The Principle of DNA Sequencing

The purpose of sequencing is to determine the order of the nucleotides of a gene. For sequencing, we don't start from gDNA (like in PCR) but mostly from PCR fragments or cloned genes.

1. The sequencing reaction :
There are three major steps in a sequencing reaction (like in PCR), which are repeated for 30 or 40 cycles.


1. Denaturation at 94°C :

During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).


2. Annealing at 50°C :

In sequencing reactions, only one primer is used, so there is only one strand copied (in PCR : two primers are used, so two strands are copied). The primer is jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.


3. extension at 60°C :

This is the ideal working temperature for the polymerase (normally it is 72 °C, but because it has to incorporate ddNTP's which are chemically modified with a fluorescent label, the temperature is lowered so it has time to incorporate the 'strange' molecules. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, come loose again and don't give an extension of the fragment.

The bases (complementary to the template) are coupled to the primer on the 3'side (adding dNTP's or ddNTP's from 5' to 3', reading from the template from 3' to 5' side, bases are added complementary to the template).

When a ddNTP is incorporated, the extension reaction stops because a ddNTP contains a H-atom on the 3rd carbon atom (dNTP's contain a OH-atom on that position). Since the ddNTP's are fluorescently labeled, it is possible to detect the color of the last base of this fragment on an automated sequencer.

Figure 7 : The different steps in sequencing. (pdf file of this picture)Animated picture of sequencing (344 kB) Because only one primer is used, only one strand is copied during sequencing, there is a linear increase of the number of copies of one strand of the gene. Therefore, there has to be a large amount of copies of the gene in the starting mixture for sequencing. Suppose there are 1000 copies of the wanted gene before the cycling starts, after one cycle, there will be 2000 copies : the 1000 original templates and 1000 complementary strands with each one fluorescent label on the last base, after two cycles, there will be 2000 complementary strands, three cycles will result in 3000 complementary strands and so on.

Figure 8 : The linear amplification of the gene in sequencing.
1. Separation of the molecules :
After the sequencing reactions, the mixture of strands, all of different length and all ending on a fluorescently labelled ddNTP have to be separated; This is done on an acrylamide gel, which is capable of separating a molecule of 30 bases from one of 31 bases, but also a molecule of 750 bases from one of 751 bases. All this is done with gel electrophoresis. DNA has a negative charge and migrates to the positive side. Smaller fragments migrate faster, so the DNA molecules are separated on their size.

Figure 9 : The separation of the molecules with electrophoresis.(pdf file of this picture)Animated picture of gel electrophoresis (159 kB)


1. Detection on an automated sequencer :
The fluorescently labelled fragments that migrate trough the gel, are passing a laser beam at the bottom of the gel. The laser exites the fluorescent molecule, which sends out light of a distinct color. That light is collected and focused by lenses into a spectrograph. Based on the wavelength, the spectrograph separates the light across a CCD camera (charge coupled device). Each base has its own color, so the sequencer can detect the order of the bases in the sequenced gene.

Figure 10 : The scanning and detection system on the ABI Prism 377 sequencer. (pdf file of this picture)Animated picture of scanning and detection system (182 kB)

Figure 11 : A snapshot of the detection of the molecules on the sequencer.

1. Assembling of the sequenced parts of a gene :
For publication purposes, each sequence of a gene has to be confirmed in both directions. To accomplish this, the gene has to be sequenced with forward and reverse primers. Since it is only possible to sequence a part of 750 till 800 bases in one run, a gene of, for example 1800 bases, has to be sequenced with internal primers. When all these fragments are sequenced, a computer program tries to fit the different parts together and assembles the total gene sequence.

Figure 12 : The assemblage of the gene.

More information in power point and pdf format.

Inventor of a DNA Sequencing Technique Is Disputed

A small biotechnology company has emerged to claim that it invented a seminal technique crucial to biotechnology research. And the government says it will consider, nearly a quarter-century after the invention was made, whether it awarded the patent to the wrong party.

The United States Patent and Trademark Office has started a proceeding to determine the rightful inventor of the technique, automated DNA sequencing: scientists at the California Institute of Technology, who hold the patent, or those at Enzo Biochem, the small company.

If Enzo were to win the patent rights it could mean significant revenue for the company and could hurt Applied Biosystems, which licenses the patent in question from Caltech and dominates the DNA sequencing business. Applied Biosystems machines were the main ones used in the Human Genome Project to determine man’s genetic blueprint.

Applied Biosystems, a unit of Applera, recorded $540 million in sales of DNA sequencing machines and chemicals in the fiscal year ended June 30, accounting for 29 percent of its revenue. Caltech is estimated to have earned tens of millions of dollars from that and related patents.

Executives at Enzo, which is based in New York, say the company filed for a patent in June 1982, a few months before the Caltech scientists said they conceived of their invention. But Enzo’s application was continually rejected, delayed and amended in the patent office and remained unknown until now.

“We had to fight and fight with them,” said Eugene C. Rzucidlo, a New York patent lawyer who represents Enzo. “It’s only now that the patent office granted our claims and set up a proceeding to find who the real first inventor is.”

Patent law was changed in the 1990s to eliminate such “submarine patents,” which can lead to infringement by companies that develop a product on their own, never knowing about a patent that suddenly surfaces. But Enzo’s application is old enough to fall under the old rules.

If it were to get a patent it would last for 17 years from the date it is issued. Caltech’s patent was issued in 1998 and expires in 2015.

Spokeswomen for Caltech and Applied Biosystems said their organizations did not know enough yet to comment. But Edward R. Reines, a Silicon Valley patent lawyer who has represented Applied Biosystems, accused Enzo of trying to mine the patent system for money.

“Enzo appears to be attempting to claim credit for the invention of modern DNA sequencing 25 years after the fact when they have not brought a meaningful DNA sequencing product to market,” said Mr. Reines, who is with Weil, Gotshal & Manges.

Enzo executives disputed that, saying the company, founded in 1976, had long sold reagents for use in genetic analysis, though not sequencing machines. Enzo reported a net loss of $15.7 million in its last fiscal year on revenue of $39.8 million.

The Caltech sequencer attached a different color of fluorescent dye to each of the four chemical units of DNA, allowing the DNA sequence to be read by a machine. It made sequencing faster and set the stage for the Human Genome Project. The inventors include Leroy E. Hood, a biologist, and Michael W. Hunkapiller, who later ran Applied Biosystems.

The Caltech patents have withstood challenges before. A whistleblower lawsuit filed by a competitor of Applied Biosystems, said federal funds had been used in the invention, entitling the government to certain discounts and other rights. The government declined to pursue the lawsuit. And a former Caltech scientist filed a lawsuit claiming to be one of the inventors but lost in court.

R. Danny Huntington, a Washington lawyer who specializes in interference proceedings, said there were only about 100 such cases a year and they could take one or two years to resolve. The party with the earlier patent application date wins about two times out of three, he said.

While that would seem to favor Enzo, the patent office declaration of the interference criticizes Enzo’s patent claims for being unusually numerous, “erratically numbered” and “extraordinary in their flagrant disregard” of certain rules. The application is still not public, but lawyers said it contained about 1,200 claims.