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Sanger Dideoxy Method Developed by Fredrick Sanger Utilizes DNA replication Uses dideoxyribonucleoside triphosphates (ddNTP) The Sanger Dideoxy Method was developed by Fredrick Sanger who won the 1980 nobel prize in chemistry for it. The method utilizes DNA replication to determine the sequence of the DNA strand. http://www.nlm.nih.gov/visibleproofs/media/detailed/vi_a_208b.jpg
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DNA SequencingDNA SequencingMimi Chen & Joanne KimMimi Chen & Joanne Kim
http://www.science-scholars.com/ibexampreparation.htm
Sanger Dideoxy MethodSanger Dideoxy Method
• Developed by Fredrick Developed by Fredrick SangerSanger
• Utilizes DNA replicationUtilizes DNA replication• Uses Uses
dideoxyribonucleoside dideoxyribonucleoside triphosphates (ddNTP)triphosphates (ddNTP)
http://www.nlm.nih.gov/visibleproofs/media/detailed/vi_a_208b.jpg
Dideoxyribonucleoside TriphosphatesDideoxyribonucleoside Triphosphates
http://www.uic.edu/classes/bios/bios100/lectures/ddNTP.jpg
DNA PreparationDNA Preparation
• Single strandedSingle stranded• PrimerPrimer• Reaction TubeReaction Tube
http://8e.devbio.com/images/ch04/0405fig6.jpg
Theory
• [ddNTP] < [dNTP]– DNA polymerase can’t
differentiate• Different lengths of DNA
strands
http://core.iddrc.org/molecular-genetics/?page_id=28
Analysis• Polyacrylamide gel
electrophoresis• 4 separate lanes of gel• Gel against x-ray film
exposed by radioactive decay
• Nucleotide sequence read by autoradiogram
http://www.sciencephoto.com/images/download_lo_res.html?id=712101064
Human Genome Project
• Different fluorescent dye colours• 1 reaction mixture • Polyacrylamide gel electrophoresis• Gel capillary tube• Electopherogram
http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
http://www.genelink.com/images/Seqfull.jpg