DNA Sequencing Mimi Chen & Joanne Kim

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