Sanger “dideoxy” method: Difference between revisions
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The Sanger dideoxy method of [[ | The Sanger dideoxy method of [[DNA|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|DNA polymerase]], [[Deoxyribonucleoside triphosphates|deoxyribonucleoside triphosphates]] ([[DNTP|dNTP]]’s) and their respective derivatives [[Dideoxyribonucleoside triphosphates|dideoxyribonucleoside triphosphates]] ([[DdNTP|ddNTP]]’s) which differ from the dNTP’s in that instead of a 3’ OH group they have a 3’ H which is integral to their function as chain terminators. This is because with the 3’ H prevents chain elongation as [[Phosphodiester bond|phosphodiester bonds]] cannot be created with the next ribonucleoside<ref>Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd</ref>. | ||
A DNA primer binds to the 5' end of the single-stranded DNA molecule to be sequenced (the template strand) to allow the DNA polymerase enzyme 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|ddCTP]], [[DdGTP|ddGTP]], [[DdATP|ddATP]], [[DdTTP|ddTTP]]) each containing one of the DNA bases, so ddCTP contains the base ‘C’ ([[Cytosine|Cytosine]]) etc, complementary to a [[Guanine|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<ref>Alberts, Bruce, et al, 2008, Molecular Biology of the Cell, 5th edition, New York, Garland Science</ref>. | |||
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|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<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. | |||
=== References === | |||
<references /> | |||
Latest revision as of 11:58, 5 December 2017
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 integral 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[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 polymerase enzyme 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, complementary 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[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[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
- ↑ Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd
- ↑ Alberts, Bruce, et al, 2008, Molecular Biology of the Cell, 5th edition, New York, Garland Science
- ↑ Hames,B.D, Hooper,N.M, 2000,Instant notes Biochemistry, 2nd edition, New York, BIOS Scientific Publishers Ltd