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Western University
Biochemistry 2280A
Eric Ball

Brandl – Lecture 7 Notes DNA sequencing: o The genetic blueprint, like the story in a book, is almost fully contained within the sequence of the letters o Definitive characterization of a DNA requires determination of its sequence o The sequence identifies all potential disease causing genes and drug targets o Two methods developed in the early 1970s to sequence DNA o Chemical sequencing (Maxam and Gilbert) o Dideoxy or chain termination sequencing (Sanger) Chain termination sequencing:  Procedure relies on the enzymatic extension of a primer using the DNA of interest as the complementary template – done in the presence of base specific chain terminators  A primer (a short oligonucleotide complimentary to the DNA of interest) is annealed to the template DNA  The normal substrate for DNA synthesis have the OH at the 3 position – that is the position that gets extended in the polymerization reaction – normal site for the next base to be added – 2’ deoxynucleotide  The key to sequencing is the 2’ 3’ dideoxynucleoside triphosphate – lacks the 3’ hydroxyl – it cannot be extended in a polymerization reaction so they act as chain terminators in a polymerization reaction  A series of 4 extension reactions are setup – each of these contains one chain terminating 2,3 dideoxynucleotide triphosphate: ddATP, ddGTP, ddCTP, or ddTTP. o The ddNTP is present at a ratio relative to its counterpart dNTP (e.g. ddATP/ dATP) such that random incorporation of the ddNTP will occur ~ once per every 500bp  The 4 reactions are separated by denaturing polyacylamide gel electrophoresis and examined by autoradiography  A similar strategy is used for automated sequencing with the exception that each of the 4 ddNTPs is tagged with a different fluorescent label, which allows its unique detection Synthetic Oligonucleotides:  Many applications of recombinant DNA technology require the use of synthetically made single stranded DNAs o Short fragments (~25 bases) of single stranded DNA with defined sequence can be made synthetically (often referred to as primers or oligos) o These uses include - hybridization probes, primers for DNA sequencing, PCR and mutagenesis  The technology available today allows the automated synthesis of single stranded DNAs of several hundred bases Materials Required To Sequence A DNA:  DNA to be sequenced (template)  Oligonucleotide primer than anneals to the template  DNA polymerase – will extend that primer exactly like replication  The dNTPs (G, A, T, C) – same as a regular replication reaction  The ddNTPs (small amount, 0.2% of dNTP) Extension Rx in the Presence of ddATP:  Materials: normal deoxyribonucleoside triphosphate precursors (dATP, DCTP, dGTP, and dTTP), oligonucleotide primer for DNA polymerase, single stranded DNA molecule to be sequenced, small amount of ddATP  If ddATP comes in at position, that chain will stop o At each base where a dA should be inserted into the growing chain, there is a 1/500 chance a ddA will be inserted and the chain terminated  A collection of DNA molecules is made that all have the same 5’ end bu
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