Molecular markers
DNA sequencing
Maxam-Gilbert DNA sequencing



Topic Completed: 1 February 2011

Revised: 15 February 2019

Copyright: 2008-2018, PathologyOutlines.com, Inc.

PubMed Search: Maxam-Gilbert DNA sequencing


Rodney E. Shackelford, D.O., Ph.D.
Page views in 2018: 507
Page views in 2019 to date: 318
Table of Contents
Background | Limitations | Videos
Cite this page: Shackelford R. Maxam-Gilbert DNA sequencing. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/molecularpathdnaseqmaxam.html. Accessed June 24th, 2019.
Background
  • 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
    1. Both purines (A + G)
    2. Preferentially at A (A > G)
    3. The pyrimidines (C + T)
    4. At cytosines only
  • The resulting DNA fragments are then denatured, run side by side in slab gel electrophoresis, autoradiographed and analyzed

  • Advantages:
    1. Purified DNA can be read directly
    2. Homopolymeric DNA runs are sequenced as efficiently as heterogeneous DNA sequences
    3. Can be used to analyze DNA protein interactions (i.e. footprinting)
    4. Can be used to analyze nucleic acid structure and epigenetic modifications to DNA

  • Disadvantages - this method is not commonly used today because:
    1. It requires extensive use of hazardous chemicals
    2. It has a relatively complex set up / technical complexity
    3. It is difficult to "scale up" and cannot be used to analyze more than 500 base pairs
    4. The read length decreases from incomplete cleavage reactions
    5. It is difficult to make Maxam-Gilbert sequencing based DNA kits

  • Modifications are used to analyze protein DNA interactions (Wikipedia: DNA Footprinting [Accessed 30 May 2018]) and DNA secondary structure, to locate rare bases (such as Hoogsteen base pairs, Wikipedia: Hoogsteen Base Pair [Accessed 30 May 2018]) and base modifications and to resolve ambiguities that arise in dideoxynucleotide sequencing
Limitations
  • 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
Videos

Maxam-Gilbert sequencing method

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