[8]
DIRECT c D N A CLONING AND SCREENING OF MUTANTS
73
[8] D i r e c t C o m p l e m e n t a r y D N A Cloning a n d S c r e e n i n g of M u t a n t s Using P o l y m e r a s e C h a i n R e a c t i o n By YAWEN L. CHIANG
Introduction Conventional cDNA cloning and screening for mutations can be tedious and time consuming. In the early 1970s a few papers described DNA amplification using polymerase1-3 and Khorana et al. 2 suggested the fundamental theory behind the polymerase chain reaction (PCR). In 1984, K. Mullis developed the PCR and filed a patent in 1985 on the "process for amplifying nucleic acid sequences"; the patent was issued in 1987. In 1986, K. Mullis, J. Larrick, and I discussed the possibility of using the technique for direct cDNA cloning and for screening mutants for the variable region of immunoglobin heavy and light chains. We successfully developed the method, establishing a new approach for cloning antibody cDNA. The PCR amplifies defined regions of the genome by using oligomerdirected primer extension. The technique depends on the DNA replication enzyme (DNA polymerase) and the chain reaction made during the exponential amplification procedure. The reaction is efficient; only small numbers of cells and minute amounts of DNA are needed. Thus it is feasible to clone an antibody gene directly from small numbers of hybridoma cells. Because the PCR is very sensitive, there are numerous contamination considerations (such as the stability and control of reagents, primer design, and reaction sensitivity) that must be addressed. Many new methods and instruments for the PCR have been developed during the past few years. We will continue to find novel applications of the PCR--it is a powerful technique for modern molecular biology. Principle of Polymerase Chain Reaction Used for Direct cDNA Cloning The PCR is based on repeated cycles of high-temperature template denaturation, oligonucleotide primer annealing, and polymerase-depeni K. Kleppe, E. O h t s u k a , R. Kieppe, I. Molineux, and H. G. K h o r a n a , J. Mol. Biol. 56, 341 (1971). 2 H. G. K h o r a n a , K. L. Agarwal, H. Buchi, M. H. Caruthers, N. K. Gupta, K. Kleppe, A. K u m a r , E. O h t s u k a , U. L. RajBhandary, J. H. v a n de Sande, V. Sgaramella, T. Terao, H. W e b e r , a n d T. Y a m a d a , J. Mol. Biol. 72, 209 (1972). 3 A. Panet and H. G. K h o r a n a , J. Biol. Chem. 249, 5213 (1974).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright © 1992by AcademicPress, Inc. All fights of reproduction in any form reserved.
74
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[8]
dent extension. One can synthesize large quantities of a particular target DNA sequence in vitro. 4-6 By using reverse transcriptase, RNA can be transcribed into cDNA, which is then amplified using the Taq DNA polymerase. In our experiments, we also added the appropriate cloning site sequences with PCR primers to facilitate easy cDNA cloning into expression vectors. Because of the amplification capabilities and extreme sensitivity of the PCR, minute changes of nucleotides in clones and cell lines can be detected by using direct sequencing 7 immediately following cDNA amplification. Chamberlain et al.S detected the deletion of the Duchenne muscular dystrophy locus via multiplex DNA amplification, and Wrischnick et al. 9 also used direct sequencing of enzymatically amplified DNA to determine the length mutations in human mitochondrial DNA. The revolutionary changes in molecular biology, resulting from the development of sequencing and PCR techniques, have allowed us to efficiently clone the immunoglobulin heavy and light chain variable regions from hybridoma cells. The development of our reproducible experiments, combined with the knowledge of the immunoglobulin constant region sequences provided by Kabat et al., l° has made it possible to produce manmade antibodies without using conventional hybridoma techniques. (The immunoglobulin constant region is named as such because of its characteristic of conserved sequences.) Experimental Section Materials a n d M e t h o d s
A summary of the method is outlined in Fig. 1. A murine hybridoma cell line, 269-10F7 (IgG 1, K), that produces anti-human C5a monoclonal antibodies 11was used. From N-terminal amino acid microsequencing data, 4 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Science 230, 1350 (1985). 5 S. J. Scharf, G. T. Horn, and H. A. Erlich, Science 233, 1076 (1986). 6 K. B. Mullis and F. A. Faloona, this series, Vol. 155, p. 335. 7 U. B. Gyllensten and H. A. Erlich, Proc. Natl. Acad. Sci. U.S.A. 85, 7652 (1988). 8 j. S. Chamberlain, R. A. Gibbs, J. E. Rainer, P. N. Nguyen, and C. T. Caskey, Nucleic Acids Res. 16, 11141 (1988). 9 L. A. Wrischnick, R. G. Higuchi, M. Stoneking, H. A. Erlich, N. Arnheim, and A. C. Wilson, Nucleic Acids Res. 15(2), 529 (1987). l0 E. A. Kabat, T. T. Wu, M. Reid-Miller, H. M. Perry, and K. S. Gottesman (eds), "Sequences of Protein of Immunological Interest," 4th Ed., U.S. Department of Health and Human Services, Washington, D.C., 1987. 1i y . L. Chiang, R. Sheng-Dong, M. A. Brow, and J. W. Larrick, BioTechniques 7, 360 (1989).
[8l
DIRECT cDNA CLONINGAND SCREENINGOF MUTANTS
75
Mouse hybridoma cells
~ single-step extraction RNA reverse transcription cDNA
plificetionusing PCR using PCR primers: YC1 and YC2
cDNA:
L
vadable region of Ig
YCA and YCB
L
light chain
VL(mouse)
variable region of Ig heavy chain V. (mouse)
~ /
cloning into human constant region gene expression cassettes
VL C
-~
'
7 V H
(m°use)
CH(hUman)
mouse/human chimericAb FIG. 1. Schematic summary of the method used to make mouse/human chimeric antibody (Ab).
we determined that 269-10F7 belongs to mouse K light chain subgroup III and mouse IgG~ heavy chain subgroup IA. From the database of Kabat e t al., ~o stretches of invariant N-terminal amino acid residues for both heavy and light chains were recognized. Oligonucleotide primers for the D N A sequence coding for this region were designed and prepared for PCR. The primer sequences were determined according to the usual codon of the specific immunoglobulin subgroups. Depending on the experimental design, additional nucleotide sequences for convenient cloning sites can be used if necessary. We applied the rules and considerations for synthesizing oligomers and created two 5' end primers (sense primer), YC1 and YCA. YC1 was used for the immunoglobulin light chain gene. It is a 39-bp oligomer, 5'-
76
ISOLATION, SYNTHESIS,DETECTIONOF DNA AND RNA
[8]
CCCGAATTCGACATTGTGCTGACCCAATCTCCAGCTTCT-3'. YCA was used for the immunoglobulin heavy chain gene. It is a 36-bp oligomer, 5'-CCCGAATTCGATGTGCATCTTCAGGAGTCGGGACCT-3'. We then designed the 3' end primers (antisense primer) for the PCR. YC2 and YCB were synthesized. YC2 was used for the light chain gene. It is a 38-bp oligomer, 5'-CCGAATTCGATGGATACAGTTGGTGCAGCATCAGCCCG-3'. YCB was used for the heavy chain gene. It is a 39-bp oligomer, 5'-CCCGAATTCTGGATAGACAGATGGGGGTGTCGTTTTGGC-3'. By employing YC1 and YC2 in the DNA amplification reaction, the entire variable-region sequences of the immunoglobulin light chain gene for hybridoma 269-10F7 were made. Likewise, by using YCA and YCB, the entire variable-region sequences of the immunoglobulin heavy chain gene were made. These cDNA fragments will be ligated with DNA from the human light and heavy chain construct regions, and the DNA construct will be placed into an expression vector to produce mouse/human chimeric antibody. This approach to create cDNA fragments and to use it to produce antibody is much quicker than the conventional method of constructing a cDNA library containing the mouse light and heavy chain variable region. The method involves the following steps. Single-Step Preparation ofRNA. Total cellular RNA is prepared by an acid guanidinium thiocyanate-phenol-chloroform single-step extraction method. 12The RNAzoI method established by C 1NNA/BIOTECX Laboratories International, Inc. (Friendswood, TX) provides a good yield of clean R N A prepared from small amounts of tissue or cells within 3 hr. Reverse Transcription of RNA to Synthesize cDNA. First-strand cDNA synthesis is accomplished by priming the RNA with 1/.~M oligo(dT) (12- to 18-mer). A 10-/xl reaction volume contains a dried pellet of RNA, 200 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD), 0. ! mg/ml of bovine serum albumin (BSA), 0.5 mM concentrations of each dNTP (dGTP, dATP, dTTP, and dCTP), 0.25 units of RNasin, 50 mM Tris-HC1, pH 7.5, 75 m M KC1, l0 m M dithiothreitol (DTT), 3 mM MgC12, and 50 /zg/ml of actinomycin D. The reaction is carried out in a 37° water bath for 1 hr. cDNA Amplification Using the Polymerase Chain Reaction. In the same tube, cDNA amplification using the PCR is carried out in 25 to 35 cycles. The sense and antisense primers mentioned above (1/xM each), 0.5 mM dNTP, and 1 unit of Taq polymerase in reaction buffer are added and the volume brought to 50/xl. The final concentration of the reaction 12p. Chomczvnski and N. Sacchi. Anal. Biochem. 162. 156 (1987).
[8]
DIRECT c D N A CLONING AND SCREENING OF MUTANTS
77
buffer is 50 mM KCI, 1.5 mM MgC12 , 10 mM Tris-HC1 (pH 8.4), and 0.02% (w/v) gelatin. The repeated PCR cycles involve denaturing at 95° for 1 min, primer annealing at 55° for 1 min, and Taq polymerase reaction at 72° for 3 min. Polymerase Chain Reaction Product Analysis. One-tenth of the final volume is loaded onto an ethidium bromide-stained, 3% (w/v) Nusieve agarose, and 1% (w/v) Seakem agarose (FMC BioProducts, Rockland, ME) minigel and analyzed (Figs. 2 and 3).
Specific Experimental Considerations Some important practical procedures should be considered when using PCR techniques for direct cDNA cloning. It is important to design appropriate primers for the PCR reaction. Lathe ~3 reviewed some theoretical and practical considerations to be used when choosing synthetic oligonucleotide probes deduced from amino acid sequence data. Girgis et a l ) 4 generated DNA probes for peptides with highly degenerate codons using mixed primers in the PCR reaction. To minimize contamination problems, use a positive displacement pipetter and follow good laboratory procedures (including frequent changing of globes, careful aliquoting of reagents, and physically separating the preand post-PCR reactions). One needs to assess the results critically, repeat experiments to obtain consistent results, and select good positive controls. The sensitivity, stability, and control of reagents, and the selection of equipment, are important. Choose credible sources when purchasing commercially available standard reagents and equipment. All experimental parameters including annealing, extension, temperature, numbers of cycles, and enzyme-to-primer ratios must be optimized on an individual basis. One should avoid using excess DNA in the reaction. DNA from other, unrelated genes extracted from other cells should be included as a negative control. Other controls such as distilled H20 or Tris-ethylenediaminetetraacetic acid (EDTA) buffer should be used in reactions where there are no target sequences. A reaction that contains just the primer sequences should also be run. To verify the data, use different pairs of primers. To determine if there is other DNA contamination, primers and probes outside the 5' or 3' region of the PCR product should be made and checked using the Southern blot analysis data. cDNA, derived from all different types ofRNA (mRNA, tRNA, rRNA, total RNA, and viruses) and from very low copy number RNA target 13 R. Lathe, J. Mol. Biol. 11t3, 1 (1985). 14 S. L Girgis, M. Alevizaki, P. Denny, G. J. Ferrier, and S. Legon, Nucleic Acids Res. 16, 10371 (1988).
78
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A 1
2
3
[8]
4
4,92-369-246--
~p 123--
FIG. 2. PCR products from 25 cycles for the variable region of light chain. Lane 1,123-bp ladder marker; lane 2, PCR product from 106 cells; lane 3, PCR product from mouse Ig light chain cDNA clone obtained from conventional cDNA library; lane 4, no cells, only primers contained. s e q u e n c e s c a n b e a m p l i f i e d . W a n g e t al. t536 u s e d t h e r e v e r s e t r a n s c r i p t i o n r e a c t i o n p r o c e d u r e p u b l i s h e d in 1987 a n d d e v e l o p e d a G e n e A m p t h e r m o s t a b l e y T t h r e v e r s e t r a n s c r i p t a s e R N A P C R kit ( P e r k i n E l m e r C e t u s , E m e r y v i l l e , C A ) . B y u s i n g this kit a c D N A p r o d u c t u p to 1.3 k i l o b a s e s (kb) in l e n g t h c a n b e g e n e r a t e d . Other choices of methods and materials are available. A special techn o l o g y s e c t i o n h a s b e e n p u b l i s h e d ~7 t h a t d e s c r i b e s o t h e r o p t i o n s f o r t h e J5 A. M. Wang, M. V. Doyle, and D. F. Mark, Proc. Natl. Acad. Sci. U.S.A. 86, 9717 (1989). ~6E. S. Kawasaki and A. M. Wang, "PCR Technology" (H. A. Erlich, ed.), p. 89. Stockton Press, New York, 1989. 17Special Technology Section: Methods and Materials: Amplification of Nucleic Acid Sequences: The Choices Multiply, J. N I H Res. 3, 81 (1991).
[8]
DIRECT c D N A CLONING AND SCREENING OF MUTANTS
1
2
3
79
4
bp 1 3 5 3 872 603 -r-310 281 --7
FIG. 3. PCR products from 35 cycles for the variable region of heavy chains. Lane 1,300 ng +X174 HaelII marker; lane 2, PCR product from mouse Ig heavy chain cDNA clone obtained from conventional cDNA library; lane 3, PCR product from 106 cells; lane 4, no cells, only primers contained.
amplification of nucleic acid sequences as well as alternative thermal cyclers, primers and probes, thermostable DNA polymerase, reagents and reagent kits, miscellaneous products, and manufacturers. It is not necessary to use conventional M 13 subcloning for sequencing. The method of direct sequencing of the PCR product is established.9,18 Other advances in PCR have been reported elsewhere.19 18 C. Wong, C. E. Dowling, R. K. Saiki, R. G. Higuchi, H. A. Erlich, and H. H. Kazazian, Jr., Nature (London) 330, 384 (1987). 19 H. A. Erlich, D. Gelfand, and J. J. Sninsky, Science 252, 1643 (1991).
80
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[9]
Results A clear 369 bp expected band for Ig light chain cDNA fragment with the cloning site (Fig. 2) and a 390 bp expected band for Ig heavy chain cDNA fragment (Fig. 3) were obtained. The PCR products were subcloned into M13 followed by subsequent sequencing. 2° The cDNA cloning of the variable regions of heavy and light chains, were obtained by constructing a conventional cDNA library, has been published elsewhere. 11These cDNA clones were also sequenced. No mutations were found in the sequences obtained by either the cDNA clone from the conventional cDNA library or from the cDNA amplified fragments produced directly by the PCR of the hybridoma cell RNA. We are presently developing a method to synthesize chimeric mouse/ human monoclonal antibodies by using synthetic long oligomers ligated together containing just the mouse CDRs (complementarity-determining regions), and to synthesize mouse/human MAb containing fewer mouse sequences. Acknowledgments I would like to thank R. Dong and M. A. Brow for providing technical assistance, P. J. Lee for providing the amino acid sequencing, the Cetus DNA Synthesis group for supplying the oligonucleotides, and J. Larrick for helpful advice during the time we worked together at Cetus. I would also like to thank D. McLaughlin for word processing assistance and G. K. Lee for editing of the manuscript. 20 j. Vieira and J. Messing,
Gene 19, 259 (1982).
[9] C o m p l e m e n t a r y D N A S y n t h e s i s in Situ: Methods and Applications By JAMES E B E R W I N E , CORINNE SPENCER, K E V I N MIYASHIRO, SCOTT MACKLER, and RICHARD F I N N E L L
Synthesis of complementary DNA (cDNA) has traditionally been performed on RNA isolated from large amounts of cells or tissue, cDNA probes and cDNA libraries have been successfully constructed from purified RNA; however, the use of isolated RNA from a large tissue source results in the loss of cellular resolution. This is an important consideration given that some RNAs are confined to distinct cell types within a specific tissue. In such situations, the RNA from these few METHODS IN ENZYMOLOGY,VOL. 216
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
DIRECT c D N A CLONING AND SCREENING OF MUTANTS
73
[8] D i r e c t C o m p l e m e n t a r y D N A Cloning a n d S c r e e n i n g of M u t a n t s Using P o l y m e r a s e C h a i n R e a c t i o n By YAWEN L. CHIANG
Introduction Conventional cDNA cloning and screening for mutations can be tedious and time consuming. In the early 1970s a few papers described DNA amplification using polymerase1-3 and Khorana et al. 2 suggested the fundamental theory behind the polymerase chain reaction (PCR). In 1984, K. Mullis developed the PCR and filed a patent in 1985 on the "process for amplifying nucleic acid sequences"; the patent was issued in 1987. In 1986, K. Mullis, J. Larrick, and I discussed the possibility of using the technique for direct cDNA cloning and for screening mutants for the variable region of immunoglobin heavy and light chains. We successfully developed the method, establishing a new approach for cloning antibody cDNA. The PCR amplifies defined regions of the genome by using oligomerdirected primer extension. The technique depends on the DNA replication enzyme (DNA polymerase) and the chain reaction made during the exponential amplification procedure. The reaction is efficient; only small numbers of cells and minute amounts of DNA are needed. Thus it is feasible to clone an antibody gene directly from small numbers of hybridoma cells. Because the PCR is very sensitive, there are numerous contamination considerations (such as the stability and control of reagents, primer design, and reaction sensitivity) that must be addressed. Many new methods and instruments for the PCR have been developed during the past few years. We will continue to find novel applications of the PCR--it is a powerful technique for modern molecular biology. Principle of Polymerase Chain Reaction Used for Direct cDNA Cloning The PCR is based on repeated cycles of high-temperature template denaturation, oligonucleotide primer annealing, and polymerase-depeni K. Kleppe, E. O h t s u k a , R. Kieppe, I. Molineux, and H. G. K h o r a n a , J. Mol. Biol. 56, 341 (1971). 2 H. G. K h o r a n a , K. L. Agarwal, H. Buchi, M. H. Caruthers, N. K. Gupta, K. Kleppe, A. K u m a r , E. O h t s u k a , U. L. RajBhandary, J. H. v a n de Sande, V. Sgaramella, T. Terao, H. W e b e r , a n d T. Y a m a d a , J. Mol. Biol. 72, 209 (1972). 3 A. Panet and H. G. K h o r a n a , J. Biol. Chem. 249, 5213 (1974).
METHODS IN ENZYMOLOGY,VOL. 216
Copyright © 1992by AcademicPress, Inc. All fights of reproduction in any form reserved.
74
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[8]
dent extension. One can synthesize large quantities of a particular target DNA sequence in vitro. 4-6 By using reverse transcriptase, RNA can be transcribed into cDNA, which is then amplified using the Taq DNA polymerase. In our experiments, we also added the appropriate cloning site sequences with PCR primers to facilitate easy cDNA cloning into expression vectors. Because of the amplification capabilities and extreme sensitivity of the PCR, minute changes of nucleotides in clones and cell lines can be detected by using direct sequencing 7 immediately following cDNA amplification. Chamberlain et al.S detected the deletion of the Duchenne muscular dystrophy locus via multiplex DNA amplification, and Wrischnick et al. 9 also used direct sequencing of enzymatically amplified DNA to determine the length mutations in human mitochondrial DNA. The revolutionary changes in molecular biology, resulting from the development of sequencing and PCR techniques, have allowed us to efficiently clone the immunoglobulin heavy and light chain variable regions from hybridoma cells. The development of our reproducible experiments, combined with the knowledge of the immunoglobulin constant region sequences provided by Kabat et al., l° has made it possible to produce manmade antibodies without using conventional hybridoma techniques. (The immunoglobulin constant region is named as such because of its characteristic of conserved sequences.) Experimental Section Materials a n d M e t h o d s
A summary of the method is outlined in Fig. 1. A murine hybridoma cell line, 269-10F7 (IgG 1, K), that produces anti-human C5a monoclonal antibodies 11was used. From N-terminal amino acid microsequencing data, 4 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Science 230, 1350 (1985). 5 S. J. Scharf, G. T. Horn, and H. A. Erlich, Science 233, 1076 (1986). 6 K. B. Mullis and F. A. Faloona, this series, Vol. 155, p. 335. 7 U. B. Gyllensten and H. A. Erlich, Proc. Natl. Acad. Sci. U.S.A. 85, 7652 (1988). 8 j. S. Chamberlain, R. A. Gibbs, J. E. Rainer, P. N. Nguyen, and C. T. Caskey, Nucleic Acids Res. 16, 11141 (1988). 9 L. A. Wrischnick, R. G. Higuchi, M. Stoneking, H. A. Erlich, N. Arnheim, and A. C. Wilson, Nucleic Acids Res. 15(2), 529 (1987). l0 E. A. Kabat, T. T. Wu, M. Reid-Miller, H. M. Perry, and K. S. Gottesman (eds), "Sequences of Protein of Immunological Interest," 4th Ed., U.S. Department of Health and Human Services, Washington, D.C., 1987. 1i y . L. Chiang, R. Sheng-Dong, M. A. Brow, and J. W. Larrick, BioTechniques 7, 360 (1989).
[8l
DIRECT cDNA CLONINGAND SCREENINGOF MUTANTS
75
Mouse hybridoma cells
~ single-step extraction RNA reverse transcription cDNA
plificetionusing PCR using PCR primers: YC1 and YC2
cDNA:
L
vadable region of Ig
YCA and YCB
L
light chain
VL(mouse)
variable region of Ig heavy chain V. (mouse)
~ /
cloning into human constant region gene expression cassettes
VL C
-~
'
7 V H
(m°use)
CH(hUman)
mouse/human chimericAb FIG. 1. Schematic summary of the method used to make mouse/human chimeric antibody (Ab).
we determined that 269-10F7 belongs to mouse K light chain subgroup III and mouse IgG~ heavy chain subgroup IA. From the database of Kabat e t al., ~o stretches of invariant N-terminal amino acid residues for both heavy and light chains were recognized. Oligonucleotide primers for the D N A sequence coding for this region were designed and prepared for PCR. The primer sequences were determined according to the usual codon of the specific immunoglobulin subgroups. Depending on the experimental design, additional nucleotide sequences for convenient cloning sites can be used if necessary. We applied the rules and considerations for synthesizing oligomers and created two 5' end primers (sense primer), YC1 and YCA. YC1 was used for the immunoglobulin light chain gene. It is a 39-bp oligomer, 5'-
76
ISOLATION, SYNTHESIS,DETECTIONOF DNA AND RNA
[8]
CCCGAATTCGACATTGTGCTGACCCAATCTCCAGCTTCT-3'. YCA was used for the immunoglobulin heavy chain gene. It is a 36-bp oligomer, 5'-CCCGAATTCGATGTGCATCTTCAGGAGTCGGGACCT-3'. We then designed the 3' end primers (antisense primer) for the PCR. YC2 and YCB were synthesized. YC2 was used for the light chain gene. It is a 38-bp oligomer, 5'-CCGAATTCGATGGATACAGTTGGTGCAGCATCAGCCCG-3'. YCB was used for the heavy chain gene. It is a 39-bp oligomer, 5'-CCCGAATTCTGGATAGACAGATGGGGGTGTCGTTTTGGC-3'. By employing YC1 and YC2 in the DNA amplification reaction, the entire variable-region sequences of the immunoglobulin light chain gene for hybridoma 269-10F7 were made. Likewise, by using YCA and YCB, the entire variable-region sequences of the immunoglobulin heavy chain gene were made. These cDNA fragments will be ligated with DNA from the human light and heavy chain construct regions, and the DNA construct will be placed into an expression vector to produce mouse/human chimeric antibody. This approach to create cDNA fragments and to use it to produce antibody is much quicker than the conventional method of constructing a cDNA library containing the mouse light and heavy chain variable region. The method involves the following steps. Single-Step Preparation ofRNA. Total cellular RNA is prepared by an acid guanidinium thiocyanate-phenol-chloroform single-step extraction method. 12The RNAzoI method established by C 1NNA/BIOTECX Laboratories International, Inc. (Friendswood, TX) provides a good yield of clean R N A prepared from small amounts of tissue or cells within 3 hr. Reverse Transcription of RNA to Synthesize cDNA. First-strand cDNA synthesis is accomplished by priming the RNA with 1/.~M oligo(dT) (12- to 18-mer). A 10-/xl reaction volume contains a dried pellet of RNA, 200 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD), 0. ! mg/ml of bovine serum albumin (BSA), 0.5 mM concentrations of each dNTP (dGTP, dATP, dTTP, and dCTP), 0.25 units of RNasin, 50 mM Tris-HC1, pH 7.5, 75 m M KC1, l0 m M dithiothreitol (DTT), 3 mM MgC12, and 50 /zg/ml of actinomycin D. The reaction is carried out in a 37° water bath for 1 hr. cDNA Amplification Using the Polymerase Chain Reaction. In the same tube, cDNA amplification using the PCR is carried out in 25 to 35 cycles. The sense and antisense primers mentioned above (1/xM each), 0.5 mM dNTP, and 1 unit of Taq polymerase in reaction buffer are added and the volume brought to 50/xl. The final concentration of the reaction 12p. Chomczvnski and N. Sacchi. Anal. Biochem. 162. 156 (1987).
[8]
DIRECT c D N A CLONING AND SCREENING OF MUTANTS
77
buffer is 50 mM KCI, 1.5 mM MgC12 , 10 mM Tris-HC1 (pH 8.4), and 0.02% (w/v) gelatin. The repeated PCR cycles involve denaturing at 95° for 1 min, primer annealing at 55° for 1 min, and Taq polymerase reaction at 72° for 3 min. Polymerase Chain Reaction Product Analysis. One-tenth of the final volume is loaded onto an ethidium bromide-stained, 3% (w/v) Nusieve agarose, and 1% (w/v) Seakem agarose (FMC BioProducts, Rockland, ME) minigel and analyzed (Figs. 2 and 3).
Specific Experimental Considerations Some important practical procedures should be considered when using PCR techniques for direct cDNA cloning. It is important to design appropriate primers for the PCR reaction. Lathe ~3 reviewed some theoretical and practical considerations to be used when choosing synthetic oligonucleotide probes deduced from amino acid sequence data. Girgis et a l ) 4 generated DNA probes for peptides with highly degenerate codons using mixed primers in the PCR reaction. To minimize contamination problems, use a positive displacement pipetter and follow good laboratory procedures (including frequent changing of globes, careful aliquoting of reagents, and physically separating the preand post-PCR reactions). One needs to assess the results critically, repeat experiments to obtain consistent results, and select good positive controls. The sensitivity, stability, and control of reagents, and the selection of equipment, are important. Choose credible sources when purchasing commercially available standard reagents and equipment. All experimental parameters including annealing, extension, temperature, numbers of cycles, and enzyme-to-primer ratios must be optimized on an individual basis. One should avoid using excess DNA in the reaction. DNA from other, unrelated genes extracted from other cells should be included as a negative control. Other controls such as distilled H20 or Tris-ethylenediaminetetraacetic acid (EDTA) buffer should be used in reactions where there are no target sequences. A reaction that contains just the primer sequences should also be run. To verify the data, use different pairs of primers. To determine if there is other DNA contamination, primers and probes outside the 5' or 3' region of the PCR product should be made and checked using the Southern blot analysis data. cDNA, derived from all different types ofRNA (mRNA, tRNA, rRNA, total RNA, and viruses) and from very low copy number RNA target 13 R. Lathe, J. Mol. Biol. 11t3, 1 (1985). 14 S. L Girgis, M. Alevizaki, P. Denny, G. J. Ferrier, and S. Legon, Nucleic Acids Res. 16, 10371 (1988).
78
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A 1
2
3
[8]
4
4,92-369-246--
~p 123--
FIG. 2. PCR products from 25 cycles for the variable region of light chain. Lane 1,123-bp ladder marker; lane 2, PCR product from 106 cells; lane 3, PCR product from mouse Ig light chain cDNA clone obtained from conventional cDNA library; lane 4, no cells, only primers contained. s e q u e n c e s c a n b e a m p l i f i e d . W a n g e t al. t536 u s e d t h e r e v e r s e t r a n s c r i p t i o n r e a c t i o n p r o c e d u r e p u b l i s h e d in 1987 a n d d e v e l o p e d a G e n e A m p t h e r m o s t a b l e y T t h r e v e r s e t r a n s c r i p t a s e R N A P C R kit ( P e r k i n E l m e r C e t u s , E m e r y v i l l e , C A ) . B y u s i n g this kit a c D N A p r o d u c t u p to 1.3 k i l o b a s e s (kb) in l e n g t h c a n b e g e n e r a t e d . Other choices of methods and materials are available. A special techn o l o g y s e c t i o n h a s b e e n p u b l i s h e d ~7 t h a t d e s c r i b e s o t h e r o p t i o n s f o r t h e J5 A. M. Wang, M. V. Doyle, and D. F. Mark, Proc. Natl. Acad. Sci. U.S.A. 86, 9717 (1989). ~6E. S. Kawasaki and A. M. Wang, "PCR Technology" (H. A. Erlich, ed.), p. 89. Stockton Press, New York, 1989. 17Special Technology Section: Methods and Materials: Amplification of Nucleic Acid Sequences: The Choices Multiply, J. N I H Res. 3, 81 (1991).
[8]
DIRECT c D N A CLONING AND SCREENING OF MUTANTS
1
2
3
79
4
bp 1 3 5 3 872 603 -r-310 281 --7
FIG. 3. PCR products from 35 cycles for the variable region of heavy chains. Lane 1,300 ng +X174 HaelII marker; lane 2, PCR product from mouse Ig heavy chain cDNA clone obtained from conventional cDNA library; lane 3, PCR product from 106 cells; lane 4, no cells, only primers contained.
amplification of nucleic acid sequences as well as alternative thermal cyclers, primers and probes, thermostable DNA polymerase, reagents and reagent kits, miscellaneous products, and manufacturers. It is not necessary to use conventional M 13 subcloning for sequencing. The method of direct sequencing of the PCR product is established.9,18 Other advances in PCR have been reported elsewhere.19 18 C. Wong, C. E. Dowling, R. K. Saiki, R. G. Higuchi, H. A. Erlich, and H. H. Kazazian, Jr., Nature (London) 330, 384 (1987). 19 H. A. Erlich, D. Gelfand, and J. J. Sninsky, Science 252, 1643 (1991).
80
ISOLATION, SYNTHESIS, DETECTION OF D N A AND R N A
[9]
Results A clear 369 bp expected band for Ig light chain cDNA fragment with the cloning site (Fig. 2) and a 390 bp expected band for Ig heavy chain cDNA fragment (Fig. 3) were obtained. The PCR products were subcloned into M13 followed by subsequent sequencing. 2° The cDNA cloning of the variable regions of heavy and light chains, were obtained by constructing a conventional cDNA library, has been published elsewhere. 11These cDNA clones were also sequenced. No mutations were found in the sequences obtained by either the cDNA clone from the conventional cDNA library or from the cDNA amplified fragments produced directly by the PCR of the hybridoma cell RNA. We are presently developing a method to synthesize chimeric mouse/ human monoclonal antibodies by using synthetic long oligomers ligated together containing just the mouse CDRs (complementarity-determining regions), and to synthesize mouse/human MAb containing fewer mouse sequences. Acknowledgments I would like to thank R. Dong and M. A. Brow for providing technical assistance, P. J. Lee for providing the amino acid sequencing, the Cetus DNA Synthesis group for supplying the oligonucleotides, and J. Larrick for helpful advice during the time we worked together at Cetus. I would also like to thank D. McLaughlin for word processing assistance and G. K. Lee for editing of the manuscript. 20 j. Vieira and J. Messing,
Gene 19, 259 (1982).
[9] C o m p l e m e n t a r y D N A S y n t h e s i s in Situ: Methods and Applications By JAMES E B E R W I N E , CORINNE SPENCER, K E V I N MIYASHIRO, SCOTT MACKLER, and RICHARD F I N N E L L
Synthesis of complementary DNA (cDNA) has traditionally been performed on RNA isolated from large amounts of cells or tissue, cDNA probes and cDNA libraries have been successfully constructed from purified RNA; however, the use of isolated RNA from a large tissue source results in the loss of cellular resolution. This is an important consideration given that some RNAs are confined to distinct cell types within a specific tissue. In such situations, the RNA from these few METHODS IN ENZYMOLOGY,VOL. 216
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