D - ddATP ddGTP ddCTP and ddTTP(4 The polymerase reactions...

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D Consider a segment of DNA that is about 1000 base pairs long that we wish to sequence. (1) The two DNA strands are separated. Heating to 100˚C to melt the base pairinghydrogen bonds that hold the strands together does this. (2) A short oligonucleotide (ca. 18 bases) designed to be complimentary to the end of one of the strands is allowed to anneal to the single stranded DNA. The resulting DNA hybrid looks much like the general polymerase substrate shown previously. (3) DNA polymerase is added along with the four nucleotide precursors (dATP, dGTP,dCTP, and dTTP). The mixture is then divided into four separate reactions and to each reaction a small quantity different dideoxy nucleotide precursor is added. Dideoxy nucleotide precursors are abbreviated
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Unformatted text preview: ddATP, ddGTP, ddCTP, and ddTTP. (4) The polymerase reactions are allowed to proceed and, using one of a variety ofmethods, radiolabel is incorporated into the newly synthesized DNA. (5) After the DNA polymerase reactions are complete, the samples are melted and run ona gel system that allows DNA strands of different lengths to be resolved. The DNA sequence can be read from the gel by noting the positions of the radiolabeled fragments. The crucial element of the sequencing reactions is the added dideoxynuclotides. These molecules are identical to the normal nucleotide precursors in all respects except that they lack a hydroxyl group at their 3’ position (3’ OH)....
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