31-03-2012, 12:17 PM
integrated DNA technology
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Introduction
Prior to the mid-1970’s no method existed by which DNA could be directly sequenced. Knowledge about gene and genome organization was based upon studies of prokaryotic organisms and the primary means of obtaining DNA sequence was so-called reverse genetics in which the amino acid sequence of the gene product of interest is back-translated into a nucleotide sequence based upon the appropriate codons. Given the degeneracy of the genetic code, this process can be tricky at best. In the mid-1970’s two methods were developed for directly sequencing DNA. These were the Maxam-Gilbert chemical cleavage method and the Sanger chain-termination method.
Maxam-Gilbert
Allan Maxam and Walter Gilbert developed a method for sequencing single-stranded DNA by taking advantage of a two-step catalytic process involving piperidine and two chemicals that selectively attack purines and pyrimidines [1]. Purines will react with dimethyl sulfate and pyrimidines will react with hydrazine in such a way as to break the glycoside bond between the ribose sugar and the base displacing the base (Step 1).
Sanger
At about the same time as Maxam-Gilbert DNA sequencing was being developed; Fred Sanger was developing an alternative method. Rather than using chemical cleavage reactions, Sanger opted for a method involving a third form of the ribose sugars. As shown in Figure 3, Ribose has a hydroxyl group on both the 2’ and the 3’ carbons whereas deoxyribose has only the one hydroxyl group on the 3’ carbon.