- Maxam–Gilbert sequencing, also known as chemical sequencing, is one of the first DNA sequencing methods ever developed. Introduced in 1977 by Allan Maxam and Walter Gilbert, this method provided a chemical approach to determining the nucleotide sequence of DNA and served as a foundational tool in the early years of molecular biology.
- Though it was eventually overshadowed by the more practical and safer Sanger sequencing method, Maxam–Gilbert sequencing played a crucial historical role in the emergence of genomics.
- This technique is based on the selective chemical cleavage of DNA at specific nucleotide bases. The process begins with the radioactive labeling of one end of a DNA fragment, typically using phosphorus-32. The labeled DNA is then split into four separate reactions, each containing a different chemical mixture that modifies specific bases: guanine (G), adenine and guanine (A+G), cytosine (C), or cytosine and thymine (C+T). After base-specific modification, the DNA backbone is cleaved at the site of the modified base using a strong alkali such as piperidine. The result is a set of DNA fragments of varying lengths, each ending at a known base.
- These fragments are then separated by high-resolution polyacrylamide gel electrophoresis. Because the DNA was labeled only at one end, the position of each band on the gel directly reflects the distance from the labeled end to the cleavage site. By comparing the banding patterns across the four lanes (one for each chemical reaction), the DNA sequence can be deduced manually from bottom to top, reading one base at a time.
- Maxam–Gilbert sequencing has several advantages, particularly in its early years. It does not require DNA polymerase or primers, making it useful for sequencing double-stranded DNA or fragments with unknown ends. It was especially useful in the initial stages of sequencing small viral genomes or plasmids, and for studying sequences with complex secondary structures where enzymatic approaches might be hindered.
- However, the method has significant drawbacks that led to its decline. It involves the use of hazardous chemicals, including hydrazine, dimethyl sulfate, and concentrated alkalis, which pose risks to laboratory personnel and require strict safety precautions. The procedure is also technically complex and labor-intensive, particularly because of the need for radioactive materials and the manual interpretation of sequencing gels. Moreover, the method is difficult to scale and automate, making it unsuitable for high-throughput sequencing projects.
- With the advent of Sanger sequencing, which is safer, more accurate, and easier to automate, Maxam–Gilbert sequencing quickly fell out of favor. Today, it is rarely used in routine molecular biology work, although it remains a part of the historical development of DNA sequencing technology. It provided critical proof-of-concept that DNA could be sequenced reliably and served as an important stepping stone toward the development of modern, high-throughput sequencing techniques.
