The main difference between Maxam Gilbert and Sanger sequencing is that the Maxam-Gilbert sequencing is the chemical method of DNA sequencing based on the nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides. But, on the other hand, the Sanger sequencing is the chain termination method, which interrupts elongation of DNA sequences by incorporating dideoxynucleotides into the sequences. Furthermore, Maxam Gilbert sequencing uses a large amount of hazardous chemical, including radioactive material and hydrazine. However, Sanger sequencing uses less hazardous chemicals.
Maxam Gilbert and Sanger sequencing are the two conventional DNA sequencing methods developed in the mid-1970s. Generally, they are responsible for determining nucleotide bases in a molecule of DNA.
Key Areas Covered
1. What is Maxam Gilbert Sequencing
– Definition, Process, Importance
2. What is Sanger Sequencing
– Definition, Process, Importance
3. What are the Similarities Between Maxam Gilbert and Sanger Sequencing
– Outline of Common Features
4. What is the Difference Between Maxam Gilbert and Sanger Sequencing
– Comparison of Key Differences
Conventional Methods, DNA Sequencing, Maxam Gilbert Sequencing, Sanger Sequencing
What is Maxam Gilbert Sequencing
Maxam Gilbert sequencing is one of the two conventional DNA sequencing methods developed by Allan Maxam and Walter Gilbert in 1977–1980. Also, it is known as the chemical method of DNA sequencing.
Basically, this method involves the terminal labeling of DNA molecules with chemical agents followed by the modification of bases of DNA in four different chemical reactions. Subsequently, DNA is cleaved in the point of attachment of modified A+ G, G, C + T, and C bases. Following, radioactive fragments are synthesized extending from the labeled end to the position of the nucleotide base. In the end, the PAGE separates the point of breaking, producing four different cleavage patterns for each of the four bases of DNA.
The steps of Maxam Gilbert Sequencing are:
- Radioactive labeling of the 5′ end by a kinase reaction using gamma-32P ATP and purification
- Four chemical Treatments for A+ G, G, C + T, C bases (Depurination of purines (A+G) by formic acid, Methylation of guanine (G) by dimethyl sulfate, Hydrolyization of pyrimidines (C+T) by hydrazine, and Inhibition of the hydrazine reaction for thymine by the addition of sodium chloride, hydrolyzing only cytosine (C)
- Cleavage of the modified DNA by hot piperidine; (CH2)5NH at the position of the modified base producing a series of labeled fragments from the radiolabeled end to the first ‘cut’ site.
- Size fractionation of the fragments on a PAGE (polyacrylamide gel electrophoresis).
- Visualization by autoradiography, inferring the sequence.
Basically, the two main important features of the Maxam Gilbert sequencing is that it is both sensitive and specific. Therefore, it can provide a good distinction between bases. Still, it takes a few days to analyze a sequence with 200-300 bases. On the other hand, it uses both radioactive elements and hydrazine, which is a neurotoxin.
What is Sanger Sequencing
Sanger sequencing is the second conventional DNA sequencing method developed by Frederick Sanger and colleagues in 1977. Significantly, it was commercialized by Applied Biosystems.
Generally, Sanger sequencing is also known as the chain termination method based on the incorporation of chain-terminating dideoxynucleotides (ddNTPs) through in vitro DNA replication by DNA polymerase. Significantly, ddNTPs lack a 3′-OH group responsible for the formation of a phosphodiester bond with the incoming nucleotide, causing DNA polymerase to cease extension of DNA with the incorporation of the modified ddNTP. Also, these ddNTPs are labeled either radioactively or fluorescently, allowing the detection of the base.
The steps of Sanger sequencing are:
- Division of the DNA sample into four separate sequencing reactions with ddATP, ddCTP, ddGTP, and ddTTP.
- Fluorescent labeling (ddATP with green dye, ddCTP with blue dye, ddGTP with yellow dye, and ddTTP with red dye)
- Carrying out separate PCR reactions with the corresponding ddNTP. Here, the dideoxynucleotide concentration should be approximately 100-fold lower than that of the corresponding deoxynucleotide.
- Heat denaturation and separation of amplicons in a denaturing polyacrylamide-urea gel. Four reactions run in one of four individual lanes (lanes A, T, G, C).
- Visualization and the determination of the DNA sequence.
Significantly, Sanger sequencing is a greatly simplified DNA sequencing method. Therefore, the advent of the method gave a boost to DNA sequencing, letting to a more rapid accumulation of sequence data for various genes and organisms. However, it does not uses many hazardous chemicals in the process. Still, the sensitivity of the Sanger sequencing method is comparatively low.
Similarities Between Maxam Gilbert and Sanger Sequencing
- Maxam Gilbert and Sanger sequencing are the two conventional methods of DNA sequencing.
- Also, they are the methods of first-generation sequencing.
- Significantly, both are time-consuming and cumbersome when compared to automated sequencing.
- Also, they allow the analysis of short DNA fragments up to 500 bases.
- However, both strands of the DNA fragment can be sequenced.
- Generally, Their principles led to the development of next-generation sequencing methods.
Difference Between Maxam Gilbert and Sanger Sequencing
Maxam Gilbert sequencing refers to the method of DNA sequencing based on nucleotide specific partial chemical modification and subsequent DNA cleavage. In contrast, Sanger sequencing refers to the process of selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
Maxam Gilbert sequencing was developed by Allan Maxam and Walter Gilbert in 1977–1980 while Sanger sequencing was developed by Frederick Sanger and colleagues in 1977.
Maxam Gilbert sequencing is the chemical method of sequencing, while Sanger sequencing is the chain-termination method.
Maxam Gilbert sequencing uses a large amount of hazardous chemical, including radioactive material and hydrazine, while Sanger sequencing uses less hazardous chemicals.
Sensitivity and Specificity
Maxam Gilbert sequencing is highly sensitive and highly specific, while Sanger sequencing is less sensitive and less specific.
Maxam Gilbert sequencing is one of the two conventional DNA sequencing methods. Generally, it uses various chemicals for the specific modification of nucleotide bases on the DNA strand. Ultimately, the cleavage of DNA at the modified sites allows the determination of bases. Significantly, this method is more sensitive and specific. However, it uses hazardous chemicals. In contrast, Sanger sequencing is the second conventional DNA sequencing method, which is widely using. Usually, it uses labeled ddNTPs to terminate the chain growth during DNA replication at each of the four nucleotides. Finally, the separation of the terminated amplicons on a gel allows the determination of the DNA sequence. However, this method is less specific and less sensitive with respect to the first method. Still, it uses less hazardous chemicals. Therefore, the main difference between Maxam Gilbert and Sanger sequencing is the method and importance.
1. “DNA Sequencing.” Integrated DNA Technologies. Available Here.
1. “Maxam-Gilbert sequencing en” By Incnis Mrsi. The original can be viewed here: Maxam-Gilbert sequencing.svg. (CC BY-SA 3.0) via Commons Wikimedia
2. “Sanger-sequencing” By Estevezj – Own work (CC BY-SA 3.0) via Commons Wikimedia