Maxam-Gilbert DNA Sequencing
Author: Rodney E. Shackelford, D.O., Ph.D., Tulane University (see Reviewers page)
Revised: 14 February 2011, last major update February 2011
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.
● In 1976-77, Allan Maxam and Walter Gilbert developed a gel-based DNA sequencing method, also called "chemical sequencing", that used 4 base-specific chemical cleavage reactions to cut 32P-end labeled double-stranded DNA fragments (Proc Natl Acad Sci USA 1977;74:560)
● The base-specific cuts occur at a small proportion of either (a) both purines (A+G), (b) preferentially at A (A>G), (c) the pyrimidines (C+T), or (d) at cytosines only
● The resulting DNA fragments are then denatured, run side-by-side in slab gel electrophoresis, autoradiographed and analyzed
● Advantages: (a) purified DNA can be read directly, (b) homopolymeric DNA runs are sequenced as efficiently as heterogeneous DNA sequences, (c) can be used to analyze DNA-protein interactions (i.e., footprinting), (d) can be used to analyze nucleic acid structure and epigenetic modifications to DNA
● Disadvantages: This method is not commonly used today because: (a) it requires extensive use of hazardous chemicals, (b) it has a relatively complex set-up/technical complexity, (c) it is difficult to "scale-up", and cannot be used to analyze more than 500 base pairs (d) the read-length decreases from incomplete cleavage reactions, and (e) it is difficult to make Maxam-Gilbert sequencing based DNA kits
● Modifications are used to analyze protein-DNA interactions (footprinting) and DNA secondary structure, to locate rare bases (such as Hoogsteen base pairs) and base modifications, and to resolve ambiguities that arise in dideoxynucleotide sequencing
● Initially, the useful DNA sequencing read-length from a gel was about 100 bp
● This was significantly increased by using 35S labeled DNA and by using gels with narrower lanes, gel gradient systems, gel-to-plate binders and temperature control systems to reduce band distortions
● However, despite all improvements, these limitations remain:
● (1) Gel electrophoresis is limited to 700-900 bp, with 400-500 bp more commonly attained
● (2) The first 15-40 bp are often difficult to interpret (Nucleic Acids Res 2007;35:6227)
● (3) Sequencing techniques based on slab gel electrophoresis require cumbersome gels, buffers, time spent loading and running the gels, autoradiography and analysis; all lower the amount of DNA that can be sequenced
● To overcome the limitations of slab gel electrophoresis and the manual reading of DNA sequences, other innovations were introduced
End of Molecular Pathology > DNA sequencing > Maxam-Gilbert DNA Sequencing
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