Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
Chemical Synthesis, Determination of Nucleotide Sequence, and DNA Amplification
DNA Sequencing Methods

Comprehensive information about a DNA molecule can be obtained only by determining its nucleotide sequence. Thus, by sequencing a Gene, it is often possible to determine its function by comparing its nucleotide sequence with those of genes whose Functions are already known. Without nucleotide sequence data, molecular cloning research is impossible. Sequencing of a given DNA fragment can be carried out either by the chemical method developed by A. Maxam and W. Gilbert or by the enzymatic method proposed by F. Sanger, but currently the so-called dideoxynucleotide method is the most widely used.

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The Dideoxynucleotide Method of DNA Sequencing

A dideoxynucleotide is an artificially synthesized nucleotide lacking the 2'- and 3'-hydroxyl groups at the carbon atoms of the sugar ring (Fig. 5.11, A). A normal deoxynucleotide, which is a standard component of DNA, lacks only the 2'-hydroxyl group (Fig. 5.11, B). Chain elongation during METABOLISM/36.html">DNA Replication occurs As a result of The addition of the next nucleoside triphosphate to the 3'-hydroxyl group of the last nucleotide of the growing chain (Fig. 5.12). If this next incoming unit is a dideoxynucleotide, DNA Synthesis halts because the subsequent nucleotide cannot form a phosphodiester bond (Fig. 5.13). The arrest of DNA synthesis is the key step in the dideoxy method, but to perform sequencing in full, A number of conditions must be met.

Fig. 5.11. A. Dideoxynucleotide (2'- and 3'-hydroxyl groups are absent in the ring). B. Deoxynucleotide (only the 2'-hydroxyl group is absent).

The first step of the standard dideoxy sequencing Procedure involves the Hybridization of a synthetic 17–20 nucleotide-long oligonucleotide with a specific region of one of the cloning vector strands adjacent to the insert. This oligonucleotide serves as a primer, supplying a 3'-hydroxyl group to initiate synthesis. The primer solution is distributed among four tubes, each containing four deoxynucleotides: dATP, dCTP, dGTP, and dTTP (one of which is isotopically labeled) and one of the four dideoxynucleotides (ddATP, ddCTP, ddGTP, or ddTTP). The concentration of each dideoxynucleotide is adjusted so that it becomes incorporated at all positions along the mixture of growing chains, rather than just at the first position encountered. (Recall that following the incorporation of a dideoxynucleotide, chain growth terminates immediately, so each chain ends with a 3'-dideoxynucleotide.) Upon completion of enzymatic synthesis mediated by DNA polymerase, each tube contains a unique set of oligonucleotides, each containing the primer sequence (Fig. 5.14).

Fig. 5.12. DNA synthesis under normal conditions. An incoming deoxyribonucleotide (deoxyribonucleoside triphosphate; dNTP) pairs with the complementary nucleotide of the template strand. A phosphodiester bond is formed between the 3'-hydroxyl group of the last nucleotide in the growing chain and the $\alpha$-phosphate group of the added nucleotide.

Next, formamide is added to the tubes to ensure strand Separation, and Polyacrylamide gel Electrophoresis is performed in four lanes (corresponding to the number of tubes). This allows single-stranded DNA fragments to be separated even if they differ in length by just a single nucleotide. An autoradiograph reveals a set of bands corresponding to the labeled DNA fragments, the comparison of which allows direct "reading" of The nucleotide sequence of the DNA segment being sequenced. In the example shown in Fig. 5.15, the first six NUCLEOTIDES of this segment, starting from the 5' end, are AGCTGC. The "fastest" band (the radioactively labeled fragment at the very bottom of the gel) corresponds to the shortest fragment and is located in the ddATP lane; the subsequent bands are located in the ddGTP, ddCTP, ddTTP lanes, and so on, respectively. Most autoradiographs clearly resolve between 250 and 350 bands. The primer sequence is located at a fixed distance (10–20 nucleotides) from the site where the cloned DNA is inserted, making it easy to identify THE START OF the cloned fragment.

Fig. 5.13. Arrest of DNA synthesis following the incorporation of a dideoxynucleotide at the end of the growing chain. A phosphodiester bond between the terminal dideoxynucleotide and the next nucleotide cannot be formed due to the absence of the 3'-hydroxyl group.

DNA Sequencing Using an M13 Phage-Based Vector

Various approaches are used to determine the nucleotide sequence of cloned DNA. One of the earliest was based on using the E. coli phage M13 as a vector. The DNA of this phage is a single-stranded circular molecule. When E. coli is infected, a double-stranded replicative form of the phage DNA is first produced, and single-stranded circular molecules—which are subsequently packaged into virions—are synthesized on this double-stranded molecule as a template. Cells infected with M13 do not undergo lysis; new single-stranded M13 DNA molecules are continuously formed within them, which pass through The Cell membrane, acquire a protein coat, and are released into the environment. M13 DNA contains a non-essential region that can be replaced with a desired DNA fragment while preserving the infectivity of the recombinant Viral Particles. The M13 system has the following advantages: the isolated double-stranded replicative form can function as a plasmid, whereas the single-stranded phage DNA can be used as a template for DNA sequencing.

Fig. 5.14. Primer extension during chain synthesis in the presence of dideoxynucleotides. Each of the four tubes produces a unique set of oligonucleotides of varying lengths, incorporating the primer sequence. A certain number of full-length DNA molecules are also formed in the process. dNTP — deoxyribonucleoside triphosphate

Fig. 5.15. Schematic representation of an autoradiograph obtained by dideoxy DNA sequencing. The contents of each of the four tubes containing one of the dideoxynucleotides (ddATP, ddCTP, ddGTP, or ddTTP) were loaded into separate wells. The nucleotide sequence is read from the autoradiograph from bottom to top. It is shown on the right side of the figure.

All of this makes it possible to use phage M13 as a combined system for DNA Cloning and sequencing. Typically, the desired DNA fragment of approximately 500 bp is inserted into the polylinker, which is part of the modified lacZ' gene cloned into the RF-DNA of phage M13. Competent E. coli cells are transformed with the recombinant viral DNA and plated onto Agar containing the X-Gal substrate. Its Hydrolysis by $\beta$-galactosidase yields a blue-colored product. White (colorless) and blue colonies appear on the plates. The former correspond to cells infected with M13 phage containing an insert that disrupted the reading frame of the lacZ' gene, whereas the latter correspond to cells infected with M13 phage with a functional lacZ' gene lacking an insert. Phage particles are isolated from the white colonies, and single-stranded DNA with the insert is extracted from them (Fig. 5.16). To sequence the latter, the isolated DNA is annealed with a primer that hybridizes to the sequence near the insert; then dideoxy sequencing, electrophoresis, and autoradiography are performed to "read" the nucleotide sequence of the insert. Other strategies are used to sequence large DNA fragments (approximately 2000 bp). One of them is as follows: this fragment is inserted into an appropriate plasmid vector and a detailed restriction map is constructed. Overlapping fragments of the insert ranging from 100 to 500 bp in length are identified, each is subcloned into M13 DNA, sequenced, and the nucleotide sequence of the entire original fragment is reconstructed. To ensure the accuracy of the obtained result and the correct identification of any given nucleotide, both strands must be sequenced multiple times. Sequencing of both strands is facilitated by the fact that each of the subcloned fragments of the original DNA can be inserted into M13 DNA in opposite orientations. As a result, the primer initiates the Synthesis of the first strand in one case and the second strand in the other.

Fig. 5.16. Use of bacteriophage M13 for cloning and sequencing. A. Insertion of a DNA fragment into the double-stranded replicative form of M13 DNA. B. Sequencing of complementary strands of the cloned DNA fragment using the same primer (P1). Arrows indicate the orientation of the insert in the vector.

Primer-Mediated Walking ("Primer Walking") For sequencing very long DNA fragments (>5000 bp), the approach described above can no longer be used because the number of M13 vectors containing overlapping subcloned sequences increases significantly. To solve this problem, Methods for sequencing double-stranded plasmid DNAs without The Need for subcloning were developed. The plasmid DNA containing the desired insert is isolated and annealed with a synthetic oligonucleotide primer that hybridizes to a sequence in one of the vector DNA strands located near the insert. Dideoxy sequencing is then performed to identify the first 250–350 nucleotides of the insert. Based on these data, a second oligonucleotide primer is synthesized, complementary to a segment of the insert located approximately 300 nucleotides away from the binding site of the first primer, and the next 250–350 nucleotides are sequenced. A third primer is synthesized in a similar manner, and the nucleotide sequence of the subsequent 250–350 nucleotides is determined (Fig. 5.17). This procedure, known as primer walking, is continued until the entire fragment has been sequenced. The second strand is sequenced in a similar manner, starting from a primer that hybridizes to this strand near the insert.

Unfortunately, mispairing of a primer of a given length with more than one site within the insert can yield ambiguous results. To avoid this, primers of at least 24 nucleotides in length are used, and annealing conditions are strictly maintained. It was precisely in this manner that DNA fragments cloned in bacteriophage $\lambda$ (~20 kb) or in a cosmid vector (~40 kb) were sequenced.

Some steps of this process have recently been automated, enabling routine sequencing of fragments several tens of thousands of Base Pairs in length. In many cases, primers added to the reaction mixture in different tubes are labeled with distinct fluorescent Dyes having different fluorescence emission wavelengths. The contents of all four tubes are then combined and electrophoresed in a single lane. The lane is scanned with a laser beam, and THE POSITION OF each fluorescent band is recorded. All data are fed into a computer, which compares them and displays the nucleotide sequence on the screen.

Fig. 5.17. DNA sequencing by primer walking. 1. Initiation of DNA strand synthesis using a primer (P1) complementary to a plasmid region located near the insert. 2. Sequencing of a cloned DNA fragment 250–350 nucleotides in length. 3. Selection of a second primer complementary to the terminal region of the already sequenced sequence, approximately 20 nucleotides long. 4. Sequencing of the next segment of the cloned DNA using the second primer (P2). 5. Selection of a third primer complementary to the terminal region of this 20-nucleotide segment. 6. The third primer (P3) is used to sequence the next segment of the cloned DNA. This procedure is repeated until the entire insert has been sequenced.