Chapter20CarboxylicAcidsandNitriles-facultyat.ppt

* * * * * * * * * * * * * * * * * * * * * * * * * * * Why was the discovery of Taq polymerase the key to polymerase chain reaction? Taq polymerase is a faster DNA polymerase than that found in mammals. Taq polymerase has a lower error rate than other DNA polymerases. Taq polymerase needs no primer. Taq polymerase does not denature at temperatures of over 90° C, allowing for automated replication. All of these * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 28.6 DNA Sequencing The order of the bases along DNA contains the genetic inheritance. Determination of the sequence is based on chemical reactions rather than physical analysis DNA is cleaved at specific sequences by restriction endonucleases For example, the restriction enzyme AluI cleaves between G and C in the four-base sequence AG-CT Note that the sequence is identical to that of its complement, (3?)-TC-GA-(5?) Other restriction enzymes produce other cuts permitting partially overlapping sequences of small pieces to be produced for analysis * Analytical Methods The Maxam–Gilbert method uses organic chemistry to cleave phosphate linkages at with specificity for the adjoining heterocycle The Sanger dideoxy method uses enzymatic reactions The Sanger method is now widely used and automated, even in the sequencing of genomes * The Sanger Dideoxy and Nucleotides The fragment to be sequenced is combined with: A) A small piece of DNA (primer), whose sequence is complementary to that on the 3? end of the restriction fragment B) The four 2?-deoxyribonucleoside triphosphates (dNTPs) The solution also contains small amounts of the four 2?,3?-dideoxyribonucleoside triphosphates (ddNTPs) Each is modified with a different fluorescent dye molecule * Dideoxy Method - Analysis The product is a mixture of dideoxy-terminated DNA fragments with fluorescent tags These are separated according to weight by electrophoresis and identified by their specific fluorescence * 28.7 DNA Synthesis DNA synthesizers use a s