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CHAIVER 1 DNA Sequencing Hugh G. Grifin and Annette M. Grifin 1. Introduction Methods to determine the sequenceo f DNA were developed in the late 1970s (1,2) and have revolutionized the science of molecular genetics. The DNA sequences of many different genes from diverse sources have been determined, and the information is stored in interna- tional databanks such as EMBL, GenBank, and DDBJ. Many scientists now accept that sequence analysis will provide an increasingly use- ful approach to the characterization of biological systems. Projects are already underway to map and sequencet he entire genome of organisms such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, and Homo sapiens. In the recent past, large-scale sequencing projects such as these were often dismissed as prohibitively expensive and of little short-term benefit, while DNA sequencing itself was seen as a repetitive and unintellectual pursuit. However, this view is now changing and most scientists recognize the importance of DNA sequence data and perceive DNA sequencing as a valuable and often indispensable aspect of their work. Recent technological advances, especially in the area of automated sequencing, have removed much of the drudgery that used to be associated with the technique, and modern innovative computer software has greatly simplified the analysis and manipulation of sequence data. Large-scale sequencing From Methods m Molecular Biology, Vol. 23. DNA Sequencmg Protocols Edited by. H. and A. Gnffm Copyright Q1993 Humana Press Inc., Totowa, NJ 1 2 Griffin and Griffin projects, such as the Human Genome Project, produce the DNA sequences of many unknown genes. Such data provide an impetus for molecular biologists to apply the techniques of reverse genetics to produce probes and antibodies that can be used to identify the gene product, its cellular location, and its time of appearancei n the developing cell (3). A function can be assigned by mutant analysis or by comparison of the deduced amino acid sequence with proteins of known function. Therefore, DNA sequencing can act as a catalyst to stimulate future research into many diverse areas of science. The two original methods of DNA sequencingd escribed in 1977 (1,2) differ considerably in principle. The enzymatic (or dideoxy chain termination) method of Sanger (I) involves the synthesis of a DNA strand from a single-stranded template by a DNA polymerase. The Maxam and Gilbert (or chemical degradation) method (2) involves chemical degradation of the original DNA. Both methods produce populations of radioactively labeled polynucleotides that begin from a fixed point and terminate at points dependent on the location of a particular base in the original DNA strand. The polynucleotides are separated by polyacrylamide gel electrophoresis, and the order of nucleotides in the original DNA can be read directly from an autoradiograph of the gel (4). Although both techniques are still used today, there have been many changes and improvements to the original methods. While the chemical degradation method is still in use, the enzymatic chain termination method is by far the most popular and widely used technique for sequence determination. This process has been automated by utilizing fluorescent labeling instead of radioactive labeling (Chapters 33-37), and the concepts of polymerase chain reaction (PCR) technology have been harnessed to enable the sequencing reaction to be “cycled” (Chap- ters 21, 26, and 34). Other recent innovations include multiplexing (Chapter 28), sequencing by chemiluminescence rather than radioac- tivity (Chapter 29), solid phases equencing (Chapter 25), and the use of robotic work stations to automate sample preparation and sequencing reactions (Chapter 38). 2. Maxam and Gilbert Method In the original Maxam and Gilbert method (2) a fragment of DNA is radiolabeled at one end and then partially cleaved in four different chemical reactions, each of which is specific for a particular base or DNA Sequencing 3 type of base. This results in four populations of labeled polynucleotides. Each radiolabeled molecule extends from a fixed point (the radiolabeled end) to the site of chemical cleavage, which is determined by the DNA sequence of the original fragment. Since the cleavage is only partial, each population consists of a mixture of molecules, the lengths of which are determined by the base composition of the original DNA fragment. The four reactions are electrophoresed in adjacent lanes through a polyacrylamide gel. The DNA sequence can then be determined directly from an autoradiograph of the gel. The original method has been improved over the years (5). Additional chemical cleavage reactions have been devised (6), new end-labeling techniques developed (7,8), and shorter, simplified protocols have been produced (Chapter 32). The main advantage of chemical degradation sequencing is that sequence is obtained from the original DNA molecule and not from an enzymatic copy. It is therefore possible to analyze DNA modifications such as methylation, and to study protein/DNA interac- tions. Chemical sequencing also enables the determination of the DNA sequence of synthetic oligonucleotides. However, the Sanger method is both quicker and easier to perform and must remain the method of choice for most sequencing applications. 3, Sanger Method The Sanger (or chain termination) method (I) involves the synthesis of a DNA strand from a single-stranded template by a DNA polymerase. The method depends on the fact that dideoxynucleotides (ddNTPs) are incorporated into the growing strand in the same way as the conventional deoxynucleotides (dNTPs). However, ddNTPs differ from dNTPs becauset hey lack the 3’-OH group necessaryf or chain elongation. When a ddNTP is incorporated into the new strand, the absence of the hydroxyl group prevents formation of a phosphodiester bond with the succeeding dNTP and chain elongation terminates at that position. By using the correct ratio of the four conventional dNTPs and one of the four ddNTPs in a reaction with DNA polymerase, a population of polynucleotide chains of varying lengths is produced. Synthesis is initiated at the position where an oligonucleotide primer anneals to the template, and each chain is terminated at a specific base (either A, C, G, or T depending on which ddNTP was used). By using the four different ddNTPs in four separate reactions the com- 4 Griffin and Griffin plete sequence information can be obtained. One of the dNTPs is usually radioactively labeled so that the information gained by elec- trophoresing the four reactions in adjacent tracks of a polyacrylamide gel can be visualized on an autoradiograph. The original method used the Klenow fragment of DNA polymerase I to synthesize the new strands in the sequencing reactions, and this enzyme is still used today (Chapter 12). Other enzymes such as Sequenase (Chapter 14), T7 polymerase (Chapter 13), and Taq polymerase (Chapter 15) are also widely used. Each enzyme has its own particular properties and qualities, and the choice of polymerase will depend on the type of template and the sequencing strategy employed. 4. Templates for DNA Sequencing The polymerase reaction requires single-stranded template. This is usually achieved by utilizing Ml3 phage that can produce large amounts of just one strand of DNA as part of its normal replicative cycle. Double stranded (replicative form) Ml3 can also be isolated, and this is used to clone the DNA fragment to be sequenced. The qualrty of DNA sequenced ata achieved using Ml3 template is extremely good and many researchers prefer to subclone to Ml3 prior to sequencing. Sequencing reactions can be performed directly on plasmid DNA, the double stranded molecule being denatured prior to sequencing. Recent innovations in DNA purification techniques and the availability of improved polymerases have greatly enhanced the quality of data produced by plasmid sequencing methods (Chapters 14, 18, and 19). Sequence determination can also be performed directly on cosmid clones (Chapter 21), lambda clones (Chapter 20), and on PCR prod- ucts (Chapters 23-25). In particular, the advento f cycle sequencing( Chap- ter 26) has vastly increased the range of templates that can be used. 5. Sequencing Strategies A lot of sequencing performed is confirmatory sequencing to check the orientation or the structure of newly constructed plasmids, or to determine the sequence of mutants. This type of sequencing can be easily achieved by subcloning a restriction fragment into Ml3 and sequencing using the universal primer. Alternatively, a custom-designed oligonucleotide primer can be synthesized and sequencing performed without the need for any subcloning. DNA Sequencing 5 The determination of long tracts of unknown sequence, however, requires careful planning and the utilization of one of a variety of strategies including: the shotgun approach, directed sequencing strategies, and the gene walking technique. A random, or shotgun, approach involves subcloning random fragments of the target DNA to an appro- priate vector such as Ml3 (Chapter 7). Sequencesf rom these recombi- nants are determined at random until the individual readings can be assembled into a contiguous sequence. This is achieved using a sequence assembly computer program (9,lO). The disadvantage of this method is the redundancy in the sequence data obtained, each section of DNA being sequenced several times over. However, the strategy benefits from making no prior assumptions about the DNA to be sequenced, such as base composition or the presence of certain restriction sites. Directed strategies usually involve the construction of a nested set of deletions of the fragment to be sequenced. Progressive deletions of the fragment are generatedw ith a nuclease,e achd eletion being approx- imately 200-300 bp. Following deletion the fragments are recloned into Ml3 or a plasmid vector adjacent to the universal primer site. The subclones are then sequenced in order of size, with the sequence of each clone overlapping slightly with the one before. In this way, a large tract of contiguous sequencei s determined on one gel. The disadvantage is the labor and time involved in constructing the deletions. Several methods are available for deletion construction including the use of exonuclease III (Chapter 8), T4 DNA polymerase (Chapter 9), and DNase I (Chapter 10). It is essential to sequence both strands of the DNA and this usually entails generating two sets of deletions. Perhaps the simplest method of sequencing is the gene walking technique (Chapter 11). This involves the initial sequencing of approx- imately 200-400 bp of the end of a cloned fragment using the universal primer (the sequence of the other end can be achieved with the reverse primer). This sequence information is then used to design a new oligonucleotide primer, which will provide the sequence of the next 200-400 bp, and so on across the entire length of the insert. This method is the least labor intensive because no deletion construction or generation of random clones is necessary, and template DNA can be made in the one batch since the template is the same for all sequencing reactions. However, the delay involved in synthesizing a new oli- Griffin and Griffin gonucleotide primer before the next reaction can be performed may considerably prolong the time taken to sequence a long tract of DNA. The cost of oligonucleotide synthesis may also be prohibitive. 6. Automation in DNA Sequencing One of the major advances in sequencing technology in recent years is the development of automated DNA sequencers. These are based on the chain termination method and utilize fluorescent rather than radioactive labels. The fluorescent dyes can be attached to the sequencing primer, to the dNTPs, or to the terminators, and are incorporated into the DNA chain during the strand synthesis reaction mediated by a DNA polymerase (e.g., Klenow fragment of DNA polymerase I, Sequenase, or Taq DNA polymerase). During the electrophoresis of the newly generated DNA fragments on a polyacrylamide gel a laser beam excites the fluorescent dyes. The emitted fluorescence is collec- ted by detectors and the information analyzed by computer. The data are automatically converted to nucleotide sequence. Several such instruments are now commercially available and are becoming increasingly popular (11; Chapters 33-37). Other aspects of the sequencing procedure that are being automated include template preparation and purification, and the sequencing reactions themselves. Robotic workstations are currently being developed to perform these tasks (Chapter 38). 7. Cycle Sequencing Cycle sequencing is a new and innovative approach to dideoxy sequencing. Its advantages over conventional sequencing techniques are that the reactions are simpler to set up, less template is required, the quality and purity of template are not as critical, and virtually any single- or double-stranded DNA can be sequenced (including lambda, cosmid, plasmid, phagemid, M13, and PCR product). In this method, a single primer is used to linearly amplify a region of template DNA using Taq polymerase in the presence of a mixture of dNTPs and a ddNTP. Either radioactive or fluorescent labels can be used, making cycle sequencing technology as relevant to automated processes as it is to manual methods (Chapters 26, 34-36). As in conventional dideoxy sequencing methods, cycle sequencing involves the generation of a new DNA strand from a single-stranded DNA Sequencing template, synthesis commencing at the site of an annealed primer, and terminating on the incorporation of a ddNTP. The difference is that the reaction occurs not just once but 20-30 times under the control of a thermal cycler (or PCR machine). This results in more and better sequence data from less template. The process of denaturing a double- stranded molecule is eliminated, with denaturation occurring automatically in the thermal cycler. The development of cycle sequencing techniques has made a major contribution to DNA sequencing meth- odology, improving the reliability and efficiency of DNA sequence determination and eliminating time-consuming steps. 8. Aim of This Book The purpose of this book is to provide detailed practical procedures for a number of DNA sequencing techniques. Although protocols for DNA sequencing methods are available elsewhere, there was a need for a book that comprehensively covered the vanguard techniques now being applied in this rapidly evolving field. Each contribution is written so that a competent scientist who is unfamiliar with the method can carry out the technique successfully at the first attempt by simply following the detailed practical procedures that have been described by each author. Even the simplest techniques occasionally go wrong, and for this rea- son a “Notes” section has been included in most chapters. These notes will indicate any major problems or faults that can occur, their sources, and how they can be identified and overcome. Since the purpose of this book is to describe practical procedures and not to go into great depth regarding theory, a comprehensive reference section is included in most chapters, enabling the reader to refer to other publications for more detailed theoretical discussions on the various techniques. References 1. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing wrth chain- termmator inhibitors. Proc. Natl. Acad. Sci USA 74, 5463-5467. 2. Maxam, A. M and Gilbert, W (1977) A new method for sequencing DNA Proc. Natl. Acad. Scl USA 74,560-564. 3. Barrell, B. (1991) DNA sequencmg: present limitations and prospects for the future. FASEB J 5,40-45 4. Sambrook, J., Frrtsch, E F., and Maniatrs, T. (1989) Molecular Cloning. ,4 Laboratory Manual 2d ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Griffin and Griffin 5 Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base- specific chemical cleavages. Meth. Enzym. 65,499-560. 6. Ambrose, B J. B. and Pless, R. C. (1987) DNA sequencing. Chemical methods. Meth. Enzymol. 152,522-538. 7. Volckaert, G. (1987) A systematic approach to chemical DNA sequencing by subcloning in pGV451 and derrved vectors. Meth Enzym. 155,23 l-250 8 Eckert, R. L. (1987) New vectors for rapid sequencing of DNA fragments by chemical degradation. Gene 51,247-254. 9. Dolz, R. (1993) Fragment assembly programs, in DNA sequencmg: Computer Analysis ofSequence Data, (Griffin, A. M. and Grrffm, H. G., eds.), Humana Press, Totowa, NJ. (Ch. 2). 10 Staden, R. (1992) Managing sequencing projects, m DNA sequencing. Com- puter Analysis of Sequence Data, (Griffin, A. M. and Griffin, H. G., eds.), Humana Press, Totowa, NJ. (Ch 17). 11 Hunkapiller, T , Karser, R J., Kopp, B F , and Hood, L. (1991) Large-scale and automated DNA sequence determination. Science 254,59-67 &IAE’TER 2 Ml3 Cloning Khicles Their Contribution to DNA Sequencing Joachim Messing 1. Introduction For studies in molecular biology, DNA purification has been essential, in particular for DNA sequencing, probing, and mutagenesis. The amplification of DNA in E. coli by cloning vehicles derived from M13mp or pUC made expensive physical separation techniques like ultracentrifugation unnecessary.A lthough today the polymerase chain reaction is a valuable alternative for the amplification of small DNA pieces (I), it cannot substitute for the construction of libraries of DNA fragments. Therefore, E. coli has served not only as a vehicle to amplify DNA, but also to separate many DNA molecules of similar length and the two DNA strands simultaneously. For this purpose, a bacteriophage like M 13 can be used. The various viral cis- and truns- acting functions are critical not only for strand separation, but also to separate the single-stranded DNA from the E. coli cell by an active transport mechanism through the intact cell wall. Although it may have been somewhat surprising to some how many changes in its DNA sequence the phage tolerated, manipulations of this amplification and transport system have been extended today even to the viral coat proteins for the production of epitope libraries (2). Much of the work is now more than a decade old, but experience has confirmed the usefulness of some simple biological paradigms. Tech- From Methods m Molecular Biology, Vol 23’ DNA Sequencmg Protocols Edlted by. H and A Gnffm Copyright 01993 Humana Press Inc , Totowa, NJ 9 10 Messing niques that were new and limiting fifteen years ago included automated oligonucleotide synthesis and the use of thermostable enzymes, which add a critical dimension to molecular biology today. Neither necessarily replaces the previous techniques, but they create greater flexibility, enormously accelerate scientific investigations, and even make certain analyses possible for the first time. However, DNA sequencing of larger contigs (several overlapping sequences that can be linked) have benefited from economizing on the synthesis of new oligonucleotides (3,4). Even in the absence of automated oligonucleotide synthesis in the earlier years, the concept of a universal primer could be developed by alternative techniques. 2. Development of DNA Sequencing Techniques: A Discussion In 1974, at one of the first meetings on the use of restriction endo- nucleases in molecular biology some of these ideas became clear, Work on the chemical synthesis of a tRNA gene was presented, and the initial work on sequencing phage $X174 using restriction fragments as primers for the plus-minus method was discussed. At that time, oligonucieotide synthesis required a major effort and could not easily be generally applied. Restriction fragments offered an alternative. They could be used as primers for DNA synthesis in vitro and for marker-rescue experiments to link genetic and physical maps of viruses like SV40, both forerunners for DNA sequencing and site- directed mutagenesis. There were several reasons to use @X 174 as the first model in developing DNA-sequencing techniques and determining the sequence of an entire autonomous genome. First, it was one of the smallest DNA viruses; it is even smaller than M13. Second, the mature virus consists of single-stranded DNA, eliminating the need to separate the two strands of DNA for template preparation; this is even more critical if one wishes to use double-stranded restriction fragments as primers. Third, a restriction map was superimposed on the genetic map by marker-rescue experiments (5). The latter feature still serves today as a precondition for other genomes. Restriction sites were critical as signposts along the thousands of nucleotides and provided the means to dissect the double-stranded replicative form or RF of @X174 in small, but ordered pieces that permitted the DNA sequencing effort