The Forgotten DNA Sequencing Method

A Tale Of Two Methods

In 1977, Allan Maxam and Walter Gilbert developed a technique of DNA sequencing radically different from the methods seen today. While most modern-day DNA sequencing techniques rely on enzymes and other biological reagents such as DNA polymerase and primers, their sequencing method relied on an assortment of inorganic chemicals that modified the DNA in precise ways.

This new form of sequencing, called Maxam-Gilbert or chemical degradation sequencing would become popular, winning the principal investigator, Walter Gilbert, a Nobel Prize in 1980 along with Frederick Sanger and Paul Berg.

It is a shame that a Nobel Prize can only be shared between three people, which is why Allan Maxam had to miss out.

However, as the recipients of the 1980 Nobel Prize hinted, there was another sequencing method developed in the same year: Sanger sequencing, and while you probably haven't ever heard of Maxam-Gilbert sequencing, Sanger sequencing is a household name (among molecular biologists).

Why is this? What caused Maxam-Gilbert sequencing to fade into the history books while Sanger sequencing dominates many applications today?

What is Maxam-Gilbert Sequencing?

Maxam-Gilbert sequencing is simple compared to modern day sequencing methods. It involves relatively few steps and does not require expensive equipment. Put simply, it involves cutting up the DNA with chemical reagents and then using gel electrophoresis to see where each reagent cut. This gives us the information needed to determine the DNA sequence.

Step 1:
Use phosphate-32 to radiolabel the 5' end of each DNA strand. By doing this, we can visualize the DNA strands we want with autoradiography. By doing this first instead of later, we avoid labeling strands that we do not want to see.

Step 2:
Denature the DNA. To do this step, dimethyl sulfoxide is often used at 90 degrees Celsius. This can often be done with a hot water bath and careful temperature control.

Step 3:
Place the single stranded DNA into four tubes containing reagents. Each of these reagents modifies the DNA at specific sites. The concentrations of reagents are carefully calculated so that each strand has, on average, one cleavage.

Formic Acid: Modifies (A+G) purines by depurinating them (removing the nitrogenous base)
Dimethyl Sulfate: Methylates Guanines and some adenosines
Hydrazine: Hydrolyzes pyrimidines (C+T)
Hydrazine + NaCl: Only hydrolyzes Cytosine

Then we add piperidine to cleave the DNA at the mutated sites.

Step 4:
Now we use gel electrophoresis to sequence these strands. However, we cannot use the traditional agarose gel electrophoresis to differentiate these fragments. Some of the fragments differ by as little as 1 bp, and that difference is important to capture. So, instead, we use polyacrylamide gel electrophoresis or PAGE.

PAGE is more dangerous than traditional gel electrophoresis as polyacrylamide is considered toxic compared to the harmless agarose. However, it has a much higher resolution thanks to the thinness of the gel and the presence of urea which keeps the DNA single-stranded throughout the gel.

Step 5:
Analyze the results. Thanks to the radiolabeling of the DNA fragments, only fragments that begin with the original start of the sequence are actually visible. This simplifies the process of analysis since we know the lines at the bottom correspond to the first base and up. By looking at which bands are present (and which aren't), we can deduce the sequence of DNA bases.

Disadvantages of Maxam-Gilbert Sequencing

Maxam-Gilbert sequencing was great for early microbiologists that did not have the easy access to enzymes that we enjoy today. However, these inorganic compounds do not come without drawbacks of their own. Who could have guessed that chemicals that break down DNA could be harmful to humans?

Hydrazine is a neurotoxin that can cause organ damage
Dimethyl Sulfate is a corrosive carcinogen
Piperidine is toxic to the skin and can cause extreme burns
Phosphate-32 is an acute beta-emitter with a short half-life of only 14 days

Given the option between working with harmless biological reagents or hospitalizing inorganic ones, I think it is easy to imagine what biologists prefer.

Another drawback of chemical degradation sequencing is that it is slow. It can take days to sequence just a couple hundred bases. The simplicity of interpreting the gel was also its flaw. The fact that it required the human eye to deduce the results made it hard to automate. Sanger sequencing, on the other hand, identified each base using colored flashes, which could be read by sensors.

Lacking the scale of NGS methods or the ease of use and safety of Sanger methods, Maxam-Gilbert sequencing was left without a niche of its own. Once the cost-efficiency and accessibility of other methods increased, the intrinsic disadvantages of chemical degradation rendered it obsolete.

Conclusion

Maxam-Gilbert sequencing exposes the constantly changing nature of scientific methodologies. While a few, such as PCR, have solidified themselves as titans, unlikely to disappear anytime soon, many are just a couple years away from being rendered obsolete.

This is obviously a good thing, if a method is replaced, it is because a more suitable alternative came about. However, it is also important to remember the methods that have been forgotten to time. Sometimes their quirks are useful to new tasks.

Maxam-Gilbert sequencing is used to do DNA foot-printing, for instance. This is involves identifying DNA-protein interactions at specific loci. When proteins bind to a locus, they protect the DNA fragment from the chemical reagents involved in Maxam-Gilbert sequencing. When the resulting fragments are run through a gel, scientists can use this fact to identify where the protein was bound.

Put simply, methods come and go, it is how science works. Despite this, holding on to the memory of certain forgotten methods can prove useful in the future!

Sources

Yay sources

The evolution of next-generation sequencing technologies - PMC
Maxam–Gilbert Sequencing: Easy Explanation & 3 Modern Uses
Polyacrylamide Gel Electrophoresis (PAGE): Principle and Procedure | Microbe Online
Maxam–Gilbert Sequencing - Laboratory Notes
Sanger vs Maxam Gilbert Sequencing- A Technical Comparison – Genetic Education