Maxam-Gilbert method

Maxam-Gilbert sequencing, also known as chemical sequencing, is a method for determining the sequence of nucleotides in a piece of DNA. Developed in 1977 by Allan Maxam and Walter Gilbert, this technique was among the first methods to be used for DNA sequencing. Despite being largely supplanted by the more efficient and less hazardous Sanger sequencing method and subsequent next-generation sequencing technologies, Maxam-Gilbert sequencing played a crucial role in the early days of molecular biology and genetic research.

Overview

Maxam-Gilbert sequencing involves the partial chemical modification of nucleotides in a DNA molecule, followed by cleavage at specific bases. The method requires radioactive labeling at one 5' end of the DNA, chemical treatment to modify and cleave specific bases, electrophoresis on a polyacrylamide gel to separate the resulting fragments, and autoradiography to visualize the pattern of DNA fragments. The sequence of the DNA can be inferred by analyzing the pattern of fragments generated from reactions targeting each of the four nucleotides.

Procedure

The Maxam-Gilbert sequencing process can be broken down into several key steps:

1. Labeling: The DNA fragment to be sequenced is labeled at one end with a radioactive phosphorus isotope (^32P). 2. Chemical Modification: The labeled DNA is treated with chemicals that modify specific bases. These chemicals include dimethyl sulfate (for purine modification), hydrazine (for pyrimidine modification), and others for specific base modifications. 3. Cleavage: The modified DNA is then subjected to conditions that cleave the DNA at sites adjacent to the modified bases. 4. Electrophoresis: The resulting DNA fragments are separated by size using denaturing polyacrylamide gel electrophoresis. 5. Autoradiography: The gel is then exposed to X-ray film for autoradiography, which visualizes the radioactive DNA fragments.

Advantages and Disadvantages

The Maxam-Gilbert method offers several advantages, including the ability to sequence very small amounts of DNA and the flexibility to sequence specific regions of DNA without the need for cloning. However, it also has significant disadvantages, such as the use of hazardous chemicals, the requirement for radioactive labeling, and being more labor-intensive and error-prone compared to other sequencing methods.

Decline in Use

With the advent of Sanger sequencing in the late 1970s, which is simpler and safer, the use of Maxam-Gilbert sequencing has declined significantly. The development of next-generation sequencing technologies has further reduced the relevance of this method in modern genetic research.

Legacy

Despite its decline in use, the Maxam-Gilbert method was instrumental in the early development of molecular biology and genetics. It was used in some of the first genome sequencing projects and contributed to our understanding of DNA structure and function.

Maxam-Gilbert method Resources
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