DNA Sequencing Mimi Chen & Joanne Kim http://www.science-scholars.com/ibexampreparation.htm DNA Sequencing Mimi Chen & Joanne Kim

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

Dideoxyribonucleoside Triphosphates The short form for dideoxyribonucleoside triphosphates is ddNTP. In the textbook dideoxyribonucleoside triphosphates are referred to as didexoy analogues, basically it is a nucleotide triphosphate who’s sugar doesn’t have a hydroxyl group of the 2’ and 3’ carbon. It is not able to bond to the next nucleotide, because the next nucleotide would need that hydroxyl group on Carbon 3’ to create a phosphodiester bond with it http://www.uic.edu/classes/bios/bios100/lectures/ddNTP.jpg

DNA Preparation Single stranded Primer Reaction Tube first the DNA is treated so it becomes a single stranded radioactively labeled primer is added to one end copies of the DNA template is added to four reaction tubes, each tube contains DNA polymerase All four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP & dTTP) one type of dideoxyribonucleoside triphosphate (ddATP, ddGTP, ddCTP, or ddTTP) http://8e.devbio.com/images/ch04/0405fig6.jpg

Theory [ddNTP] < [dNTP] Different lengths of DNA strands DNA polymerase can’t differentiate Different lengths of DNA strands dideoxy analogues make up a small part of the reaction tube compared to normal nucleotides so different lengths of the complementary stand will be build before analogue comes in the dideoxy analogue stops growth of the chain thus different lengths of the complementary DNA strand will be build 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 since they are different lengths polyacrylamide gel electrophoresis can be used to separate them after replication is done, contents of four reaction tubes are loaded onto four separate lanes of a gel since fragments differ by one base pair each, sequence can be read right off the gel in ascending order gel is placed against x-ray film radioactive decay expose x-ray film thus sequence of nucleotides is read http://www.sciencephoto.com/images/download_lo_res.html?id=712101064

Human Genome Project Different fluorescent dye colours http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml Human Genome Project Different fluorescent dye colours 1 reaction mixture Polyacrylamide gel electrophoresis Gel capillary tube Electopherogram Human Genome Project used similar technique each of the 4 ddNTPs have a different fluorescent dye colour only one reaction mixture with many copies of the DNA to be sequenced Strands separated by polyacrylamide gel electrophoresis, to the accuracy of 1 nucleotide These DNA chains are put through a capillary tube containing gel, where the shorter ones fall faster At the bottom of the capillary, a laser beam initiates the dye, producing a colour, which is inputted into the computer by a photocell These are recorded on the computer in a graph called electopherogram http://www.genelink.com/images/Seqfull.jpg