1156 Nucleic Acids Research, Vol. 19, No. 5
A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors T.A.Holton* and M.W.Graham Calgene Pacific Pty. Ltd, 16 Gipps St., Collingwood, Victoria 3066, Australia Submitted December 7, 1990 Difficulties are frequently encountered when cloning PCR products. This is presumably a consequence of the synthesis of 'ragged' ends on DNA amplified by Taq polymerase. Enzymic modification of amplified DNAs with, for example, the Klenow fragment of DNA polymerase I (1) can overcome this problem to some extent, but cloning efficiencies are often still low. Alternatively, restriction sites can be incorporated into the PCR primers for subsequent digestion and cloning, but these sites often prove difficult to cut (2), and the presence of internal sites within the amplified product can create additional problems. We describe here a simple and efficient means of directly cloning PCR generated DNAs that requires no enzymic modification of the PCR product. Our approach is based on the observation that Taq polymerase can add a single non template-directed deoxyadenosine (A) residue to the 3' end of duplex PCR products (3). We have developed a novel approach to the design of vectors that can be used for the direct cloning of PCR products that possess such overhangs. The cloning strategy is outlined diagrammatically in Fig. 1. A blunt-ended vector is prepared, which is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase. The use of ddTTP ensures the addition of only one T residue. The vector ends thus generated have a single 3'-overhanging T residue which lacks a 3'-hydroxyl (OH) group and is therefore incapable of forming a phosphodiester bond. By contrast, the other strand can ligate to other DNAs since it contains a 5'-phosphate (P). A ddT-tailed vector can ligate directly to a PCR product that contains a 3'-overhanging A by forming a phosphodiester bond between the vector's 5'-P and the PCR product's 3'-OH group from the overhanging A. In order to test this strategy a ddT-tailed vector was prepared from pBluescript KS- (Stratagene). Five micrograms of plasmid DNA was digested to completion with EcoRV according to manufacturers directions (Boehringer), the enzyme was heatkilled (65°C for 10 mins.), and the plasmid DNA purified by ethanol precipitation. Tailing was performed using a terminal transferase kit (Boehringer). The linear DNA was tailed in a 40 Id reaction in 25 mM Tris-HCl (pH 6.6), 200 mM K cacodylate, 250 jig/ml BSA, 1.5 mM CoC12, 10 /AM ddTTP (Pharmacia), using 100 units of terminal transferase, for 1 hr at 37°C. The DNA was purified by phenol/chloroform extraction, followed by ether extraction and endol precipitation. The PCR fragments were derived by amplification with Taq polymerase (Cetus) of a plasmid cDNA library made from mRNA isolated from petunia *
To whom
correspondence should be addressed
petals. The primers used were a redundant oligonucleotide (designed to prime from within the cDNA insert) and the reversesequencing primer. This reaction generated a complex mixture of DNAs, with several discrete bands visible over a background smear. The amplified products were size selected (250-500 bp) on an agarose gel and ligated into the EcoRV-cut ddT-tailed pBluescript vector, using an equimolar ratio of vector to insert, at a final DNA concentration of 10 isg/ml. Cells were transfomied and plated for blue/white selection on media containing X-gal. Ninety percent of transformants produced white colonies and subsequent analysis indicated that 80% of these had inserts witiin the expected size range. Sequence analysis across the cloning sites of several of these clones has shown the existence of an additional T residue, confirming that the PCR products were cloned in the predicted manner (data not shown). We have used a similar approach to clone PCR fragments generated using a variety of templates, including single-stranded cDNA, genomic DNA and cloned DNA.
REFERENCES 1. Wilfiams,J.F. (1989) Amplifications 3, 19. 2. Kaufman,D.L. and Evans,G.A. (1990) BioTechniques 9, 305-306. 3. Clarke,J.M. (1988) Nucl. Acids Res. 16, 9677-9686. BLUNT VECTOR
~~iz
Oh ddTW
ddT-TAILED VECTOR d' pIm PCR PRODUCT
LIGATION
+
|T4 DNALp
Fre 1. Schematic representation of cloning with ddT-tailed vectors. Blunt vector ends are indicated by open bars at the top of the diagram. Each end contains a 5'-P (P) and a 3'-OH (OH), which could be generated either with a blunt-cutting resttiction enzyme, or by making a 'sticky' site flush-ended. Vector ends prepared by ddT-tailing with terminal transferase are shown below this. These ends contain single T residues which cannot form a phosphodiester bond since they lack a 3'-OH group (indicated by Td or dT). The shaded bar below represents a PCR product containing a single overhanging 3'-A and Its associated 3'-OH group. The ddT-tailed vector can ligate to the PCR product through formation of phosphodiester bonds (indicated by solid lines) between the 5'-P of the vector and the 3'-OH group of the overhanging A from the PCR product.
A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors T.A.Holton* and M.W.Graham Calgene Pacific Pty. Ltd, 16 Gipps St., Collingwood, Victoria 3066, Australia Submitted December 7, 1990 Difficulties are frequently encountered when cloning PCR products. This is presumably a consequence of the synthesis of 'ragged' ends on DNA amplified by Taq polymerase. Enzymic modification of amplified DNAs with, for example, the Klenow fragment of DNA polymerase I (1) can overcome this problem to some extent, but cloning efficiencies are often still low. Alternatively, restriction sites can be incorporated into the PCR primers for subsequent digestion and cloning, but these sites often prove difficult to cut (2), and the presence of internal sites within the amplified product can create additional problems. We describe here a simple and efficient means of directly cloning PCR generated DNAs that requires no enzymic modification of the PCR product. Our approach is based on the observation that Taq polymerase can add a single non template-directed deoxyadenosine (A) residue to the 3' end of duplex PCR products (3). We have developed a novel approach to the design of vectors that can be used for the direct cloning of PCR products that possess such overhangs. The cloning strategy is outlined diagrammatically in Fig. 1. A blunt-ended vector is prepared, which is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase. The use of ddTTP ensures the addition of only one T residue. The vector ends thus generated have a single 3'-overhanging T residue which lacks a 3'-hydroxyl (OH) group and is therefore incapable of forming a phosphodiester bond. By contrast, the other strand can ligate to other DNAs since it contains a 5'-phosphate (P). A ddT-tailed vector can ligate directly to a PCR product that contains a 3'-overhanging A by forming a phosphodiester bond between the vector's 5'-P and the PCR product's 3'-OH group from the overhanging A. In order to test this strategy a ddT-tailed vector was prepared from pBluescript KS- (Stratagene). Five micrograms of plasmid DNA was digested to completion with EcoRV according to manufacturers directions (Boehringer), the enzyme was heatkilled (65°C for 10 mins.), and the plasmid DNA purified by ethanol precipitation. Tailing was performed using a terminal transferase kit (Boehringer). The linear DNA was tailed in a 40 Id reaction in 25 mM Tris-HCl (pH 6.6), 200 mM K cacodylate, 250 jig/ml BSA, 1.5 mM CoC12, 10 /AM ddTTP (Pharmacia), using 100 units of terminal transferase, for 1 hr at 37°C. The DNA was purified by phenol/chloroform extraction, followed by ether extraction and endol precipitation. The PCR fragments were derived by amplification with Taq polymerase (Cetus) of a plasmid cDNA library made from mRNA isolated from petunia *
To whom
correspondence should be addressed
petals. The primers used were a redundant oligonucleotide (designed to prime from within the cDNA insert) and the reversesequencing primer. This reaction generated a complex mixture of DNAs, with several discrete bands visible over a background smear. The amplified products were size selected (250-500 bp) on an agarose gel and ligated into the EcoRV-cut ddT-tailed pBluescript vector, using an equimolar ratio of vector to insert, at a final DNA concentration of 10 isg/ml. Cells were transfomied and plated for blue/white selection on media containing X-gal. Ninety percent of transformants produced white colonies and subsequent analysis indicated that 80% of these had inserts witiin the expected size range. Sequence analysis across the cloning sites of several of these clones has shown the existence of an additional T residue, confirming that the PCR products were cloned in the predicted manner (data not shown). We have used a similar approach to clone PCR fragments generated using a variety of templates, including single-stranded cDNA, genomic DNA and cloned DNA.
REFERENCES 1. Wilfiams,J.F. (1989) Amplifications 3, 19. 2. Kaufman,D.L. and Evans,G.A. (1990) BioTechniques 9, 305-306. 3. Clarke,J.M. (1988) Nucl. Acids Res. 16, 9677-9686. BLUNT VECTOR
~~iz
Oh ddTW
ddT-TAILED VECTOR d' pIm PCR PRODUCT
LIGATION
+
|T4 DNALp
Fre 1. Schematic representation of cloning with ddT-tailed vectors. Blunt vector ends are indicated by open bars at the top of the diagram. Each end contains a 5'-P (P) and a 3'-OH (OH), which could be generated either with a blunt-cutting resttiction enzyme, or by making a 'sticky' site flush-ended. Vector ends prepared by ddT-tailing with terminal transferase are shown below this. These ends contain single T residues which cannot form a phosphodiester bond since they lack a 3'-OH group (indicated by Td or dT). The shaded bar below represents a PCR product containing a single overhanging 3'-A and Its associated 3'-OH group. The ddT-tailed vector can ligate to the PCR product through formation of phosphodiester bonds (indicated by solid lines) between the 5'-P of the vector and the 3'-OH group of the overhanging A from the PCR product.
