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Biochemical Genetics, Vol. 42, Nos. 11/12, December 2004 (

C 2004)

A Simple and Highly Efficient Cloning Method

That Employs PCR to Directly Create a Fusion
Between Insert and Vector
Vincent Su1 and Ban-Dar Hsu1,2

Received 15 May 2003—Final 15 October 2003

We have developed a very simple procedure for gene cloning using compound
primers. This approach permits the insertion of a DNA fragment in a single
step using a PCR-based cloning protocol, which employs annealing rather than
ligation to create the recombinant in the plasmid. This method has been tested
successfully to clone a Phalaenopsis gene coding for P450. A potential application
of this protocol for constructing a cDNA library is also proposed.

KEY WORDS: PCR; gene cloning; compound primer; cDNA library.

INTRODUCTION
Splice overlap extension (SOE) polymerase chain reaction (PCR) is used for
splicing together genes (Higuchi et al., 1988; Horton et al., 1989). Since the ends
of the “internal” primers contain complementary sequences, when these PCR
products are mixed, denatured, and allowed to anneal, the complementary ends
of the DNA hybridize, serving as primers for the extension of the complementary
strand. The result is that the two genes are spliced together. This fused gene can
then be amplified by a second PCR (Villarreal and Long, 1991; Zhong and Baja,
1993).
Based on this concept, there have been a few protocols described for the
incorporation of PCR products into vectors. One method utilizes the formation of
sticky end sequences compatible with those generated on the plasmid (Ailenberg
and Silverman, 1996). It uses four primers: two primers are designed to contain

1 Department of Life Science, National Tsing Hua University, Hsin-Chu 30055, Taiwan.
2 To whom correspondence should be addressed; e-mail: bdhsu@life.nthu.edu.tw.
401
0006-2928/04/1200-0401/0

C 2004 Springer Science+Business Media, Inc.
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402 Su and Hsu

the desired sequence of the sticky end plus the sequence of the gene, and the
other two primers contain the same sequence as the first two primers without the
additional bases. The primers are paired to create two PCR products containing
the additional bases in a staggered 5 position. Following melting and reannealing,
the newly formed products contain the additional sequences as sticky overhangs
compatible with the sticky ends of the plasmid. Another method uses chimeric
DNA/RNA primers composed mostly of DNA, but including a 5 region composed
of one or more ribonucleotides (Coljee et al., 2000; Donahue et al., 2002). When
PCR is performed using a polymerase that lacks reverse transcriptase activity, the
result is double-stranded DNA molecules flanked by single-stranded RNA tails.
Linking one or more PCR products together via these tails, followed by ligation
of the PCR products to one another, creates chimeric genes.
Although these procedures have been successful in some instances, long and
complicated steps limit their usage. In this report, we describe a method for cloning
DNA fragments, which, though based on a similar principle, is highly efficient and
requires minimal manipulation prior to the transformation. Using this method, we
have successfully cloned the Phalaenopsis P450 gene with high efficiency.

MATERIALS AND METHODS

Vector and Phalaenopsis Genomic DNA Preparation

The cloning vector used in this study was pUC18. Total DNA of the flower of
Phalaenopsis (Dtps. Taisuco Sun) was isolated using a commercial reagent ac-
cording to the manufacturer’s instructions (Plant DNAZOL, Life Technologies/
Gibco-BRL, Cleveland).

PCR Product Preparation

Table I lists the primer sequences used for the experiments. For producing a PCR
product with blunt ends, each 100 µL PCR mixture contained 50 pmol of each
primer, 1 × Pfu buffer [10 mM (NH4 )2 SO4 , 20 mM Tris (pH 8.8), 10 mM KCl,
2 mM MgCl2 , 0.1% Triton X-100, 1 mg/mL bovine serum albumin], 0.3 mM each

Table I. Sequences of Primers Used in This Study

Primers Sequences

18-p450 5 -ggccagtgccaagcttgcatgcctgccggcaccactcctctctgttcc-3
p450-3 5 -agcaacaaattcatttctttcttatattataagc-3
pUC18 A 5 -aggtcgactctagaggatccccggg-3
S18-5 5 -accggatccccgcgttgctggcgtttttcc-3
S18-3 5 -aggggatccggaagagtatgagtattcaac-3
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Gene Cloning Using Compound Primers 403

dNTP, 5–10 ng DNA template, and 2 U Pfu polymerase (Stratagene). The reaction
cycling conditions were 94◦ C for 10 min (94◦ C for 1 min, 55◦ C for 1 min, 66◦ C
for 5 min) × 40 cycles, and were done in a Perkin–Elmer GeneAmp PCR System
2400. PCR products were gel purified from a 0.8% agarose gel using the QiaQuick
gel extraction kit (Qiagen, Valencia, CA).

Restriction Digestion and Ligation

For self-ligation of blunt ends, approximately 500 ng DNA samples were combined
in a 30 µL reaction that included 3 µL of 10× Roche ligation buffer (660 mM
Tris-HCl, 50 mM MgCl2 , 10 mM dithioerythritol, 10 mM ATP, pH 7.5), 15%
PEG, and 1U T4 DNA ligase (Roche). Reactions were incubated for 16 h at 25◦ C.
BamHI (New England Biolabs) digestions were carried out according to
the manufacturer’s directions. For self-ligation of DNA with sticky ends, approxi-
mately 100 ng DNA samples were combined in a 30 µL reaction that also included
3 µL of 10× Roche ligation buffer (660 mM Tris-HCl, 50 mM MgCl2 , 10 mM
dithioerythritol, 10 mM ATP, pH 7.5) and 1U T4 DNA ligase (Roche). Reactions
were incubated for 16 h at 4◦ C.

Bacterial Transformation
Following the ligation reaction, maximum efficiency JM109 bacteria (New
England Biolabs) were transformed with a constant amount of DNA, and plated
on ampicillin containing LB agar plates.

RESULTS
The experimental approach is described in Fig. 1 using a P450 gene from
Phalaenopsis and a pUC18 plasmid as an example. The end of 18-P450 primer
contains a complementary sequence of pUC18 (shown in gray), and so does the
P450 PCR product (Fig. 1c). When the PCR product and the circular pUC18 are
mixed, melted and reannealed, their complementary ends hybridize. The P450
PCR product then can serve as a primer for the extension of the complemen-
tary strand and finally bring about a fusion between P450 and pUC18 (Fig. 1e).
This fused P450 and pUC18 sequence can be amplified by another PCR using a
pUC18A primer, plus the P450 3 primer added at the beginning. The resultant
blunt-end PCR product is gel-purified followed by self-ligation using the DNA
ligation kit (Fig. 1f).
The feasibility of the procedure has been tested and the results are shown in
Fig. 2a of PCR product 18-P450 (Fig. 1c, 1.8 kb) and Fig. 2b of PCR product
pUC18+P450 (Fig. 1e, 4.5 kb). To assess the efficiency of this splicing process,
the band shown in Fig. 2b was gel purified. Following the self-ligation reaction,
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404 Su and Hsu

Fig. 1. The strategy for gene cloning using compound

primers. The experimental approach uses a Phalaenop-
sis P450 gene and pUC18 as an example. (a) The Pha-
laenopsis genome isolated using a commercial reagent.
(b) and (c) The PCR products 18-P450 are amplified
using primers 18-p450 and p450 3 . The end of 18-p450
primer contains a complementary sequence of pUC18
(shown in gray). (d) When the 18-P450 PCR prod-
ucts and pUC18 are mixed, melted, and reannealed, the
complementary ends hybridize. 18-P450 PCR products
serve as primers for the extension of the complemen-
tary strand. (e) P450 and pUC18 are combined. This
fused P450 and pUC18 sequence can then be amplified
by a second PCR using primers pUC18A and p450 3 .
(f) The blunt-end PCR products are used directly for
self-ligation and transformed to competent bacteria.
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Gene Cloning Using Compound Primers 405

Fig. 2. The agarose gels of PCR products and the restriction analysis of plasmids. Two
expected amplified products 18-P450 (1.8 kb) and pUC18+P450 (4.5 kb) are shown in
(a) and (b), respectively. (c) Plasmid pS18 containing a BamHI site shows appropriate
restriction products extracted from bacteria in comparison with that of pUC18. Molecular
size in kilobase (kb) is marked on the left.

maximum efficiency JM109 bacteria competent cells were transformed with 30 µL

ligation mixture, and plated on ampicillin containing LB agar plates. Since it is
possible that the colonies appeared represent the transformants containing only
pUC18, a PCR-based screening procedure was devised for analyzing the presence
of P450. Ten colonies were randomly selected from a total of 120 colonies. They
were inoculated directly into a PCR reaction mix designed to yield P450-specific
PCR product. The target gene was found in all the samples analyzed (data not
shown). By following the traditional ligation protocol using T4 DNA ligase, the
efficiency of incorporation of P450 into pUC18 was about 10-fold lower than that
obtained by using our PCR-based method. The result clearly shows this PCR-based
method brings about a highly efficient fusion of P450 and pUC18.
PCR efficiency declines exponentially as the amplicon length increases. In
order to accommodate longer gene and improve the efficiency of PCR in our
protocol, a smaller plasmid can be employed. As shown in Fig. 3, a 1.6-kb plasmid
pS18 derived from pUC18 (2.7 kb) was constructed. A BamHI site is added to the
end of each of the two primers to further improve the self-ligation efficiency of the
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406 Su and Hsu

Fig. 3. General scheme for constructing the small plasmid

pS18. The PCR products pS18 are amplified using primers
S18-5 and S18-3. A BamHI site is added to the end of each
of the two primers to improve the self-ligation efficiency of
the PCR product.

PCR product. The newly constructed pS18 is still functional. It can be transformed
into competent cells and the restriction product is shown in Fig. 2c.

DISCUSSION
In this report, we describe a procedure that allows the creation of recombinant
DNA molecules with high efficiency by simply mixing PCR products together. The
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Fig. 4. A hypothetical application of the new method for

cDNA library construction. cDNAs are first generated by
using the conventional oligo-dT primers but with an exten-
sion of a complementary sequence of pS18 plasmid (shown
in gray). When these cDNAs and pS18 are mixed, melted,
and reannealed in a PCR step, their complementary ends hy-
bridize, serving as primers for the extension of the comple-
mentary strand. This results in a fusion between the two. The
presence of a complementary segment at the end of cDNAs
facilitates their fusion with the vector.

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408 Su and Hsu

distinctive feature of this method is the use of a compound primer that contains
a complementary sequence of a specific vector. The length of this overlapped
sequence is crucial. It should meet the general requirements as a PCR primer of
the target gene on the one end and of the vector on the other end. Our method also
relies on the use of the DNA polymerase that has a proofreading (i.e., exonuclease)
function (Pfu polymerase), so that mutation is safely eliminated during PCR cycles.
In addition, the products generated are DNA fragments with blunt ends that match
exactly the primer sequence, unlike the Taq DNA polymerase which adds a single
A overhang. Following gel purification, the blunt-end PCR products then can be
used directly for self-ligation using the T4 DNA ligase. If the sequence of the target
gene is known, an appropriate restriction enzyme that has no cutting site within
the sequences of the target gene and plasmid can be selected, and recognition sites
of it can be added to the two primers used at the step of Fig. 1e (pUC18A and p450
3 in our example). The final PCR products with sticky ends are then created by
enzyme digestion. This can further increase the efficiency of self-ligation. Another
modification of this protocol is the use of a smaller plasmid as shown in Fig. 3.
A reduction in the length of amplicon by 1 kb may be quite helpful for the PCR
reaction in some instances.
Among the many potential applications, this technique could be used in the
construction of cDNA library. A hypothetical use is illustrated in Fig. 4. In a
previous report by Okayama and Berg (1982), the use of oligo-dT primers that
are directly connected to a cloning vector was described. However, the limited
efficiency of cDNA generation and cloning are the major drawbacks of the method.
In our strategy (Fig. 4), cDNAs can first be generated by using conventional oligo-
dT primers but with an extension of a complementary sequence of pS18 plasmid
(shown in gray). When these cDNAs and pS18 are mixed, melted and reannealed
in a PCR step, their complementary ends hybridize, serving as primers for the
extension of the complementary strand. This results in a fusion between the two.
The presence of a complementary segment at the end of cDNAs facilitates their
incorporation into the vector.
In conclusion, our new protocol is fast, easy, reliable, and inexpensive, thereby
offering many advantages over other conventional techniques. It is likely that
additional applications of this method will continue to emerge.

REFERENCES
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Donahue, W. F., Turczyk, B. M., and Jarrell, K. A. (2002). Rapid gene cloning using terminator primers
and modular vectors. Nucleic Acids Res. 30:e95.
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Gene Cloning Using Compound Primers 409

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