Maxam-Gilbert sequencing

Maxam-Gilbert sequencing determines a DNA nucleotide sequence by base-selective chemical cleavage of end-labelled molecules, followed by polyacrylamide gel electrophoresis and autoradiography. Allan M. Maxam and Walter Gilbert's 1977 paper uses fragment lengths to locate attacked bases along the labelled strand. Sanger sequencing instead creates endpoints during DNA synthesis; the chemical method starts with the strand whose sequence is wanted.^[ref]

The label is crucial: each visible fragment shares one identifiable end, making its length a positional ruler. The paper allows either a 5′ or a 3′ terminal label and demonstrates separately resolved, 5′-labelled strands. Chemical modification must be partial, so that many molecules retain different cleavage positions. Destroying every susceptible site on every molecule would erase much of the distant sequence from the labelled fragments.^[ref]

The original four displays preferentially reveal guanine, preferentially reveal adenine, reveal cytosine and thymine together, and reveal cytosine alone. The purine displays use unequal reactivity rather than four perfectly exclusive reactions. Comparing parallel lanes supplies redundant information: a signal in the cytosine/thymine display but not the cytosine display identifies thymine. A sequence of four interior cleavage positions assigned A, C, T and G in order of increasing distance from a 5′ label identifies $5^{\prime }\text{-ACTG-}{3}^{\prime }$ at those positions. The nucleotide sequence of the opposite strand requires the reverse complement and its orientation.

A subtle experimental point prevents an off-by-one interpretation: the observed fragment ends immediately before the base destroyed by the chemical attack. The base assigned to a band is the attacked base, not an intact last base carried by that fragment. The 1977 example used a 64-base-pair lac operon fragment and denaturing gels; the authors read 62 bases of each strand and cross-checked the complementary sequences.^[ref]

An original Poisson distribution model illustrates why partial cleavage matters. If the number of attacks on a molecule has mean $\lambda$, thenAt $\lambda =0.1$, about $90.48\%$ are unhit, $9.05\%$ singly hit and $0.47\%$ multiply hit. At $\lambda =1$, the multiply-hit fraction rises to $26.42\%$. This independent-site model is an illustration, not a fitted description of the paper's sequence-dependent reaction rates. Gel resolution, incomplete specificity and multiple hits can all limit the recovered sequencing read.