World Journal
of Microbiology
& Biotechnology
11, 478-480
Comparative study of three methods cloning PCR products
for
Y. Abed, C. Ballet* and P. De Micco The direct sequencing of the products of polymerase chain reactions (PCR) still presents difficulties and often requires special manipulations, such as the generation of excess single-stranded DNA using asymmetric PCR. Several alternative methods involve cloning PCR products into vector DNA suitable for sequencing analysis. Three of these methods have been compared in the present study. The two direct cloning methods, TA/cloning and the PCR-script system, initially gave large numbers of false positives (60% and 55%, respectively) but the number of false positives was reduced (to 35% and 3x%, respectively) by modifying the protocols used. However, ligation of the termini of the digested PCR product in the corresponding digested vector was the most efficient and consequently the most reliable method for routine cloning. Key words: Cloning,
DNA, ligation,
PCR product,
The polymerase chain reaction (PCR) has made it possible to produce a large amount of a specific DNA from a complex source using a small amount of starting material (Saiki et al. 1988). The characterization of the amplified product can be used in screening clinical specimens for disease-related mutations. Unfortunately, the direct sequencing of PCR products still presents difficulties, particularly when multiple PCR products and artefacts are generated by a single reaction (Clark 1988). The alternative methods that have been developed involve cloning the PCR products into vector DNA which is suitable for sequencing reactions. However, many of these methods have not proven to be straightforward and have often been associated with large numbers of false positives. In the present study, three cloning methods were compared for potential reliability in routine analyses: T/A cloning (Stratagene); the PCR-script system (Stratagene); and restriction digestion of sites incorporated in the termini of PCR primers. A 366-bp fragment from the 65-kDa protein-encoding gene of several Mycobacterium tuberculosis strains
was
then
cloned.
The authors are with the Laboratoire de Microbiologic. HBpital Salvator, 249 bld Sainte Marguerite. 13009, Marseille, France; fax: 33 91 74 60 44 ‘Corresponding author. @ 1995 Rapid Science
Publishers
Materials
and Methods
A4ycobactti~m ttlberculosis strains of clinical origin were grown on Middlebrook broth (Difco). Mycobacterial DNA was extracted using the Chelex technique (De Lamballerie et al. 1992) and amplified using a pair of primers, TBl (5’ GAG ATC GAG CTG GAG GAT CC 3’) and TB2 (5’ AGC TGC AGC CC4 AAG GTG TT 3’) containing BarnHI and F’stI restriction sites (Hance et al. 1989). For the PCR, 50 ~1 reaction mixture, containing 50 mM KCl, 10 mM Tris/HCl (pH 8.4), 3.0 mM MgCl,, 100 pg gelatin/ml, 1.25 mM of each deoxynucleotide triphosphate (Boehringer Mannheim), 0.25 /iM of each primer, 5 ~1 extracted DNA and 1.25 units Tuq polymerase (Perkin-Elmer), was subjected to 35 PCR cycles, with denaturation at 94°C for I min and primer annealing and extension at 70°C for I min. After the last cycle, samples were maintained at 72°C for 10 min. The amplified products were separated using 1.2% agarose horizontal gels in TAE buffer (0.04 M Trisiacetate and 0.01 M EDTA, pH 8.0). The gels were
stained in ethidium bromide solution and visualized with a U.V. transilluminator.
Each cloning
method
was tested
10 times.
T/A Cloning Method Ligation was performed in a mixture of I ~1 ligation buffer, 2 ~1 (25 ng/& linearized PCR II Phagemid (Stratagene, La Jolla, CA) 1 ,~l 15% dilution (in sterile distilled water) of PCR product, I ,ul (2 units) T4-DNA ligase (Boehringer Mannheim) and 6~1 sterile distilled water. The mixture was incubated overnight at 12°C. Competent INVctF’ cells (Stratagene), 100 ~1, were then incubated with I ~1 ligation product for 30 min on ice. After a heat-pulse of
Cloning PCR products Table
1. Mean
results
of 10 cloning
tests
performed Colonies (c.f.u./lOO
Method
PCR-script T/A cloning Ligation *True
by each
of restricted recombinants
product were
those
that
were
ampR,
of three
methods.
produced pi ceils)
Colony
phenotype
(%)
Recombination’ (%I
/IGal-
/IGal+
32 41
48 53
52 47
45 40
96
98
2
97
/?gai-
and positive
60 s at 42°C. 450 ,uI pre-warmed SOC medium (IO mM M&I,, 10 mM MgSO, and 2 mM glucose with 20 g tryptone, 5 g yeast extract and 0.5 g NaCI/I) were added and the culture was then aerated for I h at 37°C. Transformed cells (100 ~1) were plated onto the appropriate antibiotic-containing agar plate (LB with 50 pg ampicillin/ml) which had been spread with 20 ~1 10% (w/v, in dimethyl formamide) X-gal (5 bromo-4-chloro-3-indolyl-j-ogalactose). Plates were incubated for 24 to 48 h at 37°C (Mead et al. 1991).
on agarose
electrophoresis.
pyl b-o-thiogalactopyranoside. 18 to 36 h.
Results When gel
were
incubated
at 37°C
for
and Discussion plasmid
method
Plates
(Holmes
electrophoresis,
DNA
was
& Quigley it was
isolated 1981) apparent
by and
the
subjected that
less
mini-boiling to agarose than
half
of
PCR-Script System For the PCR-Script system, 2 ~1 PCR product were added to 50 ng Srfl-digested PCR-Script SK( + ) Phagemid in a 10 ~1 volume containing 1 ,uI 10 X universal buffer and rATP (0.5 mM final concentration). Four units of T4 DNA ligase (Boehringer Mannheim) and Srfl were then added to the mixture. The mixture was incubated at room temperature for 1 h and then the reaction was terminated by heat treatment at 65°C for 10 min. Competent cells (100 ~1) were incubated with 2 ~1 ligation product for 30 min on ice, and then subjected to a heat-pulse (45 s at 42°C). The cells were placed on ice for an additional 2 min, then 1 ml SOC medium was added and the cells aerated for 20 min at 37°C. Transformed cells (100 ~1) were plated onto plates, containing appropriate antibiotics (LB with 80 pg methicillin and 20 fig ampicillin/ml), which had been spread with 20 ~1 10% (w/v, in dimethyl formamide) X-gal. The plates were incubated at 37°C for 24 to 48 h (Bauer et al. 1992).
Insertion of Termini-digested PCR Product The PCR product was purified, twice with phenol/chloroform/ isoamyl alcohol (25 : 24: 1, by vol.) and once with chloroform/ isoamyl alcohol (24 : 1, v/v). After ethanol precipitation, the pellet was washed with 70% ethanol and resuspended in 25 ,uI TE buffer (10 mM Tris/HCI, pH 8.0, 1 mM EDTA) and 20 ,uI were digested with BamHI and PstI (Boehringer Mannheim) in 30~1 of mixture containing 3 ,uI 10 X universal buffer and 5 units of each enzyme. The reaction was incubated for 90 min at 37°C. At the same time, the plasmid was digested with the same enzymes. After electrophoresis on agarose gel, both insert and digested vector were purified using GeneClean (Bio 101, La Jolla, CA). Ligation was performed in a mixture of 3 ,~l (100 ng/ ,uI) insert, 1 /.d digested vector (100 ng/pl), 1~1 ligation buffer, 1 ,uI (2 units) T4 DNA ligase, 1~1 rATP (1 mM final concentration) and 3 ~1 sterile distilled water. The reaction was incubated overnight at 4°C. XLl-Blue competent cells (Stratagene), 100 ~1, were then incubated with 2 ~1 ligation product on ice for 30 min. After a heat-pulse (45 s at 42°C). 500 ,~l LB broth were added and the culture aerated for 1 h at 37°C. Transformed cells (100 ,uI) were plated onto LB plates containing 50 peg ampicillin, 20 pg tetracyclin and 80 ,ug fresh X-gal/ml and 20 mM isopro-
Figure 1. Ligation The photographed image, whole-band, of ligation pBlue-Script in the vector;
control with 0.8% agarose gel eiectrophoresis. gel was scanned and analysed on a Bio Scanner 3 + system (Millipore). l-product
performed without insert (recircularized vector KS +); 2-product of ligation performed with insert M-pBR328 DNA/BgII + pBR328 DNA/Hinfl.
World Journal of Microbiology 6 Biotechnology, Vol 11. 1995
479
I’. Abed, C. Ballet and l? De Micco the presumed recombinants (white colonies) obtained by the T/A cloning or PCR-script methods were truly positive (Table I). The high numbers of false positives generated by both these methods is partly due to the presence of Taq polymerase, which is known to inhibit cloning (Bennet & Mlenaar 1994). Consequently, it was necessary to test a large number of white colonies. Although both methods gave a low transformation efficiency, transformation was performed without conkolling ligation efficiency with agarose gel electrophoresis because small amounts of DNA were used in the ligation reaction. When PCR products were purified by GeneClean prior to the ligation reaction, the rate of false positives decreased from 55% to 31% using the PCR-script system and from 60% to 35% with T/ A cloning, thus confirming the inhibitor effect of Taq polymerase. However, the third method tested, in which terminus-restricted PCR products were inserted in the corresponding digested vector, was still more efficient than either direct method. Using this method, it was possible to control ligation efficiency with agarose gel electrophoresis before transformation, since 300 ng insert and 100 ng vector were ligated (Figure I). Transformation was only performed if ligation efficiency is confirmed and, consequently, the proportion of false positives obtained by this method was low (only 3%). Although this method requires a high concenkation of DNA, this should not be a problem as the starting material to be cloned is a PCR product.
480
World]oumal
of Microbiology 6 Biotechnology, Vol I I, 1995
References Bauer, J., Deely, D., Viola, J. & Weiner, M. 1992 PCR-script SK ( + ) cloning system: a simple and fast method for PCR cloning. Strategies in Mokdur Biology 5, 62-64. Bennett, B.L. & Mlenaar, A.J. 1994 Cloning of PCR products can be inhibited by Tuq polymerase carryover. BioTechniques 16, 32-3 7. Clark, J.M. 1988 Novel non-template nucleotide addition reaction catalyzed by procaryotic and eucaryotic DNA polymerase.
Nucleic Acids Research16, 9677-9686. De
Lamballerie, X., Zandotti, Micco, P. 1992 A one step using ‘Chelex 100’ suitable
C., Vignoli, C., Bollet, C. & De microbial DNA extraction method for gene amplification. Research in
Microbiology 143, 7855790. Hance, A.J., Grandchamp, B., L&y-FrCbault, V., Lecossier, D., Rauzier, J., Bocart, D. & Giquel, B. 1989 Detection and identification of mycobacteria by amplification of mycobacterial DNA.
Molecular Biology 3, 843-849. Holmes, D.S. & Quigley, M. 1981 A rapid boiling method preparation of bacterial plasmids. Analytica Biochemistry
for the 114,
193-197. Mead, D., Herrstadt, N., Marcil, R. & Smith, L. 1991 A universal method for the direct cloning of PCR amplified nucleic acid. Biol Technology 9, 65 7-663. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. & Erlich, H.A. 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491.
(Received in revised form ay 1995)
20 February 1995; accepted 25 Febru-
of Microbiology
& Biotechnology
11, 478-480
Comparative study of three methods cloning PCR products
for
Y. Abed, C. Ballet* and P. De Micco The direct sequencing of the products of polymerase chain reactions (PCR) still presents difficulties and often requires special manipulations, such as the generation of excess single-stranded DNA using asymmetric PCR. Several alternative methods involve cloning PCR products into vector DNA suitable for sequencing analysis. Three of these methods have been compared in the present study. The two direct cloning methods, TA/cloning and the PCR-script system, initially gave large numbers of false positives (60% and 55%, respectively) but the number of false positives was reduced (to 35% and 3x%, respectively) by modifying the protocols used. However, ligation of the termini of the digested PCR product in the corresponding digested vector was the most efficient and consequently the most reliable method for routine cloning. Key words: Cloning,
DNA, ligation,
PCR product,
The polymerase chain reaction (PCR) has made it possible to produce a large amount of a specific DNA from a complex source using a small amount of starting material (Saiki et al. 1988). The characterization of the amplified product can be used in screening clinical specimens for disease-related mutations. Unfortunately, the direct sequencing of PCR products still presents difficulties, particularly when multiple PCR products and artefacts are generated by a single reaction (Clark 1988). The alternative methods that have been developed involve cloning the PCR products into vector DNA which is suitable for sequencing reactions. However, many of these methods have not proven to be straightforward and have often been associated with large numbers of false positives. In the present study, three cloning methods were compared for potential reliability in routine analyses: T/A cloning (Stratagene); the PCR-script system (Stratagene); and restriction digestion of sites incorporated in the termini of PCR primers. A 366-bp fragment from the 65-kDa protein-encoding gene of several Mycobacterium tuberculosis strains
was
then
cloned.
The authors are with the Laboratoire de Microbiologic. HBpital Salvator, 249 bld Sainte Marguerite. 13009, Marseille, France; fax: 33 91 74 60 44 ‘Corresponding author. @ 1995 Rapid Science
Publishers
Materials
and Methods
A4ycobactti~m ttlberculosis strains of clinical origin were grown on Middlebrook broth (Difco). Mycobacterial DNA was extracted using the Chelex technique (De Lamballerie et al. 1992) and amplified using a pair of primers, TBl (5’ GAG ATC GAG CTG GAG GAT CC 3’) and TB2 (5’ AGC TGC AGC CC4 AAG GTG TT 3’) containing BarnHI and F’stI restriction sites (Hance et al. 1989). For the PCR, 50 ~1 reaction mixture, containing 50 mM KCl, 10 mM Tris/HCl (pH 8.4), 3.0 mM MgCl,, 100 pg gelatin/ml, 1.25 mM of each deoxynucleotide triphosphate (Boehringer Mannheim), 0.25 /iM of each primer, 5 ~1 extracted DNA and 1.25 units Tuq polymerase (Perkin-Elmer), was subjected to 35 PCR cycles, with denaturation at 94°C for I min and primer annealing and extension at 70°C for I min. After the last cycle, samples were maintained at 72°C for 10 min. The amplified products were separated using 1.2% agarose horizontal gels in TAE buffer (0.04 M Trisiacetate and 0.01 M EDTA, pH 8.0). The gels were
stained in ethidium bromide solution and visualized with a U.V. transilluminator.
Each cloning
method
was tested
10 times.
T/A Cloning Method Ligation was performed in a mixture of I ~1 ligation buffer, 2 ~1 (25 ng/& linearized PCR II Phagemid (Stratagene, La Jolla, CA) 1 ,~l 15% dilution (in sterile distilled water) of PCR product, I ,ul (2 units) T4-DNA ligase (Boehringer Mannheim) and 6~1 sterile distilled water. The mixture was incubated overnight at 12°C. Competent INVctF’ cells (Stratagene), 100 ~1, were then incubated with I ~1 ligation product for 30 min on ice. After a heat-pulse of
Cloning PCR products Table
1. Mean
results
of 10 cloning
tests
performed Colonies (c.f.u./lOO
Method
PCR-script T/A cloning Ligation *True
by each
of restricted recombinants
product were
those
that
were
ampR,
of three
methods.
produced pi ceils)
Colony
phenotype
(%)
Recombination’ (%I
/IGal-
/IGal+
32 41
48 53
52 47
45 40
96
98
2
97
/?gai-
and positive
60 s at 42°C. 450 ,uI pre-warmed SOC medium (IO mM M&I,, 10 mM MgSO, and 2 mM glucose with 20 g tryptone, 5 g yeast extract and 0.5 g NaCI/I) were added and the culture was then aerated for I h at 37°C. Transformed cells (100 ~1) were plated onto the appropriate antibiotic-containing agar plate (LB with 50 pg ampicillin/ml) which had been spread with 20 ~1 10% (w/v, in dimethyl formamide) X-gal (5 bromo-4-chloro-3-indolyl-j-ogalactose). Plates were incubated for 24 to 48 h at 37°C (Mead et al. 1991).
on agarose
electrophoresis.
pyl b-o-thiogalactopyranoside. 18 to 36 h.
Results When gel
were
incubated
at 37°C
for
and Discussion plasmid
method
Plates
(Holmes
electrophoresis,
DNA
was
& Quigley it was
isolated 1981) apparent
by and
the
subjected that
less
mini-boiling to agarose than
half
of
PCR-Script System For the PCR-Script system, 2 ~1 PCR product were added to 50 ng Srfl-digested PCR-Script SK( + ) Phagemid in a 10 ~1 volume containing 1 ,uI 10 X universal buffer and rATP (0.5 mM final concentration). Four units of T4 DNA ligase (Boehringer Mannheim) and Srfl were then added to the mixture. The mixture was incubated at room temperature for 1 h and then the reaction was terminated by heat treatment at 65°C for 10 min. Competent cells (100 ~1) were incubated with 2 ~1 ligation product for 30 min on ice, and then subjected to a heat-pulse (45 s at 42°C). The cells were placed on ice for an additional 2 min, then 1 ml SOC medium was added and the cells aerated for 20 min at 37°C. Transformed cells (100 ~1) were plated onto plates, containing appropriate antibiotics (LB with 80 pg methicillin and 20 fig ampicillin/ml), which had been spread with 20 ~1 10% (w/v, in dimethyl formamide) X-gal. The plates were incubated at 37°C for 24 to 48 h (Bauer et al. 1992).
Insertion of Termini-digested PCR Product The PCR product was purified, twice with phenol/chloroform/ isoamyl alcohol (25 : 24: 1, by vol.) and once with chloroform/ isoamyl alcohol (24 : 1, v/v). After ethanol precipitation, the pellet was washed with 70% ethanol and resuspended in 25 ,uI TE buffer (10 mM Tris/HCI, pH 8.0, 1 mM EDTA) and 20 ,uI were digested with BamHI and PstI (Boehringer Mannheim) in 30~1 of mixture containing 3 ,uI 10 X universal buffer and 5 units of each enzyme. The reaction was incubated for 90 min at 37°C. At the same time, the plasmid was digested with the same enzymes. After electrophoresis on agarose gel, both insert and digested vector were purified using GeneClean (Bio 101, La Jolla, CA). Ligation was performed in a mixture of 3 ,~l (100 ng/ ,uI) insert, 1 /.d digested vector (100 ng/pl), 1~1 ligation buffer, 1 ,uI (2 units) T4 DNA ligase, 1~1 rATP (1 mM final concentration) and 3 ~1 sterile distilled water. The reaction was incubated overnight at 4°C. XLl-Blue competent cells (Stratagene), 100 ~1, were then incubated with 2 ~1 ligation product on ice for 30 min. After a heat-pulse (45 s at 42°C). 500 ,~l LB broth were added and the culture aerated for 1 h at 37°C. Transformed cells (100 ,uI) were plated onto LB plates containing 50 peg ampicillin, 20 pg tetracyclin and 80 ,ug fresh X-gal/ml and 20 mM isopro-
Figure 1. Ligation The photographed image, whole-band, of ligation pBlue-Script in the vector;
control with 0.8% agarose gel eiectrophoresis. gel was scanned and analysed on a Bio Scanner 3 + system (Millipore). l-product
performed without insert (recircularized vector KS +); 2-product of ligation performed with insert M-pBR328 DNA/BgII + pBR328 DNA/Hinfl.
World Journal of Microbiology 6 Biotechnology, Vol 11. 1995
479
I’. Abed, C. Ballet and l? De Micco the presumed recombinants (white colonies) obtained by the T/A cloning or PCR-script methods were truly positive (Table I). The high numbers of false positives generated by both these methods is partly due to the presence of Taq polymerase, which is known to inhibit cloning (Bennet & Mlenaar 1994). Consequently, it was necessary to test a large number of white colonies. Although both methods gave a low transformation efficiency, transformation was performed without conkolling ligation efficiency with agarose gel electrophoresis because small amounts of DNA were used in the ligation reaction. When PCR products were purified by GeneClean prior to the ligation reaction, the rate of false positives decreased from 55% to 31% using the PCR-script system and from 60% to 35% with T/ A cloning, thus confirming the inhibitor effect of Taq polymerase. However, the third method tested, in which terminus-restricted PCR products were inserted in the corresponding digested vector, was still more efficient than either direct method. Using this method, it was possible to control ligation efficiency with agarose gel electrophoresis before transformation, since 300 ng insert and 100 ng vector were ligated (Figure I). Transformation was only performed if ligation efficiency is confirmed and, consequently, the proportion of false positives obtained by this method was low (only 3%). Although this method requires a high concenkation of DNA, this should not be a problem as the starting material to be cloned is a PCR product.
480
World]oumal
of Microbiology 6 Biotechnology, Vol I I, 1995
References Bauer, J., Deely, D., Viola, J. & Weiner, M. 1992 PCR-script SK ( + ) cloning system: a simple and fast method for PCR cloning. Strategies in Mokdur Biology 5, 62-64. Bennett, B.L. & Mlenaar, A.J. 1994 Cloning of PCR products can be inhibited by Tuq polymerase carryover. BioTechniques 16, 32-3 7. Clark, J.M. 1988 Novel non-template nucleotide addition reaction catalyzed by procaryotic and eucaryotic DNA polymerase.
Nucleic Acids Research16, 9677-9686. De
Lamballerie, X., Zandotti, Micco, P. 1992 A one step using ‘Chelex 100’ suitable
C., Vignoli, C., Bollet, C. & De microbial DNA extraction method for gene amplification. Research in
Microbiology 143, 7855790. Hance, A.J., Grandchamp, B., L&y-FrCbault, V., Lecossier, D., Rauzier, J., Bocart, D. & Giquel, B. 1989 Detection and identification of mycobacteria by amplification of mycobacterial DNA.
Molecular Biology 3, 843-849. Holmes, D.S. & Quigley, M. 1981 A rapid boiling method preparation of bacterial plasmids. Analytica Biochemistry
for the 114,
193-197. Mead, D., Herrstadt, N., Marcil, R. & Smith, L. 1991 A universal method for the direct cloning of PCR amplified nucleic acid. Biol Technology 9, 65 7-663. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. & Erlich, H.A. 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491.
(Received in revised form ay 1995)
20 February 1995; accepted 25 Febru-
