k.) 1990 Oxford University Press
1920 Nucleic Acids Research, Vol. 18, No.7
PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products Alan R.Shuldiner, Laurie A.Scott and Jesse Roth Diabetes Branch, National Institutes of Health, Building 10, Room 8-S-243, Bethesda, MD 20892, USA Submitted February 9, 1990
For many applications, it is often necessary to subclone PCR products into plasmid vectors. Subcloning strategies that have been employed include 1) incorporation of restriction endonuclease sites into the primers whose sticky ends may be ligated into the appropriate vector, and 2) blunt-end ligation (1). However, we and others have found that these conventional strategies often fail. We report a rapid and versatile method to directionally subclone PCR fragments in a single day without the use of DNA ligase. The first step in PCR-induced subcloning is amplification of the DNA of interest using oligonucleotides that contain at their 5'-ends a 24-nucleotide sequence that is complementary to the 3'-ends of the desired linearized plasmid vector (primers a and b, fig. 1). After filtration through an Ultrafree-MC unit (Millipore; Bedford, MA) to remove excess primers, 50 to 100 ng of the PCR product is placed into two tubes containing 50 ng of linearized plasmid, PCR buffer, dNTPs, and Taq polymerase. In addition, tube 1 contains oligos a and c, while tube 2 contains oligos b and d (fig. 1). During the first cycle of PCR, the 3'-ends of the PCR product may anneal to the 3'-ends of the plasmid (2). Extension results in the 'ligation' of the PCR insert to the complementary 3'-end of the plasmid. Subsequent PCR cycles (cycles 2 through 15) with the respective primers in each tube result in the logarithmic amplification of the linear recombinant vector. After PCR, 10 ,1l of tubes 1 and 2 are combined. Double stranded DNA is denatured in 0.2M NaOH for 5 min at room temperature, and then neutralized in 400 t1l of boiling Tris (50 mM, pH 6.6). Incubation for 3 to 6 hours at 60°C results in the reannealing of complementary heterologous strands (heterologous reannealing, fig. 1), followed by cyclization. The mixture is concentrated through an UltrafreeMC unit, and used to transform competent E. coli. Using this method, we have successfully subcloned three PCR fragments into pGem4Z (Promega Biotec, Madison, WI) typically obtaining hundreds to thousands of colonies per transformation (transformation efficiency approximately 5 x 103-5 x 104 colonies per microgram of PCR insert). We have demonstrated
by dideoxy sequence analysis that the PCR products are inserted into the vector in the predicted orientation. In our hands, PCRinduced subcloning is fast, easy and reliable, and thereby offers many advantages over existing subcloning strategies.
REFERENCES 1. Scharf,S.J., Horn,G.T. and Erlich,H.A. (1986) Science 233, 1076-1078. 2. Higuchi,R., Krummel,B. and Saiki,R.K. (1988) Nucl. Acids Res. 16, 7351 -7367.
PCR
Aplification froe
sanoic DNA
or cONA
PCR Product
Tube
Linearized
jTube
1
l
-+-
1
34
-
Plas,d
+
L~ (C9'1e 11
d
8-4
4-
(Cc les 2 thru 15)
Combine tu6es Danaturation
2
w
2
HIterologous ranl ing w cwcliantion or
Transforn E.coli
-s
1920 Nucleic Acids Research, Vol. 18, No.7
PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products Alan R.Shuldiner, Laurie A.Scott and Jesse Roth Diabetes Branch, National Institutes of Health, Building 10, Room 8-S-243, Bethesda, MD 20892, USA Submitted February 9, 1990
For many applications, it is often necessary to subclone PCR products into plasmid vectors. Subcloning strategies that have been employed include 1) incorporation of restriction endonuclease sites into the primers whose sticky ends may be ligated into the appropriate vector, and 2) blunt-end ligation (1). However, we and others have found that these conventional strategies often fail. We report a rapid and versatile method to directionally subclone PCR fragments in a single day without the use of DNA ligase. The first step in PCR-induced subcloning is amplification of the DNA of interest using oligonucleotides that contain at their 5'-ends a 24-nucleotide sequence that is complementary to the 3'-ends of the desired linearized plasmid vector (primers a and b, fig. 1). After filtration through an Ultrafree-MC unit (Millipore; Bedford, MA) to remove excess primers, 50 to 100 ng of the PCR product is placed into two tubes containing 50 ng of linearized plasmid, PCR buffer, dNTPs, and Taq polymerase. In addition, tube 1 contains oligos a and c, while tube 2 contains oligos b and d (fig. 1). During the first cycle of PCR, the 3'-ends of the PCR product may anneal to the 3'-ends of the plasmid (2). Extension results in the 'ligation' of the PCR insert to the complementary 3'-end of the plasmid. Subsequent PCR cycles (cycles 2 through 15) with the respective primers in each tube result in the logarithmic amplification of the linear recombinant vector. After PCR, 10 ,1l of tubes 1 and 2 are combined. Double stranded DNA is denatured in 0.2M NaOH for 5 min at room temperature, and then neutralized in 400 t1l of boiling Tris (50 mM, pH 6.6). Incubation for 3 to 6 hours at 60°C results in the reannealing of complementary heterologous strands (heterologous reannealing, fig. 1), followed by cyclization. The mixture is concentrated through an UltrafreeMC unit, and used to transform competent E. coli. Using this method, we have successfully subcloned three PCR fragments into pGem4Z (Promega Biotec, Madison, WI) typically obtaining hundreds to thousands of colonies per transformation (transformation efficiency approximately 5 x 103-5 x 104 colonies per microgram of PCR insert). We have demonstrated
by dideoxy sequence analysis that the PCR products are inserted into the vector in the predicted orientation. In our hands, PCRinduced subcloning is fast, easy and reliable, and thereby offers many advantages over existing subcloning strategies.
REFERENCES 1. Scharf,S.J., Horn,G.T. and Erlich,H.A. (1986) Science 233, 1076-1078. 2. Higuchi,R., Krummel,B. and Saiki,R.K. (1988) Nucl. Acids Res. 16, 7351 -7367.
PCR
Aplification froe
sanoic DNA
or cONA
PCR Product
Tube
Linearized
jTube
1
l
-+-
1
34
-
Plas,d
+
L~ (C9'1e 11
d
8-4
4-
(Cc les 2 thru 15)
Combine tu6es Danaturation
2
w
2
HIterologous ranl ing w cwcliantion or
Transforn E.coli
-s
