Sanger “dideoxy” method

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The Sanger dideoxy method of [[DNA Sequencing|DNA sequencing allows]] large volumes of [[DNA|DNA]] to be sequenced so that it is possible to even&nbsp;determine whole [[Genome|genomes]]. This method uses [[Deoxyribonucleoside triphosphates|deoxyribonucleoside triphosphates and]] [[Dideoxyribonucleoside triphosphates|dideoxyribonucleoside triphosphates]]. A [[DNA Primer|DNA primer binds]] to the 5' end of the single-stranded [[DNA|DNA]] molecule to be sequenced (the [[DNA template strand|template strand]]) to allow the [[DNA Polymerase|DNA polymerase]] [[Enzyme|enzyme]] to attach and synthesize&nbsp;a new&nbsp;[[DNA|DNA]] chain against the [[DNA template strand|template strand]] through [[Watson and Crick base pairing|Watson-Crick complementry base pairing]]. The new chain is built of [[Deoxyribonucleoside triphosphates|deoxyribonucleoside triphosphates]].<br>  
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The Sanger dideoxy method of DNA sequencing allows large volumes of DNA to be sequenced in vitro. This method uses a single stranded, DNA template strand, a specific primer of 18-20 bases, DNA polymerase , deoxyribonucleoside triphosphates(dNTP’s) and their respective derivatives dideoxyribonucleoside triphosphates (ddNTP’s) which differ from the dNTP’s in that instead of a 3’ OH group they have a 3’ H which is intergral to their function as chain terminators. This is because with the 3’ H prevents chain elongation as phosphodiester bonds cannot be created with the next ribonucleoside (Hames and Hooper, 2000:260)<ref>Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd</ref>  
  
If a [[Dideoxyribonucleoside triphosphates|dideoxyribonucleotide triphosphate is]] incorporated into the growing newly synthesised chain, it termiantes synthesis at that point. There are 4 different [[Dideoxyribonucleoside triphosphates|dideoxyribonucleoside triphosphate chain]]-terminating molecules ([[Dideoxyribonucleoside triphosphates|ddCTP]], [[Dideoxyribonucleoside triphosphates|ddGTP]], [[Dideoxyribonucleoside triphosphates|ddATP]], [[Dideoxyribonucleoside triphosphates|ddTTP]]) each containing one of the DNA bases, so [[Dideoxyribonucleoside triphosphates|ddCTP]] contains the base ‘C’ ([[Cytosine|Cytosine]]) etc. These 4 different molecules are used in 4 seperate [[DNA|DNA]] synthesis reactions on copies of the same single-stranded [[DNA template strand|DNA template strand to]] be sequenced. Each reaction produces sequences of different lengths complementary to the [[DNA template strand|template strand]], which have terminated at different points.<br>  
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<br>A DNA primer binds to the 5' end of the single-stranded DNA molecule to be sequenced (the template strand) to allow the DNA polymeras eenzyme to attach and synthesize a new DNA chain against the template strand through Watson-Crick complementary base pairing. <br>There are 4 different dideoxyribonucleoside triphosphate chain-terminating molecules (ddCTP, ddGTP, ddATP, ddTTP) each containing one of the DNA bases, so ddCTP contains the base ‘C’ (Cytosine) etc complemtary to a G on the template strand. These 4 different molecules are used in 4 separate DNA synthesis reactions on copies of the same single-stranded DNA template strand to be sequenced. When a ddNTP is incorporated into the growing newly synthesised chain, it terminates synthesis at that point so each reaction produces fragments of different lengths complementary to the template strand, which have terminated at different points. (Alberts, et al, 2008:549)<ref>Alberts, Bruce, et al, 2008, Molecular Biology of the Cell, 5th edition, New York, Garland Science</ref>
  
These [[DNA|DNA]] fragments are then separated by [[Gel Electrophoresis|gel electrophoresis which]] allows us to see how long each fragment is depending on how far through the gel it has travelled. By reading off each band on the gel the sequence of the newly synthesised strand as well as the template strand can be found out. So, the band which travelled furthest through the gel must have been the sequence which terminated at the first base. The band which was second furthest through the gel must have been terminated at the second base in the sequence. Etc. By looking at which [[Dideoxyribonucleoside triphosphates|dideoxyribonucleoside triphosphate bound]] to each of these [[DNA|DNA]] molecules you can determine the base on the [[DNA template strand|template strand at]] the position it terminated because of [[Watson and Crick base pairing|complementary base pairing]].
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<br>These DNA fragments are then separated by gel electrophoresis which allows us to determine how long each fragment is in relation to how far through the gel it has travelled by performing autoradiography. The sequence is read bottom to top so the band which travelled furthest through the gel must have been the sequence which terminated at the first base. The band which was second furthest through the gel must have been terminated at the second base in the sequence.ddNTPs can be colour coded for ease of identification. (Hames and Hooper, 2000:261)<ref>Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd</ref> By looking at which ddNTP bound to each of these DNA molecules you can determine the base on the template strand at the position it terminated because of complementary base pairing.<br>
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Revision as of 13:08, 26 October 2012

The Sanger dideoxy method of DNA sequencing allows large volumes of DNA to be sequenced in vitro. This method uses a single stranded, DNA template strand, a specific primer of 18-20 bases, DNA polymerase , deoxyribonucleoside triphosphates(dNTP’s) and their respective derivatives dideoxyribonucleoside triphosphates (ddNTP’s) which differ from the dNTP’s in that instead of a 3’ OH group they have a 3’ H which is intergral to their function as chain terminators. This is because with the 3’ H prevents chain elongation as phosphodiester bonds cannot be created with the next ribonucleoside (Hames and Hooper, 2000:260)[1]


A DNA primer binds to the 5' end of the single-stranded DNA molecule to be sequenced (the template strand) to allow the DNA polymeras eenzyme to attach and synthesize a new DNA chain against the template strand through Watson-Crick complementary base pairing.
There are 4 different dideoxyribonucleoside triphosphate chain-terminating molecules (ddCTP, ddGTP, ddATP, ddTTP) each containing one of the DNA bases, so ddCTP contains the base ‘C’ (Cytosine) etc complemtary to a G on the template strand. These 4 different molecules are used in 4 separate DNA synthesis reactions on copies of the same single-stranded DNA template strand to be sequenced. When a ddNTP is incorporated into the growing newly synthesised chain, it terminates synthesis at that point so each reaction produces fragments of different lengths complementary to the template strand, which have terminated at different points. (Alberts, et al, 2008:549)[2]


These DNA fragments are then separated by gel electrophoresis which allows us to determine how long each fragment is in relation to how far through the gel it has travelled by performing autoradiography. The sequence is read bottom to top so the band which travelled furthest through the gel must have been the sequence which terminated at the first base. The band which was second furthest through the gel must have been terminated at the second base in the sequence.ddNTPs can be colour coded for ease of identification. (Hames and Hooper, 2000:261)[3] By looking at which ddNTP bound to each of these DNA molecules you can determine the base on the template strand at the position it terminated because of complementary base pairing.


References

  1. Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd
  2. Alberts, Bruce, et al, 2008, Molecular Biology of the Cell, 5th edition, New York, Garland Science
  3. Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd
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