BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 504-506
Vol. 167, No. 2, 1990 March 16, 1990
Polymerase chain reaction (PCR) amplification
with a single specific primer
Miklos Kalman, Eva T. Kalman, and Michael Cashel Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Received January 3, 1990 A method is described for amplification of DNA fragments flanking a single known sequence that is sufficiently long to enable synthesis of a functional primer in polymerase chain reactions. 01990
Academic
Press,
D-C.
The polymerase chain reaction (1) is widely used for amplification of DNA sequences between two oligodeoxynucleotide primers of known sequence. However, it is also sometimes desirable to amplify DNA fragments that contain only a single known sequence that is long enough to enable synthesis of a functional primer in PCR reactions. Three such applications include isolation of multiple instances of consensussequencesscattered in the chromosome, chromosomal mapping of the location of insertion elements, and limited extension of chromosomal sequencesbeyond a region of known sequence.A method for accomplishing this has been described (2-5), called “inverse PCR” (l-3) and involves synthesis of two noncomplementary oligodeoxynucleotides that prime for DNA outward from the sequenced region. By endonucleasedigestion to generate cohesive ends in regions flanking the sequencedDNA and circularization by ligation, the two nonoverlapping outward primers are enabled to allow amplification of the DNA between them. A related method has used for the amplification of cDNA fragments using poly dT as one of the primers; it has been termed RACE (7,8).
MATERIALS AND METHODS The method has been tested with two different specific primers corresponding to a region of E. coli chromosomal DNA we have sequenced recently (6). Specifically, 5 pg of genomic DNA was cut with 20 U of BamHI at 37“C for 2 hrs, then purified by phenol extraction and precipitation with l/lOth volume of 3 M sodium acetate and 2.5 volumes of ethanol. The pellet was washed with 70% ethanol and resuspended in distilled water. One pg of restricted DNA was ligated with 100 pmole of double stranded linker-primer DNA overnight using T4 ligase. The sequencesof the linker-primer strands are (5’->3’): A= catccgcataggaagcattgand B= gatccaatgcttcctatgcggatg. The use of linker-primers is shown in Figure 1. The ligation mixture was precipitated under conditions that remove most of the nonligated linker DNA; namely, adding 0.5 volumes of 7 M ammonium acetate (pH=7) with 2 volumes of ethanol and Incubating for 20 min at -2O’C, followed by centrifuging for 5 min at 25’C, and washing the pellet in 70% ethanol. For the PCR reaction (100 pl), 200 pmole of linker-primer A was added together with 100 pmole of one (C,) or another (C,) specific primer C (shown in Fig. 1) synthesized as complementary to a unique portion of the chromosomal spe operon. Primer C, was a 36-mer, located 792 bp from a BamHI site jn the omega gene (5,6). Primer C, was a 27-mer located 787 bp from the same BamHI site. The PCR reaction mix also contained 10 m M tris-Cl (pH 8.4), 50 m M KCl, 2.5 m M MgCl , 0.2 m M of each dNTP, and 0.01% gelatin. After 30 cycles of PCR, with each cycle consisting of 1.3 min at 94OCand 2.5 min at 72’C, 1/20th of each PCR reaction was analyzed on 1 % agarosegels. The step for annealing and for the polymerization reaction were combined, 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
504
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 167, No. 2, 1990
RESULTS
AND DISCUSSION
A method presented here for PCR amplification of DNA flanking a single known primer sequence is summarized in Figure 1. The first step consists of restriction endonuclease digestion of chromosomal DNA to generate 5’overhanging cohesive ends with 5’phosphorylated termini, as for example with BamHI. A double stranded oligodeoxynucleotide linker is synthesized (20-mer and complementary 24-mer labeled linker-primer A and B in Figure 1) with one flush end and the other end complementary to the overhang generated by the restriction enzyme. In the example shown with BamHI cut DNA, the linker sequence contains a GATC 5’ overhang. The linker-primer DNA contains no phosphomonoesters. As a result, ligation of linker DNA to chromosomal DNA with T4 DNA ligase results in covalent attachment of only one of the strands of linker DNA to 5’termini of restricted chromosomal DNA (Fig. 1, step 1). The ligation mixture is then precipitated under conditions that remove most of the nonligated linker DNA (see Methods). An oligodeoxynucleotide primer (labeled specific primer C in Fig. 1) is synthesized complementary to the single known sequence, and the PCR reaction is run in the presence of this specific primer as well as additional linker-primer A. In the first PCR reaction, the specific primer C will be extended beyond the linker B ligation site into the linker sequence on only one strand (Fig. 1, step 2). This event generates a template that will enable priming by linker-primer A which results in synthesis of DNA complementary to the specific primer (Fig. 1, step 3). Subsequent PCR cycles amplify the DNA between linker-primer A and specific primer C. There are two potential problems. If the restriction endonuclease site is too far from the specific primer site, another enzyme is selected. If the restriction endonuclease site is too close for analysis, a partial digest (or another enzyme) is employed.
SPECIFIC PRIMER rzzm
LINKER-PRIMER o
A
BC
OB C=7A
iiu
1
step 1
AU 80
LB -A
1078
(A~+Ccaza) I step 2
-Al BO
01
872 603
4---------7zzzac
A u---------w UrroC
L...JA
I
step 3
02
Figure 1: Stepsfor PCR amplification with a single specific primer. Figure 2: Agarosegel analysisof PCR products.Lane A = OX174DNA sizestandard,cut with l&j&I and 3 fragmentssize labeled, lane B = PCR test reaction with 36-mer C, specific primer, laneC = PCR test reactionwith 27-mer C, specific primer, and lane D = PCR reactioncontrol preparedas in lanesB or C but lacking only specific primer C. 505
D
Vol. 167, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2 shows PCR amplification results of the method with two different specific primers complementary to noncontiguous neighboring regions of prokaryotic chromosomal DNA of known sequence. A control reaction lacking only the specific primer resulted in no discernable fragment amplification after agarosegel analysis (Fig. 2, lane D). In contrast, a complete PCR reaction using primer C, resulted in two major fragments amplified (Fig. 2, lane B). The upper band in lane B corresponds to the predicted 854 bp fragment (different from 792 bp because of the addition of primer sequences);its identity was verified by sequencing. The sequence of the lower band in lane B was also determined but did not allow its identification. The results of the second test reaction with specific-primer C, are shown in lane C of Fig. 2. Although several DNA fragments have been amplified, the most abundant band in lane C, migrating as slightly smaller than 872 bp size standard has also been identified as arising from the expected spe operon fragment by sequencing. We believe these results provide evidence for the workability of the method. A “single sided PCR” method has been published recently (9) that is similar, but not identical, to the method presented here. The two methods differ in three respects: 1) by generating the ends suitable for ligation by linear amplification rather than by restriction enzymes, 2) by using flush ended A and B linkerprimers, rather than more readily ligatable cohesive ends, and 3) in use of the method for DNA footprinting applications rather than cloning. REFERENCES 1. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239:487-491. 2. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Amheim, N.A. (1985) Science 230:1250-1254. 3. Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1986) Nature 324:163166. 4. Faloona, F., and Mullis, K.B. (1987) Meth. Enzymol. 155:335-350 5. Ochman, H., Ajioka, J.W., Garza, D., and Hartl, D.L. (1989) in PCR Technology: Principles and Applications for DNA Amplification, (H. A. Erlich, ed.), pp 105-l 11, Stockton Press, New York, NY 10010 6. Sarubbi, E., Rudd, K.E., Xiao, H., Ikehara, K., Kalman, M., and Cashel, M. (1989) J. Biol. Chem. 264:15074-15082. 7. Frohman, M.A., Dush, M.K., and Martin, G.R. (1988) Proc. Nat’l. Acad. Sci. U.S.A. 85:89989002. 8. Ohara, O., Dorit, R.L., and Gilbert, W. (1989) Proc. Nat’l. Acad. Sci. U.S.A. 895673-5677. 9. Mueller, P.R., and Wold, B. (1989) Science 246:780-786.
506
Vol. 167, No. 2, 1990 March 16, 1990
Polymerase chain reaction (PCR) amplification
with a single specific primer
Miklos Kalman, Eva T. Kalman, and Michael Cashel Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Received January 3, 1990 A method is described for amplification of DNA fragments flanking a single known sequence that is sufficiently long to enable synthesis of a functional primer in polymerase chain reactions. 01990
Academic
Press,
D-C.
The polymerase chain reaction (1) is widely used for amplification of DNA sequences between two oligodeoxynucleotide primers of known sequence. However, it is also sometimes desirable to amplify DNA fragments that contain only a single known sequence that is long enough to enable synthesis of a functional primer in PCR reactions. Three such applications include isolation of multiple instances of consensussequencesscattered in the chromosome, chromosomal mapping of the location of insertion elements, and limited extension of chromosomal sequencesbeyond a region of known sequence.A method for accomplishing this has been described (2-5), called “inverse PCR” (l-3) and involves synthesis of two noncomplementary oligodeoxynucleotides that prime for DNA outward from the sequenced region. By endonucleasedigestion to generate cohesive ends in regions flanking the sequencedDNA and circularization by ligation, the two nonoverlapping outward primers are enabled to allow amplification of the DNA between them. A related method has used for the amplification of cDNA fragments using poly dT as one of the primers; it has been termed RACE (7,8).
MATERIALS AND METHODS The method has been tested with two different specific primers corresponding to a region of E. coli chromosomal DNA we have sequenced recently (6). Specifically, 5 pg of genomic DNA was cut with 20 U of BamHI at 37“C for 2 hrs, then purified by phenol extraction and precipitation with l/lOth volume of 3 M sodium acetate and 2.5 volumes of ethanol. The pellet was washed with 70% ethanol and resuspended in distilled water. One pg of restricted DNA was ligated with 100 pmole of double stranded linker-primer DNA overnight using T4 ligase. The sequencesof the linker-primer strands are (5’->3’): A= catccgcataggaagcattgand B= gatccaatgcttcctatgcggatg. The use of linker-primers is shown in Figure 1. The ligation mixture was precipitated under conditions that remove most of the nonligated linker DNA; namely, adding 0.5 volumes of 7 M ammonium acetate (pH=7) with 2 volumes of ethanol and Incubating for 20 min at -2O’C, followed by centrifuging for 5 min at 25’C, and washing the pellet in 70% ethanol. For the PCR reaction (100 pl), 200 pmole of linker-primer A was added together with 100 pmole of one (C,) or another (C,) specific primer C (shown in Fig. 1) synthesized as complementary to a unique portion of the chromosomal spe operon. Primer C, was a 36-mer, located 792 bp from a BamHI site jn the omega gene (5,6). Primer C, was a 27-mer located 787 bp from the same BamHI site. The PCR reaction mix also contained 10 m M tris-Cl (pH 8.4), 50 m M KCl, 2.5 m M MgCl , 0.2 m M of each dNTP, and 0.01% gelatin. After 30 cycles of PCR, with each cycle consisting of 1.3 min at 94OCand 2.5 min at 72’C, 1/20th of each PCR reaction was analyzed on 1 % agarosegels. The step for annealing and for the polymerization reaction were combined, 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
504
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 167, No. 2, 1990
RESULTS
AND DISCUSSION
A method presented here for PCR amplification of DNA flanking a single known primer sequence is summarized in Figure 1. The first step consists of restriction endonuclease digestion of chromosomal DNA to generate 5’overhanging cohesive ends with 5’phosphorylated termini, as for example with BamHI. A double stranded oligodeoxynucleotide linker is synthesized (20-mer and complementary 24-mer labeled linker-primer A and B in Figure 1) with one flush end and the other end complementary to the overhang generated by the restriction enzyme. In the example shown with BamHI cut DNA, the linker sequence contains a GATC 5’ overhang. The linker-primer DNA contains no phosphomonoesters. As a result, ligation of linker DNA to chromosomal DNA with T4 DNA ligase results in covalent attachment of only one of the strands of linker DNA to 5’termini of restricted chromosomal DNA (Fig. 1, step 1). The ligation mixture is then precipitated under conditions that remove most of the nonligated linker DNA (see Methods). An oligodeoxynucleotide primer (labeled specific primer C in Fig. 1) is synthesized complementary to the single known sequence, and the PCR reaction is run in the presence of this specific primer as well as additional linker-primer A. In the first PCR reaction, the specific primer C will be extended beyond the linker B ligation site into the linker sequence on only one strand (Fig. 1, step 2). This event generates a template that will enable priming by linker-primer A which results in synthesis of DNA complementary to the specific primer (Fig. 1, step 3). Subsequent PCR cycles amplify the DNA between linker-primer A and specific primer C. There are two potential problems. If the restriction endonuclease site is too far from the specific primer site, another enzyme is selected. If the restriction endonuclease site is too close for analysis, a partial digest (or another enzyme) is employed.
SPECIFIC PRIMER rzzm
LINKER-PRIMER o
A
BC
OB C=7A
iiu
1
step 1
AU 80
LB -A
1078
(A~+Ccaza) I step 2
-Al BO
01
872 603
4---------7zzzac
A u---------w UrroC
L...JA
I
step 3
02
Figure 1: Stepsfor PCR amplification with a single specific primer. Figure 2: Agarosegel analysisof PCR products.Lane A = OX174DNA sizestandard,cut with l&j&I and 3 fragmentssize labeled, lane B = PCR test reaction with 36-mer C, specific primer, laneC = PCR test reactionwith 27-mer C, specific primer, and lane D = PCR reactioncontrol preparedas in lanesB or C but lacking only specific primer C. 505
D
Vol. 167, No. 2, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2 shows PCR amplification results of the method with two different specific primers complementary to noncontiguous neighboring regions of prokaryotic chromosomal DNA of known sequence. A control reaction lacking only the specific primer resulted in no discernable fragment amplification after agarosegel analysis (Fig. 2, lane D). In contrast, a complete PCR reaction using primer C, resulted in two major fragments amplified (Fig. 2, lane B). The upper band in lane B corresponds to the predicted 854 bp fragment (different from 792 bp because of the addition of primer sequences);its identity was verified by sequencing. The sequence of the lower band in lane B was also determined but did not allow its identification. The results of the second test reaction with specific-primer C, are shown in lane C of Fig. 2. Although several DNA fragments have been amplified, the most abundant band in lane C, migrating as slightly smaller than 872 bp size standard has also been identified as arising from the expected spe operon fragment by sequencing. We believe these results provide evidence for the workability of the method. A “single sided PCR” method has been published recently (9) that is similar, but not identical, to the method presented here. The two methods differ in three respects: 1) by generating the ends suitable for ligation by linear amplification rather than by restriction enzymes, 2) by using flush ended A and B linkerprimers, rather than more readily ligatable cohesive ends, and 3) in use of the method for DNA footprinting applications rather than cloning. REFERENCES 1. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Science 239:487-491. 2. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Amheim, N.A. (1985) Science 230:1250-1254. 3. Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B., and Erlich, H.A. (1986) Nature 324:163166. 4. Faloona, F., and Mullis, K.B. (1987) Meth. Enzymol. 155:335-350 5. Ochman, H., Ajioka, J.W., Garza, D., and Hartl, D.L. (1989) in PCR Technology: Principles and Applications for DNA Amplification, (H. A. Erlich, ed.), pp 105-l 11, Stockton Press, New York, NY 10010 6. Sarubbi, E., Rudd, K.E., Xiao, H., Ikehara, K., Kalman, M., and Cashel, M. (1989) J. Biol. Chem. 264:15074-15082. 7. Frohman, M.A., Dush, M.K., and Martin, G.R. (1988) Proc. Nat’l. Acad. Sci. U.S.A. 85:89989002. 8. Ohara, O., Dorit, R.L., and Gilbert, W. (1989) Proc. Nat’l. Acad. Sci. U.S.A. 895673-5677. 9. Mueller, P.R., and Wold, B. (1989) Science 246:780-786.
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