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