  of  different  DNA  fragment  that  you  
    could  synthesize  using  the  dideoxy  Sanger  Method.    
    -­‐ To  accomplish  this,  assume  that  the  box  on  the  next  page  represents  the  reaction  
    chamber  where  the  dideoxynucleotide  reactions  occur.    
    -­‐ When  you  write  out  a  DNA  fragment,  represent  a  DNA  nucleotide  using  a  normal  letter  
    (e.g.  A,  T,  C,  or  G).  To  represent  a  dideoxyribonucleic  acid  nucleotide,  write  the  letter  in  
    the  appropriate  color  to  (a)  identify  it  as  a  dideoxynucleotide  and  (ii)  to  show  it  has  a  
    fluorescent  tag  (as  shown  in  the  example  on  the  following  page).  
    -­‐ Underline  the  primer  sequence  for  each  fragment.  
     
     
     
     
    3’ – CGAATATGCGAGTCTGGCAACTCC – 5’ (TEMPLATE STRAND)
      ||||||||||||||||||||||||
      5’ – GCTTATACGCTCAGACCGTTGAGG – 3’
       
      Reaction  Chamber  Contains  
      dNTPs  (A,  C,  T,  G)  
      ddATPs  (A)  
      ddCTP  (C)  
      ddGTP  (G)  
      ddTTP  (T)  
     
      Examples:  
     
    5’ – GCTTAT – 3’
     
      5’ – GCTTATA– 3’
      5’ – GCTTATAC – 3’
      5’ – GCTTATACG – 3’
     
      5’ – GCTTATACGC– 3’
      etc…
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
    Worksheet  Questions:   GEL  
     
    1.  Draw  the  chemical  structures  of  ribose,  deoxyribose  and  dideoxyribose.  
    Products  of  
     
    RXN  Chamber  
     
    1  
     
     
     
     
     
    2.  Explain  what  would  happen  during  a  PCR  elongation  step  if  a  
    dideoxyribonucleotide  got  incorporated  into  a  growing  nucleotide  chain.    Why  
    would  this  happen?  
     
     
     
     
    3.  Based  on  the  DNA  sequence  you  are  working  with  (above),  indicate  on  the  gel  to  
    the  right  where  you  would  expect  to  find  each  of  the  fragments  you  wrote  down  on  
    the  previous  page.  Assume  that  electrophoresis  has  run  long  enough  to  separate  the  
    fragments,  but  not  so  long  that  any  of  the  fragments  have  fallen  off  the  edge  of  the  
    gel.  On  the  gel  to  the  right,  write  down  the  nucleotide  sequence  of  each  fragment  
    in  the  place  where  it  would  be  found  after  the  gel  has  been  run.    
     
    4.  As  the  gel  continues  to  run,  the  DNA  fragments  will  eventually  “fall  off”  the  end.  
    Before  they  do  so,  a  laser  at  the  bottom  of  the  gel  will  read  each  of  the  fragments  as  
    it  passes  by  and  generate  data  on  the  graph  below  corresponding  to  the  nucleotides  
    it  has  identified  (based  on  their  fluorescent  tags).  Complete  the  graph  below  using  
    appropriate  colors  to  indicate  the  nucleotide  sequence.  Also,  indicate  which  is  the  3’  
    end  and  which  is  the  5’  end  of  the  sequence  once  you  have  completed  it  below.  

    LASER  

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