DNA Sequ (1)The two DNa strands are separated. heating to 100 c to melt the base pargs ence Consider a segment of dna that is about 1000 base pairs long that we wish to seque hydrogen 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. dc TP, 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 ddATP, ddG TP, ddCTP, and ddTTP (4)The polymerase reactions are allowed to proceed and, using one of a variety of methods, radiolabel is incorporated into the newly synthesized dNA (5) After the DNa polymerase reactions are complete, the samples are melted and run on a 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) HO-P -o-C OH OH O dNTP ddNTP Thus dideoxynuclotides can be incorporated into DNA, but once a dideoxynuclotide has been incorporated further elongation stops because the resulting DNA will no longer have a free 3 OH end. Each of the four reactions contains one of the dideoxynuclotides added at about 1 7 the concentration of the normal nucleotide precursors. Thus, for example, in the reaction with added ddAtP about 1 of the elongated chains will terminate at the position of each A in the sequence. Once all of the elongating chains have been terminated there will be a population of labeled chains that have terminated at the position of each a in the sequenceDNA Sequencing 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 pairing hydrogen 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 ddATP, ddGTP, ddCTP, and ddTTP. (4) The polymerase reactions are allowed to proceed and, using one of a variety of methods, radiolabel is incorporated into the newly synthesized DNA. (5) After the DNA polymerase reactions are complete, the samples are melted and run on a 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). Thus dideoxynuclotides can be incorporated into DNA, but once a dideoxynuclotide has been incorporated further elongation stops because the resulting DNA will no longer have a free 3’ OH end. Each of the four reactions contains one of the dideoxynuclotides added at about 1% the concentration of the normal nucleotide precursors. Thus, for example, in the reaction with added ddATP about 1% of the elongated chains will terminate at the position of each A in the sequence. Once all of the elongating chains have been terminated there will be a population of labeled chains that have terminated at the position of each A in the sequence