DNA SEQUENCING: MAXAM-GILBERT, METHOD AND EXAMPLES - BIOLOGY - 2024

The DNA sequencing (deoxyribonucleic acid) is a procedure in molecular biology laboratories that allows to know the order of nucleotides in the genetic material of interest. Furthermore, the sequencing of RNA (ribonucleic acid) can also be disclosed.

This technique has been indispensable for the development of biological sciences. It is also applicable to other fields of knowledge - such as medical diagnosis and forensic investigations, for example.

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Previously, the sequencing of a DNA strand was considered a slow and expensive activity, which allowed the identification of only a few base pairs in the oligonucleotides.

Today, with all the advances in science, DNA sequencing is a routine operation in many laboratories worldwide thanks to the contribution of almost 50 years of research in this field. In terms of chain length, up to millions of base pairs can be sequenced in a very short time.

To do this, there are dozens of techniques developed that vary in price and precision. In this article, we will describe both classical and modern techniques, each with its advantages and disadvantages.

Until now, sequencing techniques allow obtaining the sequence of complete genomes, from small prokaryotes and yeasts to the human genome.

DNA structure

To understand the methods and techniques used for DNA sequencing, it is necessary to know certain key aspects of the structure and composition of the molecule.

DNA is a biomolecule found in all living things, from bacteria to large aquatic animals. Organelles - like mitochondria and chloroplasts - have a circular DNA molecule inside them. Even in some viruses, the genetic material found is DNA.

Structurally, DNA is a collection of nucleotides. Each one is made up of a carbohydrate, a nitrogenous base (A, T, C or G) and a phosphate group. The goal of DNA sequencing is to reveal the order in which the four nitrogenous bases are found in the sequence.

History

In the mid-1950s, researchers Watson and Crick described the structure of DNA using christolographic techniques. However, none of these researchers had been able to find a way to unravel the sequence.

Although there were certain predecessors, the most important event was the creation of the Sanger method, in 1977. Frederick Sanger, the father of the method, was a British biochemist, winner of two Nobel prizes for his enormous contributions to the biological sciences.

This technique is also known in the literature as "chain termination" or dideoxynucleotides. The principles of this technique and those that were developed based on its improvement and innovation will be described below.

Sanger method

The development of the Sanger method represented a crucial event in molecular biology. It involves the basic components of the DNA replication process that normally occurs in the cell, but adding a special component: dideoxynucleotides.

Main components of the reaction

- DNA polymerase: the DNA polymerase enzyme is a crucial element of the process. This molecule participates in the replication of the DNA strand and its role is the synthesis of the new strand, pairing the triphosphate deoxyribonucleotides with the complementary ones.

Recall that in DNA, thymines (T) pair with adenines (A) through two hydrogen bonds, while cytosine (C) does so with guanine (G) through three bonds.

- Nucleotides: Sanger sequencing involves two types of nucleotides, the four 2'-deoxynucleotides (abbreviated as dATP, dGTP, dCTP and dTTP) and the four dideoxynucleotides (ddATP, ddGTP, ddCTP and ddTTP).

Although dideoxynucleotides are similar to the monomers that are normally incorporated into DNA, they lack an -OH group in their structure. This makes it impossible to add a new nucleotide to the chain.

Therefore, when a special nucleotide is added - in a totally random way - to the chain in formation, the synthesis is paralyzed. Thus, at the end of the reaction, there are chains of different sizes, each one where the reaction was stopped at a different point.

Experimentally, four tests are prepared. Each contains the DNA extracted from the biological sample of interest, the normal nucleotides, and one of the four special nucleotide types. Either the special nucleotides are marked with some type of fluorescent marker (see automated sequencing below).

Reading the results

The first step is to separate each of the synthesized chains according to their size. Some will be longer than others, depending on where the special bases were incorporated.

There are different biochemical techniques that allow the separation of the components of a mixture using size as a discriminatory property. In Sanger's method, the different chains are separated by electrophoresis. In the more sophisticated variants of the technique, capillary electrophoresis is used.

Thus, the longer strands travel less than the shorter variants. This system then goes through a reader that recognizes the marker included in each dideoxynucleotide. In this way, the order of the sequence can be known.

This "first generation" technique is capable of reading DNA fragments no larger than 1 kilobase. At present, the Sanger method is used in various laboratories, generally in its modern variants. In addition, it is used to corroborate the results obtained with the most complex techniques - but less precise.

Automatic sequencing

When sequencing is required on a large scale, the process is accelerated through automation. This is a variation of the Sanger chain termination method, where the primers are labeled with fluorescent products in order to distinguish them.

Subsequently, the reaction product is run in an electrophoresis - all on a single lane. As each fragment exits the final portion of the gel, it is quickly identified by its fluorescent labeling, with an error of around 1%.

The most sophisticated systems have a system of up to 96 capillary tubes managed by a computer coupled to a robot. That is, 96 DNA samples can be tested simultaneously. Thus, the process involving electrophoresis and analysis of the results is fully automated.

In one day, these systems can sequence up to 550,000 bases. During the process, human labor is unnecessary, it only takes about 15 minutes to start the method.

Maxam-Gilbert sequencing

At the same time that Sanger published his work, two researchers named Allan Maxan and Walter Gilbert succeeded in developing another method to obtain the DNA sequence. The method gained popularity at the time, but was later displaced by the improvement of Sanger's method.

Contrary to the Sanger method, Maxan and Gilbert sequencing (or chemical sequencing, as it is also known) does not involve hybridization reactions. The methodology consists of labeling with reactive agents at one end, followed by a purification process.

One of the negative aspects of this technique lies in its enormous complexity and in the use of chemicals that are dangerous for the user. Chemical breaks are induced by the application of DMS, formic acid, hydrazine, and hydrazine with salts.

Process

The protocol begins with the labeling at the 5 'end of the strand with the phosphorus marker 32, then a chemical modification of the nitrogen base occurs and it is separated. Finally, the cleavage of the abasic region occurs.

First you shorten the string you want to sequence into smaller segments. This step is performed with restriction enzymes, which result in protruding ends.

Next, the reaction is carried out with an alkaline phosphatase, the purpose of which is to eliminate the phosphate group. Thus, a polynucleotide kinase can be used to perform the labeling.

The chain is denatured (the two strands open). Then the chemicals are applied. These cleavage reactions are done in a controlled manner and it is known what types of bonds each applied chemical breaks.

Reading the results

As in the Sanger method, the reading of the results involves the separation by size of the chains obtained in an electrophoresis system. Systems composed of polyacrylamide allow a very adequate resolution to be obtained for reading the gel.

Mass sequencing

The massive sequencing encompasses a series of novel methods, abbreviated as NGS, from the English “Next Generation Sequencing”.

The methods classified as NGS require a previous DNA amplification step (they do not work with a single molecule). Furthermore, the platforms used vary widely. The principles of the most popular methods will be described below:

Pyrosequencing

It involves monitoring the release of a pyrophosphate, which occurs each time a new nucleotide is added to the DNA strand. A system of enzymes is coupled, so that the emission of light (which is detectable by a camera) occurs each time a new nucleotide is incorporated.

The process begins with the separate incubation of each nitrogen base to verify whether or not there is light emission. Pyrosequencing can read long strands, but the error rate found is high.

Synthesis sequencing

This involves the incorporation of labeled nucleotides. These fluorescent components are added, washed, and the incorporated nucleotide is noted. Then, the nucleotide label is removed, and the synthesis of the strand can continue. In the next step, a labeled nucleotide will also be incorporated, and the aforementioned steps will be repeated.

A drawback to this technique occurs when the fluorescent markers are not completely removed. These emissions create background errors, resulting in significant errors.

Ligation sequencing

This technique differs from the others, as it does not use DNA polymerase. Instead, the key enzyme for this methodology is ligase. Here, fluorescently labeled DNA fragments are used, it is linked by the enzyme and it is detected.

The biggest problem with this technique is the short fragment length it is capable of processing.

Ion Torrent Sequencing

This technique is based on the measurement of the H ⁺ ion that is released each time a new nucleotide is incorporated. The principle is quite similar to pyrosequencing, but much cheaper.

Examples

The sequencing of the human genome

Sequencing the human genome has been one of the most promising challenges in biology, as well as being one of the most acclaimed rivalries in the history of science. In fact, for the scientists involved in the project, sequencing the genome became a competition.

In 1990 he started what was called the "human genome project", led by the famous scientist, Nobel Prize winner, James Watson. After a year, in 1991, Venter takes on the challenge of "beating" Watson and sequencing the genome before him. However, in 1992, Watson retired and the command was taken by another researcher.

In 1995 Venter announced his success in the complete sequencing of a bacterial genome by the random sequencing method. Similarly, the opposing team announced a year later the sequencing of the yeast genome.

In 2000, the degree was terminated. Both companies published their preliminary whole genome results in two of science's most prestigious journals: Nature and Science.

However, scientists continued to work on improving the proposals, and in 2006 the sequences of certain human chromosomes were completed.

Importance and applications

Knowing the order of the nucleotides of such an important molecule as DNA is valuable to biologists and related professionals. This chain of polynucleotides contains all the information necessary for the development and maintenance of all forms of life.

For these reasons, knowledge of this sequence is essential for biological research. Fundamentally, sequencing makes it possible to measure one of the most important properties of biological systems and to establish differences between them.

Sequencing is widely used by taxonomists and systematists, since certain DNA sequences allow establishing criteria to conclude whether or not two organisms belong to the same species, in addition to being able to propose hypotheses about the phylogenetic relationships between them.

Additionally, DNA sequencing has applications in medicine and diagnostics. For example, there are inexpensive and accessible systems that by means of sequencing allow evaluating the tendency to develop certain diseases (such as cancer) using the so-called single nucleotide polymorphisms (SNPs).

Investigations of the criminal and forensic type have also been enriched with sequencing techniques, which can be used as reliable evidence of the participation of a certain individual in a crime.

References

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