508
VECTORS
FOR CLONING
GENES
[44]
permit rapid recovery of the target gene from the h phage for sequence identification of selected mutations. The original h ZAP vector has also been modified for construction of a combination prokaryotic/eukaryotic expression vector. 3 This system should permit direct, high-efficiency transfection with a library of h phage particles and screening of eukaryotic cells. 3° Following phenotypic selection of the desired clones in the eukaryotic cells, the appropriate phage can be recovered by direct packaging of the h phage from the transfected cell genomic DNA. 3'31 The cDNA can then be excised into a plasmid molecule designed for rapid clone characterization and expression in E. coli cells. These procedures can also be coupled with subtractive hybridization techniques to further reduce the complexity of screening in eukaryotic cells. Modifications of the h ZAP vector currently underway will further increase its efficiency and versatility. 3o H. Okayama and P. Berg, Mol. Cell. Biol. 5, 1136 (1985). 31 S. W. Kohler, G. S. Provost, P. L. Kretz, M. J. Dycaico, J. A. Sorge, and J. M. Short, Nucleic Acids Res. 18, 3007 (1990).
[44] C l o n i n g o f C o m p l e m e n t a r y D N A I n s e r t s f r o m P h a g e D N A D i r e c t l y into P l a s m i d V e c t o r By ING-MING C H I U , KIRSTEN LEHTOMA, and MATTHEW L. POULIN Cloning vectors derived from bacteriophage h are used frequently in the construction of both cDNA and genomic DNA libraries. 1 Both hgtl0 and hgtl I have been used quite extensively for constructing cDNA libraries. 2 For hgtl 1, the cDNA inserts are cloned into the unique EcoRI site within the lacZ gene and can be expressed as fusion proteins with /3galactosidase. 2 Thus, the cDNA clones can be obtained by screening with the antibody probe generated against the protein of interest.3 Alternatively, a ligand having high affinity with the protein of interest may also be used as a probe. Moreover, cDNA libraries constructed in hgtl0 or hgtl 1 can be screened by hybridization with an oligonucleotide or DNA fragment containing the DNA sequence of interest. i j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 2 T. V. Huynh, R. A. Young, and R. W. Davis, in " D N A Cloning Techniques: A Practical Approach" (D. Glover, ed.), p. 49. IRL Press, Oxford, 1985. 3 R. A. Young and R. W. Davis, Science 222, 778 (1983).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, lnc, All rights of reproduction in any form reserved.
[44]
DIRECT CLONING OF c D N A INSERTS
509
Screening of phage DNA libraries for positive plaques is relatively easy with the phage lifting technique of Benton and Davis .4 However, isolating recombinant cDNA inserts from the phage clones of interest can be a tedious task. The burst sizes of the phages and the consequent yields of the phage DNA are often variable. Furthermore, the cDNA insert represents only a small fraction [approximately 2% for a 1 kilobase pair (kbp) insert] of the phage DNA, due to the much larger size of the phage vector arms. Thus, the desired cDNA inserts are usually subcloned from the positive phages into appropriate plasmid or M13 vectors for large-scale production. The cDNA insert isolated from the subcloning vector is then subjected to restriction enzyme mapping, Southern blotting and hybridization, or nucleotide sequencing. Principle of Method The Escherichia coli strain Y1088 commonly used for phage hgt infection carries the 6.1-kbp plasmid pMC9 endogenously, z This plasmid is a pBR322 derivative with the !.7-kbp lacZ and lacI genes inserted into the unique EcoRI restriction enzyme site. 5 We showed that the endogenous pMC9 DNA is extruded during the lysis of infected bacteria and can be copurified with the phage DNA. 6 Thus, the pBR322 portion of the pMC9 plasmid could be used directly for cloning the phage cDNA inserts. We have previously described a method in which cDNA inserts from hgt phages were cloned directly into pBR322. 6 This method bypassed the sometimes tedious and time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method can be used to clone DNA inserts derived from other phage vectors when bacteria containing endogenous pBR322 or other plasmid vectors are used as host cells. In this chapter we describe the protocol in more detail and show its application when combined with the polymerase chain reaction 7 (PCR) to characterize clones directly from single bacterial colonies. Materials
Solutions and Reagents NZY: 1% (w/v) NZ-amine, 0.5% (w/v) yeast extract, 85 mM NaC1, 10 mM MgCI2; autoclave 4 W. D. Benton and R. W. Davis, Science 196, 180 (1977). M. P. Calos, J. S. Lebkowski, and M. R. Botchan, Proc. Natl. Acad. Sci. U.S.A. 80, 3015 (1983). 6 I.-M. Chiu and K. Lehtoma, Gene Anal. Tech. Appl. 7, 18 (1990). 7 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Science 230, 1350 (1985).
510
VECTORSFOR CLONINGGENES
[44]
YT: 1% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, 0.17 M NaC1; autoclave SOC: 2% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, 10 mM NaCI, 2.5 mM KCI, autoclave; 10 mM MgClz, 10 mM MgSO4,20 mM glucose; filter sterilize PSB: 0.1 M NaC1, 10 mM Tris-HCl, pH 7.4, 10 mM MgCI2, 0.05% (w/v) gelatin; autoclave CaMg: 10 mM CaCI2, 10 mM MgCI2; filter sterilize IPTG: 100 mM isopropyl-fl-D-thiogalactoside in water; store at - 20° X-GaI: 2% (w/v) 5-bromo-4-chloro-3-idolyl-fl-D-galactoside in dimethylformamide (DMF); store at - 2 0 ° T1E0.2 : 1 mM Tris-HCl, pH 8.0, 0.2 mM ethylenediaminetetraacetic acid (EDTA) CIA: Chloroform-isoamyl alcohol (24: 1, v/v). 10 x ligation buffer: 0.66 M Tris-HCl, pH 7.5, 50 mM MgCI2, 10 mM ATP, 10 mM dithiothreitol (DTT); store at - 2 0 °
Enzymes Most of the restriction enzymes we have used were purchased from either New England BioLabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). T4 DNA ligase was obtained from Boehringer Mannheim. Lysozyme (Sigma, St. Louis, MO) was stored at 4° and dissolved immediately before use. RNase A was obtained from Boehringer Mannheim and the stock solution at 20 mg/ml was boiled for 15 min and stored at - 2 0 °. DNase I was also obtained from Boehringer Mannheim. Taq polymerase and the GeneAmp kit for PCR were obtained from PerkinElmer Cetus (Norwalk, CT).
Methods
Infection and Growth of Phage 1. Pick plaque containing a positive hgt phage clone with a pipette and transfer to a 1-dram vial. Rock overnight in 1 ml PSB. 2. In a sterile 15-ml culture tube, mix 100 /zl CaMg, 100 /xl Y1088 bacteria from an overnight culture, and 100 tzl of hgt phage from a plug or from a cleared lysate. 3. Incubate at 37° for 30 min. 4. Add the mixture to 50 ml NZY medium containing 50/xg/ml ampicillin in a 250-ml Erlenmeyer flask. Shake at 300 rpm at 37° overnight.
[44]
DIRECT CLONING OF c D N A INSERTS
511
5. Spin the bacteria culture at 3000 rpm in RT6000B centrifuge (Sorvail, Wilmington, DE) at 4 ° for 15 min to clear the lysate of bacterial debris. 6. Transfer the supernatant to a 50-ml polypropylene tube, add a few drops of chloroform to the phage lysate, and store at 4 °.
Phage D N A Extraction 1. Add 10/~g/ml DNase I and 50/zg/ml RNase A to 25 ml of phage lysate. 2. Incubate at 37 ° for 1 hr. 3. Extract the lysate twice with an equal volume of phenol. Rock on a Labquake (Labindustries, Inc., Berkeley, CA) for 15 min, then spin at 3000 rpm in the RT6000B at 20 ° for 15 min. Recover the aqueous layer, avoiding the white interface. 4. Extract the lysate once with an equal volume of CIA. 5. Add 1/20 vol of 5 M NaC1 and 2.5 vol of ethanol. Precipitate at - 8 0 ° for 1 hr. 6. Spin at 6000 rpm in an HB-4 rotor (Sorvall) at 4 ° for 30 min. 7. Dry the pellet in a Speed-Vac concentrator (Savant, Farmingdale, NY) and resuspend in 800/~1 of T1E0. 2. 8. Add 50/~g/ml RNase A. Incubate at 37 ° for 1 hr. 9. Extract once with phenol, then once with CIA. 10. Precipitate with 2.5 vol of ethanol at - 80 ° for 30 min. 11. Spin at 15,000 rpm in an MTX-150 microcentrifuge (Tomy, Peninsula Laboratories, Inc., Belmont, CA) at 4 ° for 15 min. 12. Dry the pellet in a Speed-Vac concentrator and resuspend in 200 /zl T1Eo. 2 . Ligation and Transformation 1. Digest 2.5/~g phage D N A with EcoRI at 37 ° for 3 hr. 2. Add 2 ~1 of l0 x ligation buffer and 1 unit of T4 D N A ligase in a total volume of 20/~1. Incubate at 14° for 16 hr. 3. Add 100 ng ofligated D N A to 50/zl of competent E. coli DH5o~ cells (Bethesda Research Laboratories, Gaithersburg, MD). Incubate on ice for 30 min. 4. Heat shock the cells at 37 ° for 45 sec. Incubate on ice for 2 rain. 5. Add 950/~1 SOC and shake at 225 rpm at 37 ° for 1 hr. 6. Plate 200/~1 of the cells with 20/~1 of 100 m M IPTG and 60 p.1 of 2% (w/v) X-Gal onto YT plates containing 50 t~g/ml ampicillin. Incubate at 37 ° overnight.
512
VECTORS FOR CLONING GENES
[44]
D N A Characterization by Alkaline Extraction Method 1. Pick white colonies and grow them in 20 ml of YT with ampicillin. Shake at 37 ° overnight. 2. Isolate plasmid DNA from 5 ml of the bacterial culture using the alkaline extraction method as described. 8 3. Digest plasmid DNA with appropriate restriction enzymes and analyze on an agarose gel containing 1 /~g/ml ethidium bromide. D N A Characterization by Boiling-Polymerase Chain Reaction Method I. Pick a bacterial colony from the plate and resuspend in 50/xl sterile H20. 2. Boil for 5 min. 3. Spin at 15,000 rpm in a microcentrifuge for 2 min. 4. Use 5/xl of supernatant as template for PCR. 5. Set up the PCR reaction: in a 20-/A volume, 10 mM Tris-HC1, pH 8.3, 50 mM KC1, 1.5 mM MgC12 , 0.1 mg/ml gelatin, 0.25 mM dNTP, 10 pmol each ofpBR322 EcoRI-specific oligonucleotides, 1 unit Taq polymerase, and 5/xl template DNA are overlaid with 20/xl mineral oil. 6. Reactions was subjected to 30 rounds of temperature cycling: 94° for I min, 45 ° for 1 min, 72° for 2 min, and a final 7 min/72 ° step in a Perkin-Elmer Cetus DNA thermal cycler. 7. Five microliters of each reaction product are analyzed by agarose gel electrophoresis.
Results
A phage clone containing a 1.4-kbp cDNA insert in hgtl 1 coding for human acidic fibroblast growth factor (aFGF) was purified from a brainstem cDNA library and designated hgtKL416. 9 The library was constructed by cloning the cDNA inserts into the unique EcoRI site of hgtl 1. ~0 To show that the cDNA insert from hgtKL416 could be subcloned directly, the phage DNA was digested with EcoRI and then religated without adding exogenous plasmid vector. The ligated DNA was then transformed into competent E. coli DH5a cells. The transformed cells were selected for ampicillin resistance. The endogenous pMC9 and recombinant clones were 8 H. C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979). 9 I.-M. Chiu, W.-P. Wang, and K. Lehtoma, Oncogene 5, 755 (1990). ~0F. DeFerra, H. Engh, L. Hudson, J. Kamholz, C. Puckett, and R. A. Lazzarini, Cell (Cambridge, Mass.) 43, 721 (1985).
[44]
DIRECT CLONING OF c D N A INSERTS
MI
513
25456M -25.1 kbp --4.4
-2.:5 --I.:55 -0.87 -0.60 FIG. 1. Gel electrophoresis of plasmid DNA containing the 1.4-kbp cDNA insert. XgtKL416 DNA was digested with EcoRI and ligated without the addition of any exogenous plasmid DNA. DNA was isolated from six white colonies, digested with EcoRI, and analyzed on a 1.0% agarose gel. Two of them showed the successful cloning of the 1.4-kbp cDNA of interest (lanes 2 and 4). The marker used is h DNA digested with HindIII and cbX DNA digested with HaeIII (lane M).
distinguished by blue-white colony selection in the presence of IPTG and X-Gal. The white colonies could contain either pBR322 or its derivative with the cDNA insert of interest. 6 DNA was extracted from six white colonies using the alkaline extraction method, s digested with EcoRI, and analyzed on an agarose gel. A unique DNA band of 4.4 kbp was observed in four of the six clones. Two clones (clones 2 and 4) contained a 4.4-kbp EcoRI fragment and the desired !.4-kbp cDNA insert (Fig. l, lanes 2 and 4). When the phage DNA was digested with EcoRI and incubated in the absence of T4 DNA ligase, no colonies were produced after transformation. 6 The size of the EcoRI fragments detected in all transformants analyzed (4.4 kbp) is consistent with the size of the pBR322 DNA. As a first step to verify the identity of the pBR322 plasmid vector in these six clones, the bacteria were shown to be tetracycline resistant when streaked out on YT plates containing 15/~g/ml tetracycline. In contrast, the tetracyclinesensitive pBluescript clone did not grow on the tetracycline medium (data not shown). Additional evidence was obtained by digesting the plasmid DNA with Hinfl and analyzing it on a 3% (w/v) NuSieve (FMC BioProducts, Rockland, ME) agarose gel. 6 HinfI generates l0 characteristic restriction fragments from pBR322. The data obtained from HinfI digestion of clones 2 and 4 are consistent with these clones being pBR322 derivatives containing a 1.4-kbp DNA fragment cloned into the unique EcoRI site.
514
VECTORSFOR CLONINGGENES
[44]
The insert was shown to be the same as that derived from hgtKL416 by restriction enzyme mapping as well as by Southern blotting and hybridization analysis (data not shown). The pBR322 DNA in clones 2 and 4 is most likely to be derived from pMC9, which is present in the host YI088. The endogenous plasmid DNA may be purified along with the phage DNA during the isolation process. If this is correct, we should be able to identify clones containing cDNA inserts using the PCR directly on the single bacterial colonies.ll A pair of oligonucleotide primers flanking the E c o R I site of pBR322 were synthesized for the PCR. The sequences of the clockwise and counterclockwise primers are 5'-GTATCACGAGGCCCT-3' and 5'-GATAAGCTGTCAAAC-3', respectively. For these experiments, three phage clones containing the newt bek cDNA 12were isolated from a forelimb blastema cDNA library. 13Inserts of 1.9, 1.7, and 1.3 kbp were identified by E c o R I digestion of purified phage DNA. The phage DNA was digested with E c o R I , religated, and used to transform DH5a cells as described above. Four white colonies resulting from transformation with phage DNA containing the 1.3-kbp b e k cDNA insert were transferred to H20, boiled for 5 min, and used as DNA templates for the PCR. The reaction products were analyzed on a 1% (w/v) agarose gel and visualized by staining with ethidium bromide (Fig. 2A, lanes 1-4). A pBR322 colony (Fig. 2A, lane 5) and a colony containing pBR322 with the 1.4-kbp human aFGF cDNA insert (Fig. 2A, lane 6) were used as negative and positive controls, respectively. The PCR product from the positive clone was digested with B a m H I (Fig. 2B, lane 1) or H i n c l I (Fig. 2B, lane 2) to confirm the identity of the DNA insert. Simultaneously, the 1.9- and 1.7-kbp b e k cDNA inserts were subcloned into pBR322 using the same approach (data not shown). These results showed that it is possible to clone the phage cDNA insert simply by using the endogenous pBR322 plasmid. Discussion Several methods have been designed to analyze the cDNA insert directly from the phage clone. Double-stranded h DNA can serve as a template for sequencing using the chain termination method. 14The cDNA insert can be amplified for subcloning by the PCR.12 By incorporating the T7 RNA polymerase recognition sequence and the translation initiation i1 D. Gussow and T. Clackson, Nucleic Acids Res. 17, 4000 (1989). 12M. L. Poulin, H. Onda, R. A. Tassava, and I.-M. Chiu, unpublishedresults (1991). 13C. W. Ragsdale, M. Petkovich, P. B. Gates, P. Chambon, and J. P. Brockes, Nature (London) 341, 654 (1989). 14R. J. Zagursky,K. Baumeister, N. Lopez, and M. L. Berman, Gene Anal. Tech. 2, 89 (1985).
[44]
DIRECT CLONING OF c D N A INSERTS
A
25
B
456M --23.1kbp
!
1 2
515
M
--25.1 kbp
--4.4 --2.5 --I.55 --0.87 --0.60
--4.4 --2.5 --I.55 --0.87 --0.60 --0.28
FIG. 2. Gel electrophoresis of the 1.3-kbp PCR product. (A) Phage DNA containing the 1.3-kbp newt bek cDNA insert ~2 was digested with EcoRI, religated, and transformed into DH5ct cells. White colonies (lanes 1-4) were transferred to 50 p,l of H20, boiled for 5 rain, and used as templates for the PCR in a 20-/~1 volume. Lanes 5 and 6 represent pBR322 and a pBR322 derivative containing the 1.4-kbp human aFGF cDNA insert. The PCR products (5 /zl) were analyzed on a 1% agarose gel. The arrowhead indicates the position of the expected PCR product from the bek cDNA clone. (B) The PCR product from the positive bek cDNA clone was digested with BamHI (lane 1) or HincII (lane 2) and analyzed on an agarose gel. The marker used is X DNA digested with HindIII and cbX DNA digested with HaeIII (lane M).
codon into a primer, the amplification by the PCR has allowed subsequent in vitro transcription and translation of the phage cDNA inserts into protein.15 However, it is still desirable, for economical reasons, to clone the cDNA inserts into appropriate plasmid or MI3 vectors for large-scale production or other analyses such as site-directed in vitro mutagenesis, deletional cloning for sequencing purposes, or transfection studies. A h insertion-type cDNA cloning vector, h ZAP, has been introduced. 16In E. coli, a phagemid contained within the X ZAP vector can be excised by superinfection with fl or M13 helper phage. This excision process also eliminates the need to subclone DNA inserts from the X phage into a subcloning plasmid vector. In spite of this, the majority of the cDNA libraries currently available are constructed in Xgt vectors. Thus, direct cloning of cDNA inserts from Xgt phage DNA into a plasmid is quite useful. 15 B. K. Nishikawa, D. M. Fowlkes, and B. K. Kay, BioTechniques 7, 730 (1989). 16 j. M. Short, J. M. Fernandez, J. A. Sorge, and W. D. Huse, Nucleic Acids Res. 16, 7583 (1988).
516
VECTORS FOR CLONING GENES
[44]
Escherichia coli Y1088 contains the plasmid pMC9, which represses expression of foreign genes that might be detrimental to host cells and phage growth. 3 The entire l a d gene and the amino-terminal portion of the lacZ (coding for fl-galactosidase) gene are contained in the 1.7-kbp EcoRI fragment of pMC9. 5 The lacI gene codes for the repressor of the lactose (lac) operon. The sequence between the l a d and lacZ genes represents the lac control region. The blue-white colony selection makes it possible to distinguish the parental pMC9 plasmid from the recombinant ones. We have employed this property to establish a direct method of cloning cDNA inserts from the phage DNA preparation without supplying a cloning plasmid vector exogenously. Ten to 15% of the colonies generated by this method are blue, representing the 1.7-kbp l a d and lacZ genes religated into pBR322. The molar ratio of phage DNA versus pMC9 DNA in the phage DNA preparation is rather high, as evidenced by the observation that 1 positive clone was identified per 3 to 12 white colonies analyzed6'1z (also see Figs. 1 and 2). This high molar ratio made it possible to analyze white colonies for the cDNA insert directly. This method bypassed the time-consuming procedures of preparing plasmid DNA, digesting it with restriction enzymes, and dephosphorylating it, thus significantly simplifying the subcloning process. The method is likely to be extended to the cloning of inserts from other h vectors when bacteria host contains pMC9 (i.e., Y1088, Y1089, or YI090) 2 or other plasmid vectors. More importantly, combination of this method with the PCR method, which allows the analysis of DNA directly from single bacterial colonies by boiling in H20, made it practical to clone several cDNA inserts simultaneously. Acknowledgments This work was supported in part by grants from the National Institutes of Health (R01 CA45611 and P30 CA 16058)and the March of Dimes Birth Defects Foundation(No. 6-549). I.-M.C. is a recipient of the Research Career DevelopmentAward (K04 CA01369)from the National Institutes of Health.
VECTORS
FOR CLONING
GENES
[44]
permit rapid recovery of the target gene from the h phage for sequence identification of selected mutations. The original h ZAP vector has also been modified for construction of a combination prokaryotic/eukaryotic expression vector. 3 This system should permit direct, high-efficiency transfection with a library of h phage particles and screening of eukaryotic cells. 3° Following phenotypic selection of the desired clones in the eukaryotic cells, the appropriate phage can be recovered by direct packaging of the h phage from the transfected cell genomic DNA. 3'31 The cDNA can then be excised into a plasmid molecule designed for rapid clone characterization and expression in E. coli cells. These procedures can also be coupled with subtractive hybridization techniques to further reduce the complexity of screening in eukaryotic cells. Modifications of the h ZAP vector currently underway will further increase its efficiency and versatility. 3o H. Okayama and P. Berg, Mol. Cell. Biol. 5, 1136 (1985). 31 S. W. Kohler, G. S. Provost, P. L. Kretz, M. J. Dycaico, J. A. Sorge, and J. M. Short, Nucleic Acids Res. 18, 3007 (1990).
[44] C l o n i n g o f C o m p l e m e n t a r y D N A I n s e r t s f r o m P h a g e D N A D i r e c t l y into P l a s m i d V e c t o r By ING-MING C H I U , KIRSTEN LEHTOMA, and MATTHEW L. POULIN Cloning vectors derived from bacteriophage h are used frequently in the construction of both cDNA and genomic DNA libraries. 1 Both hgtl0 and hgtl I have been used quite extensively for constructing cDNA libraries. 2 For hgtl 1, the cDNA inserts are cloned into the unique EcoRI site within the lacZ gene and can be expressed as fusion proteins with /3galactosidase. 2 Thus, the cDNA clones can be obtained by screening with the antibody probe generated against the protein of interest.3 Alternatively, a ligand having high affinity with the protein of interest may also be used as a probe. Moreover, cDNA libraries constructed in hgtl0 or hgtl 1 can be screened by hybridization with an oligonucleotide or DNA fragment containing the DNA sequence of interest. i j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 2 T. V. Huynh, R. A. Young, and R. W. Davis, in " D N A Cloning Techniques: A Practical Approach" (D. Glover, ed.), p. 49. IRL Press, Oxford, 1985. 3 R. A. Young and R. W. Davis, Science 222, 778 (1983).
METHODS IN ENZYMOLOGY, VOL. 216
Copyright © 1992 by Academic Press, lnc, All rights of reproduction in any form reserved.
[44]
DIRECT CLONING OF c D N A INSERTS
509
Screening of phage DNA libraries for positive plaques is relatively easy with the phage lifting technique of Benton and Davis .4 However, isolating recombinant cDNA inserts from the phage clones of interest can be a tedious task. The burst sizes of the phages and the consequent yields of the phage DNA are often variable. Furthermore, the cDNA insert represents only a small fraction [approximately 2% for a 1 kilobase pair (kbp) insert] of the phage DNA, due to the much larger size of the phage vector arms. Thus, the desired cDNA inserts are usually subcloned from the positive phages into appropriate plasmid or M13 vectors for large-scale production. The cDNA insert isolated from the subcloning vector is then subjected to restriction enzyme mapping, Southern blotting and hybridization, or nucleotide sequencing. Principle of Method The Escherichia coli strain Y1088 commonly used for phage hgt infection carries the 6.1-kbp plasmid pMC9 endogenously, z This plasmid is a pBR322 derivative with the !.7-kbp lacZ and lacI genes inserted into the unique EcoRI restriction enzyme site. 5 We showed that the endogenous pMC9 DNA is extruded during the lysis of infected bacteria and can be copurified with the phage DNA. 6 Thus, the pBR322 portion of the pMC9 plasmid could be used directly for cloning the phage cDNA inserts. We have previously described a method in which cDNA inserts from hgt phages were cloned directly into pBR322. 6 This method bypassed the sometimes tedious and time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method can be used to clone DNA inserts derived from other phage vectors when bacteria containing endogenous pBR322 or other plasmid vectors are used as host cells. In this chapter we describe the protocol in more detail and show its application when combined with the polymerase chain reaction 7 (PCR) to characterize clones directly from single bacterial colonies. Materials
Solutions and Reagents NZY: 1% (w/v) NZ-amine, 0.5% (w/v) yeast extract, 85 mM NaC1, 10 mM MgCI2; autoclave 4 W. D. Benton and R. W. Davis, Science 196, 180 (1977). M. P. Calos, J. S. Lebkowski, and M. R. Botchan, Proc. Natl. Acad. Sci. U.S.A. 80, 3015 (1983). 6 I.-M. Chiu and K. Lehtoma, Gene Anal. Tech. Appl. 7, 18 (1990). 7 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Science 230, 1350 (1985).
510
VECTORSFOR CLONINGGENES
[44]
YT: 1% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, 0.17 M NaC1; autoclave SOC: 2% (w/v) Bacto-tryptone, 0.5% (w/v) yeast extract, 10 mM NaCI, 2.5 mM KCI, autoclave; 10 mM MgClz, 10 mM MgSO4,20 mM glucose; filter sterilize PSB: 0.1 M NaC1, 10 mM Tris-HCl, pH 7.4, 10 mM MgCI2, 0.05% (w/v) gelatin; autoclave CaMg: 10 mM CaCI2, 10 mM MgCI2; filter sterilize IPTG: 100 mM isopropyl-fl-D-thiogalactoside in water; store at - 20° X-GaI: 2% (w/v) 5-bromo-4-chloro-3-idolyl-fl-D-galactoside in dimethylformamide (DMF); store at - 2 0 ° T1E0.2 : 1 mM Tris-HCl, pH 8.0, 0.2 mM ethylenediaminetetraacetic acid (EDTA) CIA: Chloroform-isoamyl alcohol (24: 1, v/v). 10 x ligation buffer: 0.66 M Tris-HCl, pH 7.5, 50 mM MgCI2, 10 mM ATP, 10 mM dithiothreitol (DTT); store at - 2 0 °
Enzymes Most of the restriction enzymes we have used were purchased from either New England BioLabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). T4 DNA ligase was obtained from Boehringer Mannheim. Lysozyme (Sigma, St. Louis, MO) was stored at 4° and dissolved immediately before use. RNase A was obtained from Boehringer Mannheim and the stock solution at 20 mg/ml was boiled for 15 min and stored at - 2 0 °. DNase I was also obtained from Boehringer Mannheim. Taq polymerase and the GeneAmp kit for PCR were obtained from PerkinElmer Cetus (Norwalk, CT).
Methods
Infection and Growth of Phage 1. Pick plaque containing a positive hgt phage clone with a pipette and transfer to a 1-dram vial. Rock overnight in 1 ml PSB. 2. In a sterile 15-ml culture tube, mix 100 /zl CaMg, 100 /xl Y1088 bacteria from an overnight culture, and 100 tzl of hgt phage from a plug or from a cleared lysate. 3. Incubate at 37° for 30 min. 4. Add the mixture to 50 ml NZY medium containing 50/xg/ml ampicillin in a 250-ml Erlenmeyer flask. Shake at 300 rpm at 37° overnight.
[44]
DIRECT CLONING OF c D N A INSERTS
511
5. Spin the bacteria culture at 3000 rpm in RT6000B centrifuge (Sorvail, Wilmington, DE) at 4 ° for 15 min to clear the lysate of bacterial debris. 6. Transfer the supernatant to a 50-ml polypropylene tube, add a few drops of chloroform to the phage lysate, and store at 4 °.
Phage D N A Extraction 1. Add 10/~g/ml DNase I and 50/zg/ml RNase A to 25 ml of phage lysate. 2. Incubate at 37 ° for 1 hr. 3. Extract the lysate twice with an equal volume of phenol. Rock on a Labquake (Labindustries, Inc., Berkeley, CA) for 15 min, then spin at 3000 rpm in the RT6000B at 20 ° for 15 min. Recover the aqueous layer, avoiding the white interface. 4. Extract the lysate once with an equal volume of CIA. 5. Add 1/20 vol of 5 M NaC1 and 2.5 vol of ethanol. Precipitate at - 8 0 ° for 1 hr. 6. Spin at 6000 rpm in an HB-4 rotor (Sorvall) at 4 ° for 30 min. 7. Dry the pellet in a Speed-Vac concentrator (Savant, Farmingdale, NY) and resuspend in 800/~1 of T1E0. 2. 8. Add 50/~g/ml RNase A. Incubate at 37 ° for 1 hr. 9. Extract once with phenol, then once with CIA. 10. Precipitate with 2.5 vol of ethanol at - 80 ° for 30 min. 11. Spin at 15,000 rpm in an MTX-150 microcentrifuge (Tomy, Peninsula Laboratories, Inc., Belmont, CA) at 4 ° for 15 min. 12. Dry the pellet in a Speed-Vac concentrator and resuspend in 200 /zl T1Eo. 2 . Ligation and Transformation 1. Digest 2.5/~g phage D N A with EcoRI at 37 ° for 3 hr. 2. Add 2 ~1 of l0 x ligation buffer and 1 unit of T4 D N A ligase in a total volume of 20/~1. Incubate at 14° for 16 hr. 3. Add 100 ng ofligated D N A to 50/zl of competent E. coli DH5o~ cells (Bethesda Research Laboratories, Gaithersburg, MD). Incubate on ice for 30 min. 4. Heat shock the cells at 37 ° for 45 sec. Incubate on ice for 2 rain. 5. Add 950/~1 SOC and shake at 225 rpm at 37 ° for 1 hr. 6. Plate 200/~1 of the cells with 20/~1 of 100 m M IPTG and 60 p.1 of 2% (w/v) X-Gal onto YT plates containing 50 t~g/ml ampicillin. Incubate at 37 ° overnight.
512
VECTORS FOR CLONING GENES
[44]
D N A Characterization by Alkaline Extraction Method 1. Pick white colonies and grow them in 20 ml of YT with ampicillin. Shake at 37 ° overnight. 2. Isolate plasmid DNA from 5 ml of the bacterial culture using the alkaline extraction method as described. 8 3. Digest plasmid DNA with appropriate restriction enzymes and analyze on an agarose gel containing 1 /~g/ml ethidium bromide. D N A Characterization by Boiling-Polymerase Chain Reaction Method I. Pick a bacterial colony from the plate and resuspend in 50/xl sterile H20. 2. Boil for 5 min. 3. Spin at 15,000 rpm in a microcentrifuge for 2 min. 4. Use 5/xl of supernatant as template for PCR. 5. Set up the PCR reaction: in a 20-/A volume, 10 mM Tris-HC1, pH 8.3, 50 mM KC1, 1.5 mM MgC12 , 0.1 mg/ml gelatin, 0.25 mM dNTP, 10 pmol each ofpBR322 EcoRI-specific oligonucleotides, 1 unit Taq polymerase, and 5/xl template DNA are overlaid with 20/xl mineral oil. 6. Reactions was subjected to 30 rounds of temperature cycling: 94° for I min, 45 ° for 1 min, 72° for 2 min, and a final 7 min/72 ° step in a Perkin-Elmer Cetus DNA thermal cycler. 7. Five microliters of each reaction product are analyzed by agarose gel electrophoresis.
Results
A phage clone containing a 1.4-kbp cDNA insert in hgtl 1 coding for human acidic fibroblast growth factor (aFGF) was purified from a brainstem cDNA library and designated hgtKL416. 9 The library was constructed by cloning the cDNA inserts into the unique EcoRI site of hgtl 1. ~0 To show that the cDNA insert from hgtKL416 could be subcloned directly, the phage DNA was digested with EcoRI and then religated without adding exogenous plasmid vector. The ligated DNA was then transformed into competent E. coli DH5a cells. The transformed cells were selected for ampicillin resistance. The endogenous pMC9 and recombinant clones were 8 H. C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979). 9 I.-M. Chiu, W.-P. Wang, and K. Lehtoma, Oncogene 5, 755 (1990). ~0F. DeFerra, H. Engh, L. Hudson, J. Kamholz, C. Puckett, and R. A. Lazzarini, Cell (Cambridge, Mass.) 43, 721 (1985).
[44]
DIRECT CLONING OF c D N A INSERTS
MI
513
25456M -25.1 kbp --4.4
-2.:5 --I.:55 -0.87 -0.60 FIG. 1. Gel electrophoresis of plasmid DNA containing the 1.4-kbp cDNA insert. XgtKL416 DNA was digested with EcoRI and ligated without the addition of any exogenous plasmid DNA. DNA was isolated from six white colonies, digested with EcoRI, and analyzed on a 1.0% agarose gel. Two of them showed the successful cloning of the 1.4-kbp cDNA of interest (lanes 2 and 4). The marker used is h DNA digested with HindIII and cbX DNA digested with HaeIII (lane M).
distinguished by blue-white colony selection in the presence of IPTG and X-Gal. The white colonies could contain either pBR322 or its derivative with the cDNA insert of interest. 6 DNA was extracted from six white colonies using the alkaline extraction method, s digested with EcoRI, and analyzed on an agarose gel. A unique DNA band of 4.4 kbp was observed in four of the six clones. Two clones (clones 2 and 4) contained a 4.4-kbp EcoRI fragment and the desired !.4-kbp cDNA insert (Fig. l, lanes 2 and 4). When the phage DNA was digested with EcoRI and incubated in the absence of T4 DNA ligase, no colonies were produced after transformation. 6 The size of the EcoRI fragments detected in all transformants analyzed (4.4 kbp) is consistent with the size of the pBR322 DNA. As a first step to verify the identity of the pBR322 plasmid vector in these six clones, the bacteria were shown to be tetracycline resistant when streaked out on YT plates containing 15/~g/ml tetracycline. In contrast, the tetracyclinesensitive pBluescript clone did not grow on the tetracycline medium (data not shown). Additional evidence was obtained by digesting the plasmid DNA with Hinfl and analyzing it on a 3% (w/v) NuSieve (FMC BioProducts, Rockland, ME) agarose gel. 6 HinfI generates l0 characteristic restriction fragments from pBR322. The data obtained from HinfI digestion of clones 2 and 4 are consistent with these clones being pBR322 derivatives containing a 1.4-kbp DNA fragment cloned into the unique EcoRI site.
514
VECTORSFOR CLONINGGENES
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The insert was shown to be the same as that derived from hgtKL416 by restriction enzyme mapping as well as by Southern blotting and hybridization analysis (data not shown). The pBR322 DNA in clones 2 and 4 is most likely to be derived from pMC9, which is present in the host YI088. The endogenous plasmid DNA may be purified along with the phage DNA during the isolation process. If this is correct, we should be able to identify clones containing cDNA inserts using the PCR directly on the single bacterial colonies.ll A pair of oligonucleotide primers flanking the E c o R I site of pBR322 were synthesized for the PCR. The sequences of the clockwise and counterclockwise primers are 5'-GTATCACGAGGCCCT-3' and 5'-GATAAGCTGTCAAAC-3', respectively. For these experiments, three phage clones containing the newt bek cDNA 12were isolated from a forelimb blastema cDNA library. 13Inserts of 1.9, 1.7, and 1.3 kbp were identified by E c o R I digestion of purified phage DNA. The phage DNA was digested with E c o R I , religated, and used to transform DH5a cells as described above. Four white colonies resulting from transformation with phage DNA containing the 1.3-kbp b e k cDNA insert were transferred to H20, boiled for 5 min, and used as DNA templates for the PCR. The reaction products were analyzed on a 1% (w/v) agarose gel and visualized by staining with ethidium bromide (Fig. 2A, lanes 1-4). A pBR322 colony (Fig. 2A, lane 5) and a colony containing pBR322 with the 1.4-kbp human aFGF cDNA insert (Fig. 2A, lane 6) were used as negative and positive controls, respectively. The PCR product from the positive clone was digested with B a m H I (Fig. 2B, lane 1) or H i n c l I (Fig. 2B, lane 2) to confirm the identity of the DNA insert. Simultaneously, the 1.9- and 1.7-kbp b e k cDNA inserts were subcloned into pBR322 using the same approach (data not shown). These results showed that it is possible to clone the phage cDNA insert simply by using the endogenous pBR322 plasmid. Discussion Several methods have been designed to analyze the cDNA insert directly from the phage clone. Double-stranded h DNA can serve as a template for sequencing using the chain termination method. 14The cDNA insert can be amplified for subcloning by the PCR.12 By incorporating the T7 RNA polymerase recognition sequence and the translation initiation i1 D. Gussow and T. Clackson, Nucleic Acids Res. 17, 4000 (1989). 12M. L. Poulin, H. Onda, R. A. Tassava, and I.-M. Chiu, unpublishedresults (1991). 13C. W. Ragsdale, M. Petkovich, P. B. Gates, P. Chambon, and J. P. Brockes, Nature (London) 341, 654 (1989). 14R. J. Zagursky,K. Baumeister, N. Lopez, and M. L. Berman, Gene Anal. Tech. 2, 89 (1985).
[44]
DIRECT CLONING OF c D N A INSERTS
A
25
B
456M --23.1kbp
!
1 2
515
M
--25.1 kbp
--4.4 --2.5 --I.55 --0.87 --0.60
--4.4 --2.5 --I.55 --0.87 --0.60 --0.28
FIG. 2. Gel electrophoresis of the 1.3-kbp PCR product. (A) Phage DNA containing the 1.3-kbp newt bek cDNA insert ~2 was digested with EcoRI, religated, and transformed into DH5ct cells. White colonies (lanes 1-4) were transferred to 50 p,l of H20, boiled for 5 rain, and used as templates for the PCR in a 20-/~1 volume. Lanes 5 and 6 represent pBR322 and a pBR322 derivative containing the 1.4-kbp human aFGF cDNA insert. The PCR products (5 /zl) were analyzed on a 1% agarose gel. The arrowhead indicates the position of the expected PCR product from the bek cDNA clone. (B) The PCR product from the positive bek cDNA clone was digested with BamHI (lane 1) or HincII (lane 2) and analyzed on an agarose gel. The marker used is X DNA digested with HindIII and cbX DNA digested with HaeIII (lane M).
codon into a primer, the amplification by the PCR has allowed subsequent in vitro transcription and translation of the phage cDNA inserts into protein.15 However, it is still desirable, for economical reasons, to clone the cDNA inserts into appropriate plasmid or MI3 vectors for large-scale production or other analyses such as site-directed in vitro mutagenesis, deletional cloning for sequencing purposes, or transfection studies. A h insertion-type cDNA cloning vector, h ZAP, has been introduced. 16In E. coli, a phagemid contained within the X ZAP vector can be excised by superinfection with fl or M13 helper phage. This excision process also eliminates the need to subclone DNA inserts from the X phage into a subcloning plasmid vector. In spite of this, the majority of the cDNA libraries currently available are constructed in Xgt vectors. Thus, direct cloning of cDNA inserts from Xgt phage DNA into a plasmid is quite useful. 15 B. K. Nishikawa, D. M. Fowlkes, and B. K. Kay, BioTechniques 7, 730 (1989). 16 j. M. Short, J. M. Fernandez, J. A. Sorge, and W. D. Huse, Nucleic Acids Res. 16, 7583 (1988).
516
VECTORS FOR CLONING GENES
[44]
Escherichia coli Y1088 contains the plasmid pMC9, which represses expression of foreign genes that might be detrimental to host cells and phage growth. 3 The entire l a d gene and the amino-terminal portion of the lacZ (coding for fl-galactosidase) gene are contained in the 1.7-kbp EcoRI fragment of pMC9. 5 The lacI gene codes for the repressor of the lactose (lac) operon. The sequence between the l a d and lacZ genes represents the lac control region. The blue-white colony selection makes it possible to distinguish the parental pMC9 plasmid from the recombinant ones. We have employed this property to establish a direct method of cloning cDNA inserts from the phage DNA preparation without supplying a cloning plasmid vector exogenously. Ten to 15% of the colonies generated by this method are blue, representing the 1.7-kbp l a d and lacZ genes religated into pBR322. The molar ratio of phage DNA versus pMC9 DNA in the phage DNA preparation is rather high, as evidenced by the observation that 1 positive clone was identified per 3 to 12 white colonies analyzed6'1z (also see Figs. 1 and 2). This high molar ratio made it possible to analyze white colonies for the cDNA insert directly. This method bypassed the time-consuming procedures of preparing plasmid DNA, digesting it with restriction enzymes, and dephosphorylating it, thus significantly simplifying the subcloning process. The method is likely to be extended to the cloning of inserts from other h vectors when bacteria host contains pMC9 (i.e., Y1088, Y1089, or YI090) 2 or other plasmid vectors. More importantly, combination of this method with the PCR method, which allows the analysis of DNA directly from single bacterial colonies by boiling in H20, made it practical to clone several cDNA inserts simultaneously. Acknowledgments This work was supported in part by grants from the National Institutes of Health (R01 CA45611 and P30 CA 16058)and the March of Dimes Birth Defects Foundation(No. 6-549). I.-M.C. is a recipient of the Research Career DevelopmentAward (K04 CA01369)from the National Institutes of Health.
