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FIGURE 24.07 Separation and Detection of Fragments on Gel during DNA Sequencing

(A) The products of the four separate sequencing reactions are run side by side on a polyacrylamide gel and the fragments of different sizes are separated by electrophoresis. (B) To detect the fragments, the gel is transferred to a piece of paper to give it strength, and dried so that the polyacrylamide does not stick to the film. After the gel is completely dry, a piece of photographic film is placed over the gel. The positions of the radioactive DNA fragments are revealed by the dark bands they produce on the film.

DNA fragments are separated according to size on a polyacrylamide gel.

B) Detection of bands by autoradiography

Film on gel

Film

Film

Film shows position of bands

Lay film on gel and keep in dark, then develop film

Film shows position of bands reaction, a mixture of dGTP and ddGTP are available for DNA polymerase. Sometimes DNA polymerase uses dG and sometimes ddG. The amount of ddG relative to dG is adjusted to yield a mixture of chains that are terminated by ddG at all positions where there is a G in the sequence.The reaction to generate the "G" lane on a sequencing gel contains normal deoxynucleotide triphosphates for the other three bases but both dG and ddG for guanosine. The other three sequencing reactions are set up similarly. In practice, the template, primers, radioactive dNTPs for all four bases, and DNA polymerase are all mixed together. This mixture is then distributed among four tubes, each with a different dideoxynucleotide.

This mixture of fragments is separated according to their sizes by gel electrophoresis (Fig. 24.07A). Since the largest DNA sub-fragments generated by sequencing reactions are usually only 200-300 base pairs in length, the fragments are too small to be resolved by the large pores of agarose and must be separated using a polyacrylamide gel. The shortest pieces are closer to the bottom, as they move fastest during electrophoresis. These short pieces are generated when the ddNTP is incorporated shortly after DNA polymerase begins synthesis at the primer. A series of bands is produced, each corre

DNA fragments were originally detected by radioactive labeling, although nowadays fluorescent dyes are normally used as labels.

sponding to a piece of DNA of a particular length. The lengths reveal the positions of the G bases in the original DNA. To completely sequence DNA, the same thing is done simultaneously for all four bases, A, G, T, and C. So there are four reaction mixtures, each containing a specific ratio of normal deoxynucleotides to dideoxynucleotides. Each reaction contains a series of artificially terminated chains ending in one of the four bases. All four samples are loaded side by side onto a gel. Separation by electrophoresis gives four ladders representing each of the bases (Fig. 24.07A).

Some way is needed to detect the DNA bands. Originally, radioactive nucleotide precursors or radioactively labeled primers were incorporated into the sub-fragments generated by the polymerase. After separating the sub-fragments by electrophoresis, the polyacrylamide gel is dried and a sheet of photographic film is laid on top of the gel. The radioactive bands leave a black mark on the film, thus allowing the researcher to visualize the position of the original bands (Fig. 24.07B). As before, the position of each band corresponds to a chain of DNA of a particular length and reveals the position of one base. The sequence is read off directly from the bottom, combining results from all four bases. Several hundred bases of sequence can usually be obtained from one gel. Part of a real sequencing gel is shown in Figure 24.08. Instead of radioactivity, modern DNA sequencing techniques use fluorescently labeled nucleotide precursors or primers for detection (see below).

Genetically engineered DNA polymerase with properties suitable for use in test tubes rather than cells is now used for DNA sequencing.

DNA Polymerases for Sequencing DNA

All DNA polymerases can elongate a primer that is annealed to a single-stranded DNA template. However, the characteristics needed for use in sequencing are more rigorous. First, the polymerase must have high processivity, that is, it must move a long way along the DNA before dissociating. Premature dissociation would give strands that ended at random before the dideoxynucleotide was incorporated. In addition, many DNA polymerases possess exonuclease activities (see Ch. 5). 5' to 3' exonucle-ase activity may be used to remove a strand of DNA ahead of the replication point. In contrast, 3' to 5' exonuclease activity is used to remove incorrect bases during proofreading. Such activities interfere with accurate sequencing as they might shorten the length of strands already synthesized.

In practice, no natural DNA polymerase is entirely suitable for sequencing. The first DNA polymerase used was Klenow polymerase, which is DNA polymerase I from E. coli that lacks the 5' to 3' exonuclease domain. Klenow polymerase was originally obtained by protease digestion of purified DNA polymerase I but was later made by expression of a modified gene. Because Klenow polymerase has relatively low pro-cessivity, it can only be used to sequence around 250 bases per reaction. More recently, genetically modified DNA polymerase from bacteriophage T7 has been used. This is marketed as "Sequenase" and has high processivity, a rapid reaction rate, negligible exonuclease activity and the ability to use many modified nucleotides as substrates, thus making it perfect for sequencing reactions.

Producing Template DNA for Sequencing

High quality DNA sequencing requires purified single-stranded DNA to which the sequencing primer can bind. Originally, template DNA was from the bacterial virus M13 that was engineered to contain the template sequences (Fig. 24.09). M13 virus is rod-shaped and contains a circle of single stranded DNA (ssDNA). Upon infecting an E. coli cell, the single-stranded viral DNA is converted to a double-stranded form, the replicative form (RF). After replicating itself for a while, the RF then turns its efforts

Klenow polymerase DNA polymerase I from E. coli that lacks the 5' to 3' exonuclease domain

M13 Rod-shaped bacteriophage that infects E. coli, contains a circle of single stranded DNA, and is used to manufacture DNA for sequencing replicative form (RF) Double-stranded form of the genome of a single-stranded DNA (or RNA) virus. The RF first replicates itself and is then used to generate the ssDNA (or ssRNA) to pack into the virus particles Sequenase® Genetically modified DNA polymerase from bacteriophage T7 used for sequencing DNA

Producing Template DNA for Sequencing 669

FIGURE 24.08 Autoradiograph of Real Sequencing Gel

The sequencing of two different DNA templates is shown. The two sequences each consist of four lanes that represent the four different bases. The sequence is read from the bottom of the gel toward the top. This gel was run by Kiswar Alam in the Author's laboratory.

Producing Template DNA for Sequencing 669

FIGURE 24.09 Single-Stranded DNA from Bacteriophage M13

When M13 infects E. coli, the single-stranded viral DNA is converted to a double-stranded replicative form (RF). This RF then replicates so making many double-stranded copies. After the double-stranded form becomes abundant, large numbers of single-stranded copies are made. These are eventually packaged into viral particles that are secreted into the culture medium.

FIGURE 24.09 Single-Stranded DNA from Bacteriophage M13

When M13 infects E. coli, the single-stranded viral DNA is converted to a double-stranded replicative form (RF). This RF then replicates so making many double-stranded copies. After the double-stranded form becomes abundant, large numbers of single-stranded copies are made. These are eventually packaged into viral particles that are secreted into the culture medium.

Single stranded DNA for use as a template can be generated by taking advantage of bacteriophage M13, which naturally contains ssDNA.

to manufacturing large numbers of single-stranded circles of DNA to pack into newly made virus particles.

Not only does M13 generate single-stranded DNA but it also purifies it. Unlike most viruses, M13 doesn't destroy the bacterial cells. Instead, the cells continuously secrete virus particles containing ssDNA into the surrounding medium. In addition, since the viral DNA does not integrate into the bacterial chromosome, only viral DNA gets packaged into the particles. Since the viral particles are secreted, they are easily isolated from the bacterial cells, and the DNA they contain can be extracted.

The template DNA that has unknown sequence is first cloned into the double-stranded replicative form of M13. Normally, an M13 vector that has already been engineered to contain a convenient multiple cloning site is used (Fig. 24.10). This multiple cloning site is contained within the N-terminal fragment of the lacZ gene of E. coli, which allows the use of blue/white screening to monitor insertion of the template DNA into the M13 vector (see Ch. 22 for details). Furthermore, the sequence to the side of the inserted DNA is already known and provides a starting point. This is essential, as the primer for sequencing must be complementary to a known sequence on the template strand in order to hybridize in the correct position. This engineered virus is used to infect E. coli, and virus particles containing single strands are manufactured in large quantities. Nowadays, bacterial plasmids containing the M13 origin of replication are used to manufacture single-stranded DNA. The use of intact virus is avoided and improved yields of DNA can be obtained more conveniently.

DNA to sequence

DNA to sequence

FIGURE 24.10 Sequencing Using M13-Based Vectors

The use of M13 vectors allows the easy production of single-stranded template DNA. The DNA to be sequenced is inserted into the multiple cloning site (MCS) within the M13 vector. The MCS is located within the alpha fragment of the lacZ gene. When no insert is present functional p-galactosidase is made, which turns the E. coli host blue in the presence of X-gal. When an insert disrupts lacZ, no functional p-galactosidase is made and the cells stay white. This allows simple identification of cells carrying M13 vectors that have received DNA inserts. Sequencing is carried out using primers corresponding to M13 sequences just outside the cloned DNA.

A variety of technical improvements have made DNA sequencing a little less tedious. Using double-stranded DNA (dsDNA) directly for sequencing is more convenient than generating single strands. In reality, "double stranded" DNA sequencing involves a preliminary step, either heat or alkali treatment, to denature the dsDNA into single strands. Therefore, the actual sequencing reactions use single stranded DNA just as described above.

PCR products can be used for In fact, it is now possible to completely avoid cloning DNA into either M13 or a seqiiendng cifter- sepcirating plasmid vector by using PCR to generate segments of DNA (see Ch. 23). PCR prod-

into single strands. ucts are linear double-stranded lengths of DNA and they can be directly sequenced after separation into single strands. As noted in Chapter 23, one drawback of PCR is that we need to know enough sequence on each side of the target DNA to construct primers for PCR. Hence, we cannot always avoid cloning.

Primer Walking along a Strand of DNA

Sequencing moderately long pieces of DNA was originally done by cutting the DNA into smaller segments with restriction enzymes and then sub-cloning each fragment separately into M13 or another vector. Nowadays, this has largely been replaced by primer walking (Fig. 24.11). This involves first sequencing the cloned DNA as far as possible using the primer belonging to the M13 or plasmid vector. Next, the newly obtained sequence information is used to design another primer. Sequencing is continued as far as this allows. Then another primer is made, and another, and so on until the end of the cloned DNA is reached.

Automated Sequencing

Today, the majority of sequencing is done using automated techniques. The main modification here is to use fluorescent dyes to label the DNA instead of radioactivity. Each of the four sequencing reactions is done just as before, but the DNA is labeled by primer walking Approach to sequencing a long cloned DNA molecule by using successive primers located at stages along the molecule

Automated Sequencing 671

Bind primer

Sequence

Bind primer

Bind primer

Sequence

Sequence

Sequence

Sequence

Sequence

FIGURE 24.11 Primer Walking along a DNA Molecule

FIGURE 24.11 Primer Walking along a DNA Molecule

When the DNA to be sequenced is too long to be sequenced by a single reaction, primer walking is used. First (A), the cloned DNA is sequenced as far as possible starting from a primer binding site within the vector. The sequence information obtained allows a second primer to be made that lies close to the far end of the known sequence. A second sequencing reaction with this primer provides a further stretch of sequence (B). This process is continued, using as many primers as necessary to cross the inserted DNA. Eventually, the sequence obtained corresponds to the vector (C). This tells the experimenter that the unknown DNA has been completely crossed and is now fully sequenced.

Automated sequencing relies on using four fluorescent dyes of different colors, one for each base. This allows all fragments to be run in a single gel lane, where they are scanned by a laser.

attaching a fluorescent dye to the primer before running the reactions. Although the same DNA primer is used for each of the four reactions, four fluorescent dyes of different colors are needed. The first color is used when carrying out the A-reaction, another one for G, another for T, and the fourth for C. When the sequencing gel is run, bands of four different colors are seen, a separate color for each base. In fact, since the bases are color coded, all four completed reactions can be run in the same track on the sequencing gel, as shown in Figure 24.12.

Rather than running the gel for a fixed period and then examining the bands afterwards, the gel is monitored while running. As the bands move down the gel they pass a laser and detector assembly. The laser beam scans the bands and the four different dyes fluoresce in different colors. A computer records the color of each band, and compiles the data into actual sequence. The first bands to be recorded will have run right through the gel and off the end while later bands are still passing the laser. Consequently, more bases can be read from a single sequencing reaction by the continuous flow approach. Automated sequencers have been improved by using capillary separation. This improves speed but more importantly has allowed the assembly of machines that may have as many as 96 sequencing reactions running simultaneously.

FIGURE 24.12 Automated Fluorescent DNA Sequencing

Automated sequencing uses four different fluorescent dyes, one for each of the four bases. All four reactions are run in a single lane of the gel since the four bases are easily distinguished by their colors.

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