DNA_sequencing - The labeled DNA strands are separated on...

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Fundamentals of Biochemistry DNA Sequencing
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DNA synthesis proceeds from 5’ to 3’
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DNA synthesis--detail N N N N NH 2 O H O H H H H P O O O - N NH 2 O N O H O H H H H NH O O N O H OH H H H H O P O O - O N N N N NH 2 O H O H H H H P O O O O - N NH 2 O N O H OH H H H H O
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What if……the 3’ were not there? N N N N NH 2 O H H H H H H O P - O O - O N N N N NH 2 O H OH H H H H O P - O O - O vs
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The DNA chain would terminate if a 2’,3’ dideoxy nucleotide were incorporated into the DNA
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DNA polymerase requires a primer that is base-paired to the DNA strand to be copied, and this is the starting point for DNA synthesis primer new DNA strand DNA polymerase
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By adding ddNTP to the polymerase mixture, some of the new DNA strands will be terminated at each “N” base on the DNA For example, using ddTTP: One reaction is run for each of the 4 bases. Radioactive ATP is added to label all of the strands.
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Unformatted text preview: The labeled DNA strands are separated on an acrylamide gel. In this method, 4 lanes are required for each DNA strand being sequenced. For one lane with ddCTP: Overall: A G C T A G C T What does a sequencing gel look like? Each set of sequencing reactions can only generate 50-200 bases depending. So how to sequence longer stretches of DNA? The DNA is cut up into smaller pieces which are sequenced and then analyzed for overlaps: The method has become more automated to allow for more rapid sequencing of longer DNA strands, including fluorescent dyes in the ddNTPs What does the data look like? More data:...
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