A gene is a segment of DNA that carries the information needed by the cell to construct a protein. Which protein that is, when it is made, and how damage to it can give rise to genetic disease all depend on the gene's sequence. In other words, they depend on how the building blocks of DNA, the nucleotides A, C, G, and T (adenine, cytosine, guanine, and thymine) are ordered along the DNA strand. For example, part of a gene may contain the base sequence TGGCAC, while part of another gene may contain the base sequence TCACGG. Knowing a gene's base sequence can lead to isolation of its protein product, show how individuals are related, or point the way to a cure for those people carrying it in its damaged form.
In 1977 two methods for sequencing DNA were introduced. One method, referred to as Maxam-Gilbert sequencing, after the two scientists at Harvard University who developed the technique, uses different chemicals to break radioactively labeled DNA at specific base positions. The other approach, developed by Frederick Sanger in England and called the chain termination method (also called the Sanger method), uses a DNA synthesis reaction with special forms of the four nucleotides that, when added to a DNA chain, stop (terminate) further chain growth.
By either method, a collection of single-stranded DNA fragments is produced, each fragment one base longer than the next. The length of a fragment depends on where a chemical cleaved the strand (in Maxam-Gilbert sequencing) or where a special terminator base was added (in the chain termination method). The fragments are then separated according to their size by a process called gel electrophoresis, in which the fragments are drawn through a gel material by electric current, with shorter fragments migrating through the gel faster than longer fragments. The DNA sequence is then "read" by noting which reaction produced which fragment.
All DNA sequencing protocols must incorporate a method for making the DNA fragments generated in the reaction "visible"; they must be capable of being detected. For Maxam-Gilbert sequencing a technique called end labeling is used, in which a radioactive atom is added to the ends of the DNA fragment being sequenced. The first step in this process is to use an enzyme , called a restriction endonuclease, to cut the DNA at a specific sequence. If the restriction endonuclease is Hind III, for example, the sequence AAGCTT will be cut.
The ends of the DNA segment made by the restriction endonuclease will have phosphate groups (-PO32-) at its ends. An enzyme called a phosphatase is used to remove the phosphate group. Another enzyme, called a kinase, is then used to add a radioactive phosphate in its place. This reaction will add radioactive atoms onto both ends of the DNA restriction fragment. A second restriction endonuclease is used to make a cut within the end-labeled fragment and gel electrophoresis is then employed to separate the two resulting subfragments from each other. Each subfragment now has one labeled and one unlabeled end. The subfragment whose sequence is to be determined is cut out of the gel to purify it away from the other end-labeled subfragment.
The end-labeled piece of DNA is then divided and the fragments are placed in four separate tubes. They are then treated with different chemicals that weaken and break the bond holding the base to the backbone of the DNA molecule. These chemicals are base specific. In other words, one chemical causes the "C" reaction, in which the bond holding the C base in position is broken. Another chemical breaks the bond holding the G in place (the "G" reaction). Another breaks both G and A bases from the DNA backbone (the "G+A" reaction), and a fourth breaks the bonds holding the C and T bases in place (the "C+T" reaction).
Each reaction is limited so that each DNA molecule will have only one of its base positions altered. Within the "C" reaction, for example, each DNA molecule will have only one of its C bases weakened and removed. One of the DNA molecules may have the C base closest to the end dislodged. On another DNA molecule, a C that appears 500 bases away from the end may be removed. Every C position in the population of molecules, however, is subject to treatment.
When the first step in the reaction is concluded, another reagent is then used to completely break the DNA strands at the points where the bases have been removed. In this way, a collection of DNA fragments is generated that differ in size according to the position along the strand where the break occurred.
Each reaction is electrophoresed in its own lane on a gel that separates the DNA fragments by their length. A process called autoradiography is then used to detect the separated fragments. In this technique, X-ray sensitive film is placed flat against the gel under conditions of complete darkness. Since the fragments made in the sequencing reactions are end-labeled with radioactive atoms, their emissions will expose the X-ray film at the positions where they are found on the gel. When the X-ray film is developed, bands, like a bar code, reveal the sizes of the fragments generated in each separate reaction.
Each band that has been rendered visible on the X-ray film differs from the one above or the one below it by a single base. The DNA sequence is then read from the bottom of the gel upward. A band found in the "G" reaction lane is read as a G, a band found in the "C" lane is read as a C, a band found in the lane from the "G+A" reaction but with no corresponding band of the same length in the "G" lane is read as an A, and a band in the "C+T" reaction without a corresponding base in the "C" lane is read as a T (see Figure 1).
Chain Termination Method
Because it employs fewer steps and does not require the use of restriction enzymes , the chain termination method of DNA sequencing is used in more laboratories than is the Maxam-Gilbert approach. Chain termination sequencing is a clever variation of the reaction used to replicate DNA, and requires only a handful of components. Four reaction tubes, designated "A," "C," "G," and "T," are prepared and the DNA strand whose sequence is to be determined is added into each tube. This DNA strand is called the template.
Along with the template, a short, single-stranded piece of DNA (called a primer) is added; it attaches specifically to one section of the template and serves as a starting point for synthesis of a new DNA strand. Also added are the four "building block" nucleotides, a buffer (to maintain the proper pH level), the enzyme DNA polymerase, and its cofactor magnesium, all of which are needed to extend the primer into a full-length DNA chain.
To make the DNA strands visible, a nucleotide carrying a radioactive phosphorous or sulfur atom is included in the reaction. Also included in each reaction tube is a quantity of a unique dideoxynucleotide, a modified form of a nucleotide that lacks the site at which other bases can attach during chain growth. Thus, when this nucleotide is added to the DNA chain, all further chain growth is terminated. In the "A" tube, a dideoxynucleotide form of the A base, dideoxyadenosine triphosphate, is added. In the "C" tube, dideoxycytidine triphosphate is added. Dideoxyguanosine triphosphate is added to the "G" tube, and dideoxythymidine triphosphate to the "T" tube.
When the reactions are incubated at a temperature suitable for the DNA polymerase, nucleotides that are complementary to the bases on the template are added onto the end of the attached primer. The bases A and T are complementary, as are G and C. Thus, if a T base is on the template strand, DNA polymerase will add the complementary base, A, at the corresponding position in the new, extending strand. If, by chance, the DNA polymerase adds a dideoxynucleotide, chain growth is terminated at that point.
Since dideoxynucleotides are randomly incorporated, each reaction generates a mixture of DNA molecules of variable length, but all are terminated by a dideoxynucleotide. Careful adjustment of reactant concentration will give a set of DNA molecules that terminate at each of the possible positions. The "A" tube, for instance, will contain a mixture of DNA chains, each of which ends in a different "A."
As in Maxam-Gilbert sequencing, each reaction is loaded in its own gel lane and electrophoresal autoradiography is then used to detect the fragments. The base sequence is read directly off the X-ray film from the bottom of the gel upward, noting the lane in which each band appears (see Figure 2). The bands at the bottom of the gel represent the shortest fragments and resulted from termination events closest to the primer. Bands toward the top of the gel represent longer fragments made by termination events farthest from the primer.
Automated Sequencing with Fluorescent Dyes
As originally developed, both the Maxam-Gilbert and the chain termination methods of DNA sequencing require the use of radioisotopes to visualize the fragments generated in the reactions. However, in addition to being a health risk and presenting a disposal problem, the use of radioactivity makes automation of the DNA sequencing process difficult. Machines that could automatically read DNA sequences did not become practical until fluorescent dyes were introduced as a way to label sequencing fragments.
Two approaches are used to fluorescently label the products of a DNA sequencing reaction: the dye primer method and the dye terminator method. Both are applied only to the chain termination method. The sequencing products made by these methods are electrophoresed on an instrument that uses a laser to detect the different fragments.
In the dye primer method, fluorescent dyes are attached to the 5′ ("five prime") end of the primer, which is the end opposite to that where nucleotides are added during chain growth. Four reactions are prepared containing all the components described above, but with no radioisotopes. In the "A" reaction, the primer carries a dye at one end that fluoresces green when struck with a laser. In the "C" reaction, the primer carries a dye that fluoresces blue. The "G" reaction's primer has a yellow dye on its end and the "T" reaction's primer carries a red dye.
Termination events in the "A" reaction tube result in fragments with a green dye at one end and a dideoxyadenosine at the other. Termination events in the "C" reaction result in the formation of fragments having a blue dye at one end and a dideoxycytidine at the other. Similar relationships hold in the "G" and "T" reactions. When incubation is complete, the four reactions are combined and electrophoresed in a single gel lane of an automated sequencer. A laser, shining at the bottom of the gel, excites the dyes on the DNA fragments, causing them to fluoresce as they pass. The instrument's optics system detects the fluorescent colors during electrophoresis and a computer then translates the order of colors into a base sequence (see Figure 3).
In the second approach to fluorescent DNA sequencing, the dye terminator method, dyes are attached to the dideoxynucleotides instead of to the primers. The DNA fragment in the sequencing reaction becomes dye-labeled when a dideoxynucleotide is incorporated. The dye terminator method uses a single reaction tube (rather than four) because each dideoxynucleotide is associated with a different dye. The cost and time for sequencing are therefore reduced, making this approach the preferred method used by most laboratories.
Manufacturers of DNA sequencing reagents now provide kits that contain all the components necessary for a sequencing reaction in a "master mix" format. For the dye terminator approach, a master mix can be purchased that contains DNA polymerase, the four nucleotides, buffer, magnesium, and the four dye-labeled dideoxynucleotides. The addition of template and primer to the master mix completes the reaction.
In addition to the widespread use of sequencing kits, other improvements have been made. Enhanced signal strength and improved sensitivity have been achieved through the development of stronger fluorescent dyes and the exploitation of heat-stable DNA polymerases that allow for repeated cycling of the sequencing reaction.
see also Automated Sequencer; Cycle Sequencing; DNA; DNA Polymerases; Gel Electrophoresis; Human Genome Project; Nucleotide; Restriction Enzymes.
Frank H. Stephenson
and Maria Cristina Abilock
Sanger, Frederick, S. Nicklen, and Alan R. Coulson. "DNA Sequencing with Chain-terminating Inhibitors." Proceedings of the National Academy of Sciences 74 (1977): 5463-5467.