Chemical Cleavage (Maxam and Gilbert) Method for DNA Sequence Determination

Unlike the determination of the amino acid sequence of proteins and peptides, which is based on the sequential degradation of these structures and the subsequent identification of the cleaved-off amino acid residue, DNA sequence analysis is based on the high-resolution electrophoresis on denaturing polyacrylamide gels of oligonucleotides with one common end and varying in length by a single nucleotide at the other end (Chapter 52 ). In the Maxam and Gilbert method for DNA sequencing (1 ,2 ), the four sets of oligonucleotides are obtained by treating a ³² P-end-labeled DNA fragment (Chapters under four different conditions with a reagent that modifies a particular nucleotide, followed by cleavage of the DNA molecule next to the modified nucleotide. It is for this reason that the method is also known as the partial chemical degradation method for DNA sequencing. The other, and probably most frequently used method for sequencing DNA, mainly developed by Sanger and his colleagues (3 ,4 ) is described in Chapter 53 .