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Lecture11_NABPty2012 (1)

3 3 c t a g c g t c 5 cruciform structure a c g c t a

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Unformatted text preview: same in either direction (“Radar,” “Madam, I’m Adam”). DNA base sequences can be determined by using DNA polymerase and dideoxynucleotides Synthesize DNA enzymatically in the presence of a small amount of a labeled dideoxynucleotide (e.g., ddTTP) and larger amounts of ordinary ATP, GTP, CTP and TTP, using a primer and one strand of the DNA to be sequenced as a template. DNA polymerase synthesizes the new strand in the 5’ → 3’ direction. The dideoxynucleotide interrupts DNA synthesis wherever it is incorporated. This happens at random points where the corresponding base occurs in the sequence. In this illustration, each DNA fragment ends in ddT, which is labeled with a red dye. O O O O P O P O P O-CH2 5’ primer 3’ synthesis GCCGCATGTCGTAACTT CGGCGTACAGCATTGAA 3’ template 5’ O NH O O O ddTTP (continued …) N O O DNA sequencing can be automated Repeat the procedure with ddC, ddA and ddG, using a different fluorescent dye to tag the dideoxynucleotide in each case. Then mix all the DNA fragments and separate them according to size by electrophoresis. Electrophoresis of the DNA fragments gives a “ladder” of bands, each ending in a particular dideoxynucleotide at its 3’ end and tagged with the corresponding dye. This procedure has been automated in machines that can sequence more than 3x106...
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