dna sequencing protocols

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.. The qualrty o

CHAIVER 1

DNA Sequencing

1 Introduction Methods to determine the sequence of 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 sequence the entire genome of organisms such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, and Homo sapiens In the recent past, large-scale sequenc- ing projects such as these were often dismissed as prohibitively expen- sive 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 analy- sis 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

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2 Griffin and Griffin

projects, such as the Human Genome Project, produce the DNA sequen- ces of many unknown genes Such data provide an impetus for molec- ular 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 appearance in 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 sequencing described in 1977 (1,2) differ considerably in principle The enzymatic (or dideoxy chain termina- tion) 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 degra- dation of the original DNA Both methods produce populations of radioac- tively 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 chemi- cal degradation method is still in use, the enzymatic chain termina- tion 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 phase sequencing (Chapter 25), and the use of robotic work stations to automate sample preparation and sequenc- ing 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 polynucleo- tides 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 origi- nal 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 sequenc- ing 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 synthe- sis of a DNA strand from a single-stranded template by a DNA poly- merase 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 because they lack the 3’-OH group necessary for 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 polyacryla- mide 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 poly- merase (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 sequence data 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 advent of 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 strate- gies 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) Sequences from 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 generated with a nuclease, each deletion 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 sequence is determined on one gel The disad- vantage 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 univer- sal 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 sequenc- ing 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 sequenc- ing 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 increas- ingly 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 devel- oped 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 sequenc- ing 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 auto- matically 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 proce- dures for a number of DNA sequencing techniques Although proto- cols for DNA sequencing methods are available elsewhere, there was

a need for a book that comprehensively covered the vanguard tech- niques now being applied in this rapidly evolving field Each contri- bution 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 meth- ods 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

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

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10 Messing

niques that were new and limiting fifteen years ago included auto- mated 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 frag- ments 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 alterna- tive 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 devel- oping 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 con- sists of single-stranded DNA, eliminating the need to separate the two strands of DNA for template preparation; this is even more criti- cal if one wishes to use double-stranded restriction fragments as prim- ers 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

Ml3 Cloning Vehicles 11

to proceed in a walking manner along the genome Today the restric- tion map can be replaced by any DNA sequence (e.g., STS), since the synthesis of oligonucleotides is so rapid that researchers can use the DNA sequence that was just read from a sequencing gel to design and produce an oligonucleotide to extend the sequencing gel further

in the 5’ direction Therefore, the use of oligonucleotides instead of restriction fragments in such a primer walking method would have enormously accelerated the @X174 project

At the Cleveland Conference on Macromolecules in 198 1, after a talk that I had given, the replacement of shotgun sequencing by such

a method was suggested by a colleague, who, as a pioneer in DNA syn- thesis and its automation, saw a perfect match of this emerging technol- ogy with DNA sequencing Another expert in the chemical synthesis

of DNA, Michael Smith, had more interest in the applications than improving the method, and had switched from the diester method to the phosphoramidite method (6), as well as conceiving the idea of using oligonucleotides in marker rescue (7) These innovative researchers pioneered marker rescue and established the physical and genetic map

of $X174 (5) It is clear that today’s protein engineering had its roots right there with the right people at the right time, because they also recognized that oligonucleotides could eliminate the need for strand separation for DNA sequencing (8), and developed this method until

it reached greater maturity (4,9) Even double-stranded DNA sequenc- ing with universal primers became easier with the development of the pUC plasmids (IO)

Despite all the advantages of choosing @X174 as a model system, Sanger’s group nearly picked a different single-stranded DNA phage,

fd In principle, E coli has two different types of single-stranded DNA phage, represented by $X174 and fd The first is packaged into an icosahedral head; it kills and lyses the host cell, but does not require

F pili, which are receptor sites on the surface of the cell wall encoded

by F factors Its host range is restricted to E coli C Phage like fd can only infect male-specific E cd, producing pili at their surface that are packaged in a filamentous coat and discharged from the cell with- out lysis; infected cells can continue to divide These differences are critical, but there was another reason for choosing fd originally, The major coat protein encoded by gene VIII of the phage, a very small but very abundant protein, had been sequenced by protein sequen-

12 Messing

cing methods Therefore it seemed obvious, particularly to those who had pioneered protein sequencing, to use protein sequence to check the DNA sequence The protein sequence allowed the design and synthe- sis of an oligonucleotide that would prime in vitro DNA synthesis within the coat protein gene Furthermore, the derived DNA sequence had to match the protein sequence Of course, the codon redundancy

of many amino acids made it difficult to design a unique primer, and

it might have been not too surprising that the approach did not lead to the correct DNA sequence (11) It turned out later that this was not caused by the choice of codons, but to a mistake in the protein sequence instead, Still, the paradigm of reverse genetics again has its roots right there

3 Replication Systems and Ml3

In 1974, a research group at the Max Planck Institute of Biochem- istry in Munich became interested in viruses, initially in E coli more than in mammalian cells The “Abteilung” was organized in sub- groups, and one subgroup was interested in developing in vitro reph- cation systems using both bacteriophage and small plasmids This had a strong biochemical emphasis, and the researchers rapidly began

to learn about single-stranded DNA replication Eleven years earlier, Hofschneider had isolated a similar filamentous phage from the Munich sewers that he named after a series of phage with the initial

M (12) Number 13 was the one that was studied most Looking for a different research topic than plasmids or DNA replication, the possi- bility of combining Ml3 phage production with the in vitro DNA synthesis-based method of DNA sequencing was seen

Although this might have been obvious to those familiar with phage replication, innovative methods were needed for adaptation to DNA cloning techniques The walking method for sequencing $X174 was the strategy used at that time, and some thought that it would be dif- ficult to clone large fragments into Ml3 (although the author’s record was around 40 kb) and that a walking method might therefore have a limited use Logically, the only alternative to the walking method was the use of shotgun clomng and a universal primer The replica- tive form of Ml3 could be used to clone DNA fragments of a size slightly larger than necessary for single sequencing reactions, and a universal sequence near the cloning site would be used as a primer

Ml3 Cloning Vehicles

This would shift the work from preparing primers to preparing tem- plates, which still remains more economical (13) If they were numer- ous, cloning was much faster than any biochemical technique, and with these thoughts in mind, a plan took shape to construct in vitro recombinants of phage Ml3 without using existing methods One might recall that in vitro recombinants were usually based on drug- resistance markers This led to the development of plasmid vectors with unique cloning sites that were scattered ail over the plasmid genome (14)

4 Transposons Mutagenesis Both Zinder’s and Schaller’s laboratory had in mind, and actually later used, transposons to develop f 1 and fd transducing phage (15,16) However, one could predict that such a course of experiments, although useful for plasmid cloning vehicles, would be less useful for M13 It seemed plausible, and such an experiment could demon- strate that, in contrast to $X174, filamentous phage can accommo- date additional DNA by extending the filamentous coat; infected cells can be treated like plasmid-containing cells To some degree this had already been proved since one group had already described mutants

of more than unit length (17) Another advantage of transposon mutagenesis was that insertion mutants would be naturally selected This was one of the biggest obstacles from the beginning Although plasmids and bacteriophage h were natural transducing elements, fila- mentous phage had never been shown to have this property, and it was not obvious whether insertion mutants would be viable This was difficult because it was already known that amber mutants of most viral genes not only cause abortive infection, but also lead to killing

of the host, something that does not happen when these mutations are suppressed Therefore, insertion mutants that can be used as plas- mids would kill the cell

It was thus predictable that insertion mutants had to be restricted

to noncoding regions Therefore, a decision was made to use a restric- tion enzyme that recognized at least two different sites in the intergenic region of the RF Rather than asking whether the intergenic region contains a target site for transposons, the restriction map showed that

it was possible to get at least two different insertion mutants within the intergenic region The only difficulty in such an experiment was

14 Messing

to find conditions where the restriction enzyme would cut RF only once at any of the possible target sites, so that a population of unit length RF could be ligated to the appropriate marker DNA fragment However, there was another reason not to use drug-resistance mark- ers Infected cells still divide, but very slowly Therefore, selection takes much longer than with plasmids, but it makes it very easy to distinguish infected from noninfected cells on a bacterial lawn A single infection grown on a bacterial lawn forms a turbid plaque If bacterial cells are then transfected by the calcium chloride technique

of Mandel and Higa (18), a transformed cell can be recognized as a plaque Hence, no selection technique is necessary, but the ability to distinguish between wild-type M 13 and M 13 insertion mutants would remain a problem Although it was quite plausible to think of the histochemical screen used for bacteriophage hplac by Malamy et al (19), the la& gene would have been a large insertion However, it turned out that, rather than using entire genes as markers, one could clone only the portion encoding the amino-terminal and the repress- ible control region, and provide the rest in tram by the host of the phage This became clear when Landy et al (20) wrote on the purifi- cation of an 800-bp Hind11 fragment from hplac capable of a-comple- mentation in a cell-free transcription-translation system

An informal sequence of this fragment showed that it was 789-bp long and included the first 146 codons of the ZacZ gene, but it was still necessary to assemble many components and purify several restriction endonucleases Work began after some strains and puri- fied Zac repressor were traded; this allowed the purification of the 789-bp Hind11 fragment out of about fifty other restriction fragments

by simply filtering the DNA/lac repressor complex through a nitro- cellulose filter After adding IPTG, it was possible to recover the DNA

in solution Using DNA-binding proteins for purifying and cloning promoter regions is now a well established technique, but ligating restriction fragments via blunt ends was not established at the time when this experiment was ready As described elsewhere (21), only two transformants were obtained-one of them was saved and named

M 13mp 1 Electron microscopy proved that added DNA was packaged

as a filamentous phage and produced as single-stranded DNA (22,23) Now the path took a more formal shape Not only would the histo- chemical screen work by detecting a blue among colorless plaques, but

Ml3 Cloning Vehicles 15

ATG ACC ATG ATT AC- TC A CTG GCC GTC (+ or Viral strand)

GGmAT TC (+ or viral strand)

CT TA AG (- or complementary strand)

ATG ACC ATG ATT ACE AAT TC A CTG GCC GTC (+ or Viral strand)

EcoRI Fig 1 Creation of an EcoRI sue by chemical mutagenesis By screening the nucleotides of the first ten codons of the 1ucZ gene, we found that the sequence GGATTC could erther be converted into an EcoRI site GAATTC or a BamHI sue GGATCC by a single base change Since there already was a BumHI site in gene III, but no EcoRI site m M13mp1, and I had an ample supply of EcoRI enzyme purified myself, we decided to select the GAATTC site that also changed codon GAT for asparttc acid to AAT for asparagine (24)

it could be reversed One uncertainty was how to introduce new restric- tion sites in the right region, Such a site had to be unique for M 13mp 1 and positioned not somewhere in the viral genome, but in the ZacZ

region, so that insertion mutants would not give rise to a-comple- mentation Inspection of the sequence showed that there were not many sequences in the amino-terminal region that could be converted

in a single step into a unique restriction site Attempts to use EcoRI linkers that became available at the time to “marker rescue” them did not succeed Without somebody to synthesize oligonucleotides

that were more homologous to the Zac region, it was hopeless As an alternative, a chemical mutagen was tried It was known that methy- lated G could mispair with uracil or thymine, therefore, by methylat- ing the single-stranded Ml3 DNA with nitrosomethylurea, a mutation could be introduced into the minus strand and the subsequent RF molecules (Fig 1)

Unfortunately, there was no good genetic selection for this proce- dure It would require brute-force methods of enriching EcoRI-sensi- tive RF from a transformed phage library by gel electrophoresis of linear versus circular molecules Still, it was hard to believe when this author isolated RF that was not only sensitive to EcoRI, but had exactly the predicted base change in codon 5 of the 1acZ gene (24)

16 Messing

The same mutagenesis led to two more EcoRI mutants and a mutant

RF that was resistant to BumHI, a site within gene III At that time, there was still much concern that many mutations might not be toler- ated because of changes in the protein sequence or the secondary structure of RNA Therefore, changes in the lac DNA should prob- ably occur at a higher frequency than in the viral DNA Furthermore, the aminoterminus of the 1acZ gene appeared to be more flexible since

it was demonstrated that fusion proteins retained P-galactosidase func- tion On the other hand, this author did not recognize the tremendous selection power for suppressor mutations In other words, any muta- tion that was introduced could potentially be compensated for by another mutation somewhere else The primary mutant might give a low titre, but because of the growth advantage a suppressor mutant would rapidly take over An example of such a case is M 13mp 1, Later, Dotto and Zinder (25) showed that insertion mutants at the mpl Hue111 site gave a low titre phenotype: Since M13mpl gave a normal titer, they searched for a suppressor mutation Codon 40 in gene II of

M 13mp 1 was indeed changed

5 The Need for a Universal Primer

Researchers continue to use chemical mutagens to get rid of restric- tion sites The reason for this becomes clear when they return to the use of Ml 3 in DNA sequencing The EcoRI site in M13mp2 allowed cloning by screening for colorless plaques Now one could readily purify these plaques and prepare a template for sequencing the inserted DNA Still, as with the $X174 project, a restriction fragment from the adjacent Zuc DNA needed to be purified as a primer, and such a fragment was subcloned into a plasmid for convenience (26) Such a primer fragment still needed to be denatured since it was double- stranded, and had to be cut off after the sequencing reaction to pro- duce a shift of the 3’ end in the sequencing gel

Although such a protocol could still be improved on, a more seri- ous obstacle arose suddenly from the concern over the biological con- tainment of Ml 3 recombinant DNA The NIH Recombinant Advisory Committee (RAC) thought that the conjugation proficient E coli host strains could lead to the spread of Ml3 infection and pose a risk in using Ml3 as a cloning vehicle On the other hand, using one of the

truD or tru1 mutants reduces conjugation by a factor of 106, but they

Ml3 Cloning Vehicles 17

still were infectious to M13 Since this F factor carrying the traD muta- tion was wild-type lac, a histochemical screen with the mp vectors would not be possible, and one would have to return to drug-resistant- type M 13 vectors The scientific reasoning of RAC is hard to under- stand First, nobody argued against Agrobacterium tumefaciens as a plant transformation vector, although it was conjugation proficient and could easily spread in the environment Second, F pili were never made under stress or anaerobic conditions, something that was already known as “phenocopies.” Conjugation in the human gut was in any case nearly zero Third, Ml3 infection per se reduces conjugation by

a factor of 106 It was hard to argue with so many well known scien- tists at that time, so a new series of E coli strains (JM series) all carrying the Ml5 deletion on the F’ traD36 episome was constructed Since it was a concern of NIH, it was necessary to summarize the status of all the strains for potential users in the NIH Bulletin (27)

Although DNA sequencing of eukaryotic origin by Ml3 cloning was now possible, preparation of the primer from the plasmid was still cumbersome It was clear that an oligonucleotide to replace the restriction fragment as a universal primer was needed Inquiries regarding the synthesis of a universal primer by a commercial sup- plier revealed that the cost of such an attempt made it out of the ques- tion A further attempt and timely support produced success, not only with a universal primer but also with another application of oligo- nucleotides (3)

In 1978 this author made another interesting observation, namely that in-frame insertions of linkers in the EcoRI site could still give a positive color reaction, One of these isolates, called M13mp5, could

be used to clone both EcoRI and Hind111 fragments at the same site and with the same primer for sequencing (27) The utility of creating cloning sites on top of each other was based on the universal primer concept, but in turn caused the development of multiple cloning sites (MCS) or polylinkers that are now found in all cloning vehicles and provide many additional uses (Fig 2) Therefore, work began on the synthesis of an oligonucleotide that could be inserted into the EcoRI site and generate restriction sites recognized by six basepair cutters like BamHI, AccI, SmaI, or Hind11 useful for cloning either blunt- ended fragments or fragments with sticky ends produced by four base cutters like Sau3A, TaqI, and HpaII (Fig 3) (consistent with a DNA

Fig 2 Symmetric (a) and asymmetric (b) polylinkers Two other “tricks” were used m the construction of polylmkers One type of polylmker was symmetrical where all sites except the central one occurred twice By cloning a drug-resistance marker into the central site, the polylinker could be used in a linker scanning method

of coding regions (10) and (34) The other type of polylinker was a pair, where two vectors contain an array of sites only once, but each of them m the opposite onen- tation Cloned DNA no longer could be cloned out with a single enzyme as in the first type, but DNA could be cloned by using two different sites at the same time This had the advantage that the orientation of a cloned fragment could be deter- mined By using a vector pair, both orientations can be obtained with the same pair

of restriction cuts and therefore each strand of a restriction fragment could become the viral strand of Ml3 and available as a template for sequencing Furthermore,

by using two restriction enzymes that produce 3’ and 5’ overhangs, one can either use It for cloning oligonucleotlde hbrarles or to generate umdlrectional deletions with exonuclease III

shotgun sequencing approach) This also required a renewed chemi- cal mutagenesis to eliminate the AccI and the HincII sites naturally occurring in M13 All single mutations were combined by marker rescue to give rise to M13mp7 (3) This was just the system, but did shotgun sequencing succeed?

AALL, HaeIII, etc.;

Ba.UlorExoIIIIV;

sheared and repaired DNA

Fig 3 Sticky and blunt-end cloning of small fragments mto unique cloning sites By destgning a unique sequence for the M13mp vectors that were recogmzed

by restriction enzymes that could cut a hexanucleotide sequence in various ways

by etther producing sticky ends of 4 or 2 bases, or blunt ends, the variety of DNA fragments that could be cloned next to the umversal primers were endless Note that the sequence GTCGAC was recognized by SufI, AccI, and H&II, each pro- ducing different ends In our sequencing project wtth cauliflower mosatc virus we generated small DNA fragments for shotgun DNA sequencing by cleaving CAMV with EcoRI*, Mb&, HpaII, TagI, HincII, HueIII, and A/u1 (28) Later we used DNaseI (35), sonication (30), and a combination of exonuclease III and VII to generate blunt ends (36)

Initially, lack of funds and proper laboratory facilities made a dem- onstration impossible Financial support finally arrived, however, and

a dedicated research team began producing the sequence of cauli- flower mosaic virus on the side (8031 bp) This was accomplished in a record time of three months, and the results were finally published a year later (28)

20 Messing

6 Conclusion During the same time, several researchers reported that they could not finish their work on sequencing the mitochondrial DNA if they could not use a host for Ml3 approved by the NIH guidelines This author gave them not only the JM strains, but also the newly devel- oped M13mp7 for blunt-end cloning to facilitate their work Although this author’s work was not published, having been rejected by PNAS

as trivial at the time, in the end, I recognized that there was no sense

in competing but that the scientific community should be used as a laboratory at large, and this indeed became reality (29)

Becoming overwhelmed by requests for strains and protocols, the newly developing reagent companies were turned to for help Their educational and service role helped immensely to disseminate the knowledge needed to train students and investigators in academia and industry in M 13 cloning, sequencing, and site-directed mutagen- esis (30) Along the way, the first Apple-based software on shotgun sequencing was developed (31,32), in addition to a textbook for an undergraduate course (33) A good overview of M13mp, pUC vec- tors, and helper phage has also appeared (21) As with all methods of wider scope, modifications and refinements have been produced in many laboratories as many chapters in this book show However, some principles have not changed: whenever there is a way to use a cell or parts of one as a machine, scientists seem to get ahead faster and cheaper, and that is what biotechnology is all about

Acknowledgment Most of this author’s work on Ml3 was supported by the Deutsche Forschungs Gemeinschaft, grant no 5901-9-0386, and the Depart- ments of Agriculture and Energy, grant no AC0243 lER10901

References

1 Saiki, R K., Gelfand, D H., Stoffel, S , Scharf, S J., Higuchi, R., Horn, G T., Mullis, K B., and Ehrlich, H A (1988) Primer-directed enzymatic amplifica- tion of DNA with a thermostable DNA polymerase Science 239,487- 491

2 Scott, J and Smith, G (1990) Searchmg for peptIde ligands with an epitope library Science 249,386 -390

3 Messing, J., Crea, R., and Seeburg, P H (1981) A system for shotgun DNA sequencing Nucl Acids Res 9, 309-321

4 Norrander, J., Kempe, T., and Messing, J (1983) Improved Ml3 vectors using oligonucleotide-directed mutagenesis Gene 26, 101-106

Ml3 Cloning Vehicles 21

5 Edgell, M H., Hutchison, C A., III, and Sclair, M (1972) Specific endonuclease

R fragments of bacteriophage $X174 deoxyribonucleic acid J Viral 9,574-582

6 Beaucage, S L and Caruthers, M H (1981) Deoxynucleoside phosphorami- dites-a new class of key Intermediates for deoxypolynucleotide synthesis

9 Zoller, M and Smith, M (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors an efficient and general procedure for the production of point mutations in any fragment of DNA Nucl Acids Res 10,6487-6500

IO Vieira, J and Messing, J (1982) The pUC plasmtds, an M 13mp7 derived sys- tem for msertion mutagenesis and sequencing with synthetic universal prtm- ers Gene 19,259-268

11 Sanger, F , Donelson, J E., Coulson, A R., Kossel, H., and Fischer, H (1973) Use of DNA polymerase I primed by a synthetic ohgonucleotide to determine a nucleotide sequence in phage f 1 DNA Proc Nat1 Acad Ser USA 70,1209-l 2 13

12 Hofschneider, P H (1963) Untersuchungen uber “kleine” E coli K12 Bacteriophagen M12, M13, und M20 Z Naturforschg Mb, 203-205

13 Davison, A J (1991) Experience in shotgun sequencing a 134 kilobase pair DNA molecule DNA Sequence 1,389-394

14 Bolivar, F , Rodriguez, R L., Greene, P J., Betlach, M V., Heynecker, H L , Boyer, H W , Crosa, J W , and Flakow, S (1977) Construction and characteriza- tion of new cloning vehicles II A multipurpose cloning system Gene 2,95-l 13

15 Herrmann, R., Neugebauer, K., Zentgraf, H., and Schaller, H (1978) Transpo- sition of a DNA sequence determining kanamycin resistance into the single- stranded genome of bacteriophage fd MOE Gen Genet 159, 17 1

16 Vovis, G F and Ohsumi, M (1978) The filamentous phages as transducing particles, m The Single-Stranded DNA Phages (Denhardt, D T , Dressler, D., and Ray, D S , eds ), Cold Spring Harbor Laboratory, Cold Spring Harbor,

20 Landy, A , Olchowski, E , and Ross, W (1974) Isolation of a functional lac

regulatory region Mol Gen Gene& 133,273-28 1

2 1 Messing, J (1991) Clonmg m Ml 3 phage or how to use biology at its best

Gene 100,3-12

22 Messing

22 Messing, J , Gronenborn, B., Muller-Hill, B., and Hofschnetder, P H (1977) Frlamentous cohphage Ml3 as a cloning vehtcle insertion of a HrndII frag- ment of the fat regulatory region m the Ml3 rephcattve form in vitro Pruc Natl Acad SCL USA 74,3642-3646

23 Messing, J and Gronenborn, B (1978) The filamentous phage Ml3 as carrter DNA for operon fusions in vitro, m The Single-Stranded DNA Phages (Denhardt, D T., Dressler, D., and Ray, D S., eds.), Cold Spring Harbor Labo- ratory, Cold Spring Harbor, NY, pp 449-453

24 Gronenborn, B and Messing, J (1978) Methylatron of single-stranded DNA

m vttro mtroduces new restrtctrons endonuclease cleavage sites Nature 272, 375-377

25 Dotto, G P and Zmder, N D (1984) Reduction of the mmimal sequence for mmatton of DNA synthesis by qualitative and quantttatrve changes of an ml- trator protein Nature 311,279-280

26 Heidecker, G., Messmg, J , and Gronenborn, B (1980) A versatile prrmer for DNA sequencing m the M13mp2 clonmg system Gene 10,69-73

27 Messing, J (1979) A multrpurpose cloning system based on the single-stranded DNA bacteriophage M13 Recombinant DNA Technical Bulletin, NIH Publt- canon No 79-99,2, No 2,43-48

28 Gardner, R C., Howarth, A J., Hahn, P 0, Brown-Leudi, M , Shepherd, R J., and Messmg, J (198 1) The complete nucleotide sequence of an mfectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencmg Nucl Acrds Res 9,2871-2888

29 Holden, C (1991) Briefings Science 254,28

30 Messing, J (1983) New Ml3 vectors for clonmg Methods Enzymol 101,20-78

31 Larson, R and Messing, J (1982) Apple II software for Ml3 shotgun DNA sequencmg Nucl Acids Res 10, 39-49

32 Larson, R and Messing, J (1983) Apple II computer software for DNA and protean sequence data DNA 2,3 l-35

33 Hackett, P H., Fuchs, J A , and Messing, J (1984) An introduction to recom- bmant DNA techniques Basic Experiments in Gene Manzpulution Benjamin- Cummings, Menlo Park, CA

34 Messing, J., Vieira, J , and Gardner, R (1982) Codon insertion mutagenesis to study functional domains of P-lactamase In vrtro mutagenesis Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 52

35 Messmg, J and Seeburg, P H (1981) A strategy for hrgh speed DNA sequen- cing, in Developmental Biology Using Purified Genes (Brown, D and Fox, F , eds ), ICN-UCLA Symposia on Molecular and Cellular Biology, vol 23 Aca- demic, NY, pp 659-669

36 Yanisch-Perron, C , Vieira, J , and Messing, J (1985) Improved Ml3 phage cloning vectors and host strains, nucleottde sequences of the M 13mp and pUC vectors Gene 33,103-l 19

hAP!l’ER 3

1 Introduction The bacteriophage M 13 has been developed into a cloning vector system for obtaining single-stranded DNA template required for the dideoxy chain termination method of sequencing DNA (1,2) Gen- eral aspects of bacteriophage Ml3 as a cloning vector system are reviewed in Chapter 2, and the preparation of foreign DNA fragments for Ml3 cloning is described in Chapter 7 In this chapter the prepa- ration of Ml3 vectors and the ligation of foreign DNA fragments (inserts) into Ml3 vectors are described

2 Materials

2.1 Preparation of Replicative Form (RF) Ml3 DNA

1 L-Broth: Bacto-tryptone l%, bacto-yeast extract O.S%, NaCl 1%

2 2X YT: Bacto-tryptone 1.6%, bacto-yeast extract 1%, NaClO.5%

3 M9 minimal medium: Na,HFQ, 7H20 12.8 g, IU-12P04 3 g, NaC10.5 g, NH&l 1.0 g, 20% glucose 20 mL, Hz0 to 1 L

4 BacteriophageM13: A single blue plaque from a freshly transformed plate

5 E coli JM 103 or JM 109: A colony grown on an M9 minimal agar plate

6 Solution 1: 50 mII4 Glucose, 25 mM Tris-HCI, pH 8.0, 10 rniI4 EDTA,

pH 8.0, autoclaved and stored at 4°C

From Methods m Molecular Brology, Vol 23 DNA Sequencmg Protocols

E&ted by- H and A Gnffm Copyright 01993 Humana Press Inc., Totowa, NJ

23

24 YU

7 Solution 2: 0.2M NaOH, 1% SDS, freshly mix together equal volumes

of 0.4M NaOH and 2% SDS stocks before use

8 Solution 3: 60 mL 5M Potassium acetate, 11.5 mL glacial acetic acid, 28.5 mL H20, stored at 4OC

9 TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0

10 Phenol-chloroform: Mix well equal volumes of TE (pH 8.0)-saturated phenol (nucleic actd grade) and chloroform (AR grade), stored at 4°C

m a dark glass bottle

11 3M NaAc, pH 5.2, stored at 4°C

12 Ethanol: Absolute alcohol, stored at -20°C

13 RNase: 1 mg/mL DNase-free pancreatic RNase A

2.2 Preparation of Ml3 Vectors

1 Restriction enzymes

2 10X RE buffers

3 Calf intestinal alkaline phosphatase (CIP)

4 10X CIP dephosphorylation buffer

5 Proteinase K 10 mg/mL

6 10% SDS ’

7 0.5M EDTA, pH 8.0

8 3M NaAc, pH 7.0, stored at 4°C

9 10X TBE: Tns base 108 g, Boric acid 55 g, EDTA 9 2H20 9.5 g, HZ0 to 1 L

10 0.8% Agarose gel in 1X TBE

2.3 Ligation of Inserts into Ml3 Vectors

1 T4 DNA ligase

2 10X Ligation buffer

3 10 mM ATP, stored at -20°C

4 Ml3 vectors (prepared as described in the methods section)

5 Inserts (having termini compatible with the vectors)

All enzymes and buffers are stored at -20°C Other solutions can

be stored at room temperature except when indicated otherwise

3 Methods

A number of M 13 vectors have been constructed (.2,3) and are com- mercially available from several companies Therefore, it would be more convenient to purchase the Ml3 vectors than to prepare them oneself Sometimes, however, you may need a Ml3 vector with a

special cloning site to fit your cloning strategy Thus the preparation

of Ml3 vectors is described below

Cloning into Ml3 25

3.1 Mini Preparation of RF Ml3 DNA

1 Inoculate 5 mL of L-Broth in a 20-mL sterile culture tube (e.g., universal) with one bacterial colony (e.g., JM103 or JM109) from an M9 minimal agar plate Incubate at 37OC in an orbital shaking incubator overnight

2 Add 50 l,tL of the bacteria culture to 2 mL of L-Broth in a 5-mL culture tube (e.g., bijoux) Inoculate this culture with Ml3 bacteriophage by touching a single blue plaque from a transformatton plate with a sterile toothpick and washing its end m the culture Incubate the infected cul- ture at 37°C for 4-5 h m an orbital shaking incubator

3 Transfer 1.0-l 5 mL of the culture to a microfuge tube and centrifuge at 12,OOOg for 2 min at room temperature in a microfuge Remove supernatant

to a fresh tube, being careful not to disturb the pellet If desired, the single strand Ml3 (single-stranded) DNA can be prepared from the supernatant

4 Remove any remaining supernatant by aspiration from the tube containing the bacterial pellet Resuspend the pellet by ptpeting it or vigorous vortex- ing in 100 pL of solution 1 and leave at room temperature for 5-10 mm

5 Add 200 cls, of freshly prepared solution 2 Close the tube and mix the contents by mvertmg the tube rapidly five times Do not vortex Store the tube on ice for 5 mm

6 Add 150 pL of ice-cold solution 3 Vortex the tube gently m an inver- ted position for 10 s Store on ice for 5 min

7 Centrifuge at 12,OOOg for 5 min and transfer the supernatant to a fresh tube

8 Add an equal vol of phenol:chloroform, mix by vortexing for 20-30 s Spin as in step 7 and transfer the aqueous phase (top layer) to a fresh tube

9 Add 2 vol of ethanol, mix by vortexing, and stand for 5 min at room temperature

10 Spin as m step 7 and remove the supernatant by gentle asptratton

11, Wash the pellet with 1 mL of 70% ethanol Spin for 2 mm m the same orientation of the pellet Remove the supernatant as m step 10 Vacuum dry for 3-5 mm or air-dry for 10 min

12 Dissolve the pellet m 20 pL of TE (pH 8.0) contammg RNase (20 pg/ mL) to remove RNA Vortex briefly The double-stranded RF Ml 3 DNA

is now ready for analysis by digestion with restriction enzymes

3.2 Preparation of Ml3 Vectors

3.2.1 Digestion with a Single Restriction Enzyme (RE)

1 Digest RF Ml3 DNA with a single RE in the followmg reaction:

26 YU

Incubate for 2 h Remove 2 pL of the reaction and analyze the extent of digestion by electrophoresis through 0.8% agarose gel, using undigested Ml3 DNA as a marker If digestion is mcomplete, add more RE and continue the incubation

2 When digestion is complete, extract the Ml3 DNA with phenol:chloro- form and precipitate DNA with 0.1 vol of 3M NaAc, pH 5.2, and 2.5 vol of ethanol for 1 h at -20°C or overnight if convenient

3 Recover the DNA by centrifugation at 12,OOOg for 10 min in a microfuge Wash the pellet with 70% ethanol and vacuum dry Dissolve the pellet

in 20 p.L of TE, pH 8.0 Now it is ready to use for ligation with inserts

if dephosphorylatton is not required

3.2.2 Dephosphorylation of Ml3 Vectors

4 The single RE-dtgested Ml3 vector can be dephosphorylated by treat- ment with calf intestinal alkaline phosphatase (CIP) to reduce the back- ground of nonrecombinant molecules formed by circularization of the vector during ligation

To 20 pL of linearized Ml3 vector obtained in step 3, add:

10X CIP buffer 5 pL

CIP 1 U/100 pmoles for protruding 5’ termmt

1 U/2 pmoles for blunt or recessed termim

5 Incubate for 30 min at 37OC for protruding 5’ termini vectors For blunt or recessed terminus vectors, incubate for 15 min at 37”C, then add the same amount of CIP and continue incubation for a further of 45 min at 55°C

6 At the end of the incubation period, add the following to the reaction: 10% SDS 2.5 pL (final concentration 0.5 %) 0.5M EDTA (pH 8.0) 0.5 pL (final concentration 5 n-&f)

10 mg/mL proteinase K 0.5 pL (final concentration 100 Clg/mL) Incubate for 30 min at 56OC to remove the CIP

7 Cool the reaction to room temperature, and extract with phenol:chloro- form twice Add 0.1 vol of 3M NaAc, pH 7.0, mix well and add 2.5 vol

of ethanol Mix and store at 20°C for 1 h or overnight Recover the DNA as in step 3 The final pellet is dissolved in 20 pL of TE, pH 8.0 Now it is ready to use for ligation with inserts

3.2.3 Digestion with Two Restriction Enzymes

8 If a different RE digestion is required for generating a Ml3 vector with incompatible termini (forced dtrectional cloning vector), the DNA recov- ered in step 3 above is dissolved in 10 pL of TE (pH 8.0) and steps l-3 are repeated with the second RE To check the extent of the digestion

by the second RE, set up a separate RE digestion reaction with the sec-

pH 8.0 It is ready to use for ligation with inserts

10 For vectors digested with two different REs it is not necessary to dephos- phorylate to reduce the background But, to avoid religation of the small fragment generated by RE digestion of the polycloning sites into the

M 13 vectors, it can be removed by electrophoresis through agarose gel

or polyacrylamide gel (refer to Chapter 10 in Volume 2 of this series) 3.3 Ligation of Inserts into Ml3 Vectors

1 Set up ligation reaction m the followmg order:

Ml 3 vector 1 r-1L (20 w)

10X ligation buffer 1 pL

T4 DNA ligase 5 U for blunt termini

1 U for cohesive termmi

Incubate at 14OC overnight Then store at -2OOC or use directly for transformation

2 Set up two control reactions contammg the same components except that in the place of the mserts, be sure that one control contains H,O, and the other contains an appropriate amount of a test DNA that has been successfully cloned mto the Ml3 vector on a previous occasion Incubate under the same conditions as m step 1

3 After ligation, analyze 1 pL of each ligation reaction, using the same amounts of the Ml3 vector and inserts without ligase as control, by electrophoresis through 0.8% agarose gel to check that the ligation has been successful

4 Transform 5 w of the remaining ligation sample mto competent bacte- ria of the appropriate strain of E coli, e.g., JM103 or JM109

4 Notes

1 Bacteria carrying an F’ episome (e.g., JM103 or JM109) can grow m M9 muumal medium, but much more slowly than m L-Broth and do not survive prolonged storage at 4°C It is, therefore, better to streak a master culture of the bacteria on the M9 minimal agar plate every month

RF Ml 3 DNA by analyzing RE-digested and undigested RF Ml 3 DNA

by electrophoresis through the same agarose gel Because the single- stranded M 13 DNA cannot be digested by REs commonly used in M 13 cloning, Its migration through the gel does not change after RE drges- tion, whereas the digested RF Ml3 DNA will change its migration through the gel

4 When recovermg Ml 3 DNA after centrifugation of precipitated samples, sometimes the Ml3 DNA pellet is not visible Therefore, it is necessary

to carefully remove the supernatant and leave a little bit (20-30 l.tL) behind in the tube to minimize the loss of the DNA Do likewise when washing with 70% ethanol, then vacuum dry

5 To remove CIP which may affect subsequent ligation of Ml3 vectors with inserts after dephosphorylation, an alternative method is to macti- vate the CIP by heating at 70°C for 10 min in the presence of 5 mM of EDTA, pH 8.0, and then extract with phenol:chloroform Because EDTA precipitates from solution at acid pH if its concentration exceed 5-10

mM, the 3M NaAc, pH 7.0, is used instead of the commonly used 3M NaAc, pH 5.2, for precipitation of dephosphorylated Ml 3 DNA

6 The efficiency of ligation of blunt termini is somewhat lower than for ligation of cohesive termini To improve the efficrency of the ligation

of blunt termmi, higher concentration of inserts and T4 DNA hgase is required The extent of ligation of Ml3 vectors with inserts can be checked by electrophoresis through 0.8% agarose gel, using unhgated Ml3 vectors and inserts as control Normally, the ligated Ml3 vectors with inserts will mrgrate slowly through the gel because of the increased

1 Messmg, J (1983) New Ml3 vectors for cloning Meth Enzymol 101,20-79

2 Sanger, F., Nicklen, S , and Coulson, A R (1977) DNA sequencmg with cham- terminating mhlbltors Proc Nat1 Acad Sci USA 74, 5463-5467

3 Yamsch-Perron, C , Vlelra, J., and Messing, J (1985) Improved M 13 phage cloning vectors and host strains: nucleotlde sequences of the M13mp18 and pUC19 vectors Gene 33,103-l 19

of transfection Subsequently, this method was used to introduce a variety of circular and linear DNAs into strains of E coli, and many variations in the technique have been described that are aimed at increasing the yield of transformants or transfectants The central requirements for success are the presence of multivalent cations, an incubation temperature close to O”C, and a carefully controlled heat- shock at 42OC, but a number of other compounds and procedures have been found to increase efficiency in some or all strains of E coli that have been tested The mechanisms involved in DNA transformation

of cells are not fully understood and even with the most efficient methods that are available the proportion of cells that become “compe- tent” for transformation is limited to around 10% of the total population The procedures for Ml3 transfection of E coli cells are simple, requiring the establishment of a log phase bacterial culture, the prepa-

From Methods m Molecular Bfology, Vol 23 DNA Sequenong Protocols

Edlted by Ii and A Gnffm Copyright 01993 Humana Press Inc , Totowa, NJ

31

32 Tomley

ration of competent cells and the introduction of DNA into these cells, Fresh cells can be prepared for each transfection or, alternatively, frozen aliquots of competent cells can be stored at -70°C and thawed

as required This chapter includes two alternative procedures for pre- paring competent cells, one based on the original Calcium method and a second based on that of Hanahan (2)

2 Materials

1 An E, co11 strain smtable for propagating bacteriophage Ml 3 vectors Recommended strains include JMlOl, JM103, JM107, JM109, TGl, and TG2 (see Note 1)

2 Bacterial growth media: Sterilize by autoclaving in suitable ahquots

a L-broth: 1% tryptone, 0.5% yeast extract, 200 mM NaCl

b SOB-broth: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, pH 7.0 Just before use, add Mg2+ to 20 mM from a sterile ftl- tered stock of 1M MgC12, 1M MgSO+

c H-agar: 1% tryptone, 140 rniV NaCl, 1.2% bacto-agar To pour plates, melt agar by boiling or microwavmg, cool to around 50°C and pour mto Petri-dishes on a level surface (15-20 ml/plate) Once agar has set, dry plates by storing inverted at 37°C for several hours before use

d H-top agar: 1% tryptone, 140 mM NaCl, 0.8% bacto-agar Melt agar

as above and hold at 48OC m a waterbath until needed

3 Sterile, detergent-free 1-L conical flasks fitted with porous tops or cot- ton-wool bungs and an orbital incubator capable of shaking these vig- orously (around 250 rpm) at 37°C

4 50-mL polypropylene tubes (e.g., disposable Falcon 2070 or reusable Oakridge, sterile and detergent-free), and a centrifuge, preferably refrig- erated, capable of spinning these tubes at 4,000g

5 Transformation buffers (see Note 2):

a 100 mM Calcium chloride: Store 1M CaC12 at -20°C m 1.5-mL ali- quots When needed, thaw, dilute to 15 mL, filter, and chill on ice

b TFB: 10 mM K-MES, 100 mM RbCl or KCl, 45 mMMnC12 4H20, 10

mM CaC12 2H20, 3 mM hexamminecobalt chloride Make up a 1M stock of MES, adJust the pH to 6.3 with 5M KOH, and store at -20°C

m IO-mL ahquots To make up 1 L of TFB, use one 10 mL aliquot of

IM K-MES, add all the other salts as solids, filter, and store m 15-

mL aliquots at 4OC, where tt is stable for over a year It is important that the final pH of the buffer is 6.15 f 0.1

c FSB: 10 mM potassium acetate, 100 rniV KCl, 45 mM MnC12 4H20,

10 mM CaCl, 2H20, 3 rruV hexammmecobalt chloride, 10% glyc-

Transfection of E coli Cells 33

erol Make up a 1M stock of potassium acetate, adjust the pH to 7.2 with 2M acetic acid, and store at -20°C in lo-mL ahquots To make

up 1 L of FSB, use one lo-mL aliquot of 1M potassium acetate, add the other salts as solids, glycerol to lo%, adjust the pH to 6.4 with 0.W HCl (N.B if you add too much, do not attempt to readjust the

pH with base: discard the batch and start again), filter, and store in 15mL aliquots at 4°C During storage, the pH of this buffer drifts down to 6.1-6.2 and then stabilizes

d DMSO/DTT (see Note 3): Make up 1 M potassium acetate as in step 5c, above, and store in 100~& aliquots Take a fresh bottle of high- est grade DMSO, divide into IO-mL aliquots and store m sterile, tightly capped tubes at -7OOC To make up 10 mL of DMSO/DTI’, use one lOO+L aliquot of 1 M potassium acetate, 9 mL DMSO, 1.53

g of DTT, sterilize through a filter that will withstand organic solvents (e.g., Millex SR, millipore), and store in 300-w aliquots at -20°C

6 Fresh cells/X-gal/IPTG: Make this up just before you need it, calculat- ing the volumes required for your total number of samples Per sample, take 200 pL of fresh E coli cells and gently add 40 pL of X-gal (20 mg/

mL m dimethylformamide) and 40 p.L of IPTG (24 mg/mL in water)

7 Sterile, disposable 5-mL tubes (e.g., Falcon 2054)

3 Methods All the methods relate to the preparation of competent cells from a

50 mL log-phase culture

3.1 Preparation of Log Phase Cells

1 Pick a single bacterial colony from a fresh plate, or take 500 pL of a fresh overnight culture, and transfer into 50 mL of L-broth (for Cal- cium method) or SOB-broth (for Hanahan method) in a conical flask Set up duplicates, one for preparing competent cells and the other for fresh plating cells that are required in step 3 of Section 3.4

2 Incubate flasks at 37OC with vigorous shaking until the cell concentra- tion reaches around 5 x 107/mL For most E coli strains this means an Abe,, of between 0.3 and 0.4, and will take around 3 h incubation from a single colony, around 2 h from an overnight culture

3 Transfer the contents of one flask to a sterile, precooled, 50-mL tube and store on ice for 10 mm to cool the cells

4 Pellet cells by spinning at 4000g for 10 min and carefully pour off the broth, mverting the tube to drain away the last traces Choose either method 2 or 3 for making competent cells, with all steps carried out aseptically

34 Tomley

3.2 Fresh or Frozen Competent Cells

Using Calcium Chloride

1 Suspend cells in 10 mL of cold O.lM CaCl*, store on ice for 5 min, then pellet as in step 4 of Section 3.1

2 Suspend cells gently in 2.5 mL of cold O.lM CaClz and incubate on ice until ready for use At this point cells can be dispensed m small aliquots into cooled sterile 1.5-mL tubes, snap-frozen in liquid nitrogen, and stored at -70°C until needed, when they should be thawed rapidly then immediately stored on ice

3.3 Fresh or Frozen Competent Cells

Using Hanahan Method

1 Suspend cells in 10 mL of cold TFB (for fresh) or FSB (for freezing), store on ice for 10 mm then pellet as m step 4 of Sectton 3.1,

2 Suspend cells gently in 4 mL of cold TFB or FSB and store on me

3 Add 140 pL of DMSO/DTT (for fresh) or DMSO alone (for freezing), mix immediately by gentle swirling, and store cells on ice for 15 min

4 Add a further 140 pL of DMSO/DTT or DMSO, and mix immediately Fresh cells should be stored on ice for at least 15 min then dispensed m small aliquots mto cooled 5-mL tubes For freezing cells, proceed with step 5, below

5 Dispense cells in small aliquots into cooled 5-mL tubes, snap-freeze m liquid nitrogen, and store at -70°C until needed Thaw ahquots rapidly, then store cells on ice for 15 mm prior to use

3.4 Introduction of Ml3 DNA to Competent Cells

and Generation of Progeny Plaques

1 Incubate tubes of competent cells m an rcewater bath For most pur- poses 50-pL aliquots of cells will generate sufficient plaques, but more may be used if very high numbers are required (see Notes 4 and 5)

2 Carefully add 5 pL of an Ml3 ligation containing approximately 40-

400 ng total DNA to each ahquot and swirl gently (see Note 6) Leave

m the icewater bath for 30-45 min In addition to the ligations, two transformation controls should be included, one containing 5-10 pg of

ds circular Ml3 DNA, and one containmg no DNA

3 Meanwhile, melt sufficient H-top agar (3 ml/plate, plus a few mL excess) and keep It m a waterbath at 48OC Just before moving on to step 4, below, take the second flask of cells (from step 1 of Section 3.1.) and make up sufficrent fresh cells/X-gal/IPTG mix (280 pL/plate)

4 Transfer the tubes of competent cells/ligations to a rack in a waterbath, preheated to 42°C and leave for exactly 90 s without shaking (see Note

Transfection of E coli Cells

7) Transfer rapidly back into the icewater bath and make up to 200 pL with SOB-broth (minus MgZ2+) If you think you are going to get too many plaques, then split the samples at this point, e.g., leave a 180 pL

in the first tube and transfer 20 pL to a second

5 Add 280 pL of fresh cells/X-gal/IPTG mix to each sample then, work- ing one sample at a time, add 3 mL of H-top agar, mrx quickly, and pour onto a dry H-agar plate Be careful not to mtroduce air bubbles

6 Allow plates to dry thoroughly then incubate overnight at 37°C (see Note 8) Plaques formed by wild-type Ml 3 will be blue, and those formed

by recombinants will be white (see Note 4)

4 Notes

1 Host bacteria can be stored for short periods at 4°C on mmtmal (M9) agar, which maintains the F’episome, but should be replated frequently from a master stock stored at -7OOC in L-broth plus 15% glycerol

2 For making up and storing all transformation buffers:

l use only high quality pure water, e.g., Milli-Q or equivalent;

l use sterile, detergent-free glassware or plasticware;

9 sterilize solutrons by filtration through 0.45pm pore filters, e g., Nalgene or Acrodisc

3 In the original Hanahan method, DMSO and DTT solutions were added sequentially However, the efficiency of transformation is just as high

rf they are added together (3)

4 The number of plaques that are obtained from each transfection varies enormously and is dependent on many factors, such as the amount of DNA added, the ratio of insert to vector in the ligation reaction, the efficiency of ligation, and the efficiency of transformatron As a guide, closed circular Ml3 DNA should give over lo7 plaques&g using the Hanahan method thus the control transfected with 10 pg of ds Ml3 DNA should yield over 100 plaques Vector DNA that has been linear- ized then religated to an excess of Insert with compatible ends will give anything from 102-lo4 less plaques per pg, depending on the religation efficiency, thus, each test transfection of 40-400 ng may give between

40 and 40,000 plaques For the Calcium method, the numbers of plaques are around 2-lo-fold lower Freshly prepared Calcium-treated cells can

be stored for up to 48 h in CaCl, at 4OC and the efficiency of transfec- tion increases up to six-fold over the first 24 h then declines to the original level (4)

5 The efficiency of transformation using frozen competent cells is reduced compared to freshly prepared cells but, unless very high numbers of plaques are required, frozen cells are adequate and very convenient,

36 Tomley

6 The volume of ligated DNA added to the cells should not exceed 10%

of the total volume

7 The length of time for heat-shock is calibrated for Falcon 2054 tubes and for others the heat-up time may be different

8 It is important that agar plates are thoroughly dried before top agar 1s poured onto them

1 74-1.84

4 Dagert M and Ehrlich S D (1979) Prolonged incubation m calcium chloride improves the competence of Escherichia colr cells Gene 6,23-28

Ml3 Phage Growth

1 Introduction Ml3 phages do not lyse their host, but are released from infected cells as the cells continue to grow and divide Cells infected with Ml3 have a longer replication cycle than uninfected cells, and as the infection proceeds, areas of slower-growing cells can be visualized

as turbid plaques on lawns of E coli (1) Well-separated plaques con- tain cells infected with phages derived from a single transformation event and these can be picked and regrown to provide pure stocks of recombinant phage particles and DNA

During infection of its host cell, the single-stranded DNA (ssDNA)

of the phage is converted and amplified into double stranded replica- tive forms (RF) that are intermediates in the production of progeny ssDNA These ssDNA molecules are packaged into a protein coat and extruded out of the cell and, since there is no size constraint on packaging, recombinants containing foreign DNA are readily pro- duced From liquid culture, packaged particles are readily recover- able from the broth and the ssDNA can be rapidly extracted (2,3) The method is straightforward, and because Ml3 phages replicate rapidly, both phage growth and DNA purification can be carried out

in one day provided that a fresh overnight culture of host bacteria is available

From* Methods m Molecular Slology, Vol 23: DNA Sequencrng Protocols

E&ted by H and A Griffin Copynght 01993 Humana Press Inc., Totowa, NJ

37

38 Tomley

2 Materials

1 An E coli strain suitable for propagating bacteriophage Ml3 vectors Recommended strains mclude JMlOl, JM103, JM107, JM109, TGI, and TG2

2 2X TY broth: 1% tryptone, 1% yeast, 100 mM NaCl Sterthze by auto- claving in 50-mL aliquots

3 Stertle culture tubes for growmg up 1.5 mL cultures (e.g., disposable lo-mL Falcon tubes or glass/plastic universal bottles) and an orbital incubator for shaking the tubes vigorously at 37OC

4 A mrcrofuge and 1 S-mL mrcrofuge tubes

at room temperature

c Phenol: Use high-grade redistilled phenol stored at -2OOC in aliquots

of 100 -500 mL To equtlibrate, warm to room temperature, then melt at 65°C and add hydroxyquinoline to 0.1% To the melted phe- nol, add an equal volume of 0.5M Trts-HCl, pH 8.0, mtx for a few minutes to allow phases to separate, and take off the upper layer (buffer) Do repeated extractions with O.lM Trts-HCl, pH 8.0, unttl the pH of the phenolic phase gets up to around 7.8 Extract once wrth

TE, remove the aqueous phase, then store the phenol at 4°C in a dark bottle under a layer of fresh TE (around 20 mL for 100 mL phenol) Fresh phenol should be prepared once a month

d Chloroform: Use high-grade chloroform that has not been exposed

to the air for long periods of time Mix 24.1 with isoamyl alcohol and store m a tightly capped bottle at room temperature

e 3M sodium acetate, pH 5.2: Dissolve solid sodmm acetate in water, adjust the pH to 5.2 with glacial acetic actd, dtspense into aliquots, autoclave, and store at room temperature

f Ethanol: For convenience, store in tightly capped bottles at -20°C

3 Method

1 Pick a single colony from a freshly streaked plate of a suitable E coli host strain into 10 mL of 2X TY m a conical flask and grow with shak- ing at 37°C overmght

2 Dilute the overnight culture 1 :OO in 2X TY to give sufftctent fresh cul- ture for 1.5 ml/plaque Ahquot 1.5-mL vol into sterile tubes

Liquid Culture and SSDNA Preparation 39

3 Carefully touch a sterile toothpick into a well-separated single plaque and wash the end of the toothpick in a 1.5-mL aliquot of fresh cells (see Notes 1 and 2) This IS sufficient to transfer Infected cells from the plaque to the new culture Incubate the 1.5-mL cultures with vigorous shaking at 37°C for 4.5-5.5 h (see Notes 3 and 4)

4 Transfer cultures to 1.5~mL microfuge tubes and spin for 5 min in a microfuge

5 Transfer supernatants to clean tubes making sure the pellet is undis- turbed, add 200 uL of PEG/NaCl to each, vortex well, and leave at room temperature for at least 15 min

6 Spin for 5 min in microfuge to pellet the phage particles Remove the PEG-containing supernatant with a pipet, respin the tubes for a few seconds and carefully remove all remaming traces of supernatant from around the phage pellet using a drawn-out Pasteur pipet (see Note 5) The phage pellet should be visible at the bottom of the tube

7 Add 100 pL of TE to each phage pellet and vortex vigorously to ensure that they are properly resuspended, then add 50 pL of phenol, vortex well, and leave for a few minutes Vortex again and spin for 2 mm to separate the phases

8 Carefully remove the upper aqueous layer into a fresh 1.5~mL tube, being careful to leave behind all the precipitated material at the interface

9 Add 50 pL of chloroform, vortex and spin for 1 min to separate the phases (see Note 6)

10 Carefully remove the upper aqueous layer into a fresh 1.5-mL tube, add 0.1 vol of 3M sodium acetate and 2.5 vol of absolute ethanol and pre- cipitate the DNA

11 Spin in microfuge for 5 min and remove the ethanol by aspiration, being careful not to disturb the DNA pellet that is often barely visible at this stage Add 200 l,tL of 70% ethanol, spin for 2 min, remove the ethanol very carefully, and dry the pellet either under vacuum for a few minutes or

by leaving the open tubes on the bench until dried by evaporation,

12 Dissolve the pellet in 30 pL of TE At this stage, a few microliters (2-5 pL) can be removed and run on a minigel to check the quality and yield of DNA (see Note 7) The remainder of the DNA should be stored at -20°C until required

4 Notes

1 If plaques have been picked and regrown on plates as colonies (e.g., because of the need to screen inserts by hybridization), cultures for ssDNA preps are prepared by touching the toothpicks onto the colomes and washing in the 1 S-mL fresh cells

40 Tomley

2 Once plaques have been obtamed, it is best to grow phages and purify the DNA as quickly as possible as there is an increase m plaque con- tamination and a deterioration m quahty of DNA obtamed tf plaques are stored at 4°C

3 Do not grow up 1.5mL cultures for extended perrods of time as this increases the possibtlity of selecting for mutants and also increases cell lysis and htgher levels of contaminating host chromosomal DNA

4 Do not grow at temperatures above 37°C because although the host cells ~111 grow the ytelds of Ml3 phages drop rapidly with increasing temperature

5 Make sure all the PEG is removed as this causes high backgrounds m sequencing reactions

6 The chloroform extraction can be omitted, but the highest quality tem- plates are obtamed tf rt 1s included

7 A yield of ssDNA of between 5 and 10 pg/mL ts normal If the final pellet 1s suspended in 30 pL of TE, approx 4-8 pL IS ample for obtam- ing high-quality sequence using standard dtdeoxy methods

References

1 Marvm, D A and Hohn, B (1969) Filamentous bacterial viruses Bacteriol Rev 33, 172-209

2 Bankier, A and Barrell, B G (1983) “Shotgun DNA Sequencing.” In Tech-

niques in the Life Sciences (Biochemistry), ~0185, Techniques in Nuclerc Acid Biochemistry (Flavell, R A , ed ), pp l-34, Elsevier, Amsterdam

3 Sambrook, J., Fntsch, E F., and Maniatis, T (1989) “Small-scale preparation

of single-stranded Bacteriophage Ml3 DNA.” In Molecular Closmg, A Labo- ratory Manual 2nd Ed., Cold Sprmg Harbor Laboratory Press, Cold Sprmg Harbor, NY, pp 4-29,4-30