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This Sample Protocol contains the full text of the published Unit, including expert commentary sections with critical information designed to ensure the success of your experiments.
Contributed by Elaine A.
Elion
Harvard Medical School
Boston, Massachusetts
Any two segments of DNA can be ligated together into a new recombinant molecule using the polymerase chain reaction (PCR). The DNA can be joined in any configuration, with any desired junction-point reading frame or restriction site, by incorporating extra nonhomologous nucleotides within the PCR primers. Cloning by PCR is often more rapid and versatile than cloning with standard techniques that rely on the availability of naturally occurring restriction sites and require microgram quantities of DNA. It is not necessary to know the nucleotide sequence of the DNA being subcloned by this technique, other than the two short flanking regions (~20 bp) that serve as anchors for the two oligonucleotide primers used in the amplification process. Moreover, PCR can be performed on low-abundance or even degraded DNA (or RNA) sources.
This unit describes using PCR to construct hybrid DNA molecules. The main objective is to give an overview of how PCR can be exploited to accomplish numerous cloning strategies; it is assumed that the reader is already familiar with basic molecular biology techniques including PCR amplification (UNIT 15.1) and subcloning (UNIT 3.16). The basic protocol outlines the PCR amplification and cloning strategies. A troubleshooting guide for problems most frequently encountered in PCR cloning, and three specific examples of this technique--for creating (1) in-frame fusion proteins, (2) recombinant DNA products, and (3) deletions and inversions by inverse PCR--are presented in the Commentary.
In this protocol, synthetic oligonucleotides incorporating new unique restriction sites are used to amplify a region of DNA to be subcloned into a vector containing compatible restriction sites. The amplified DNA fragment is purified, subjected to enzymatic digestion at the new restriction sites, and then ligated into the vector. Individual subclones are analyzed by restriction endonuclease digestion and either sequenced or tested in a functional assay.
5'
exonuclease proofreading activity can be used instead of Taq DNA polymerase
to reduce the amount of nucleotide misincorporation during amplification.
Pfu DNA polymerase (Stratagene) and Vent DNA polymerase (New England Biolabs)
have this activity (follow manufacturers' instructions).The main benefit of cloning by PCR is that unique restriction sites can be introduced on either side of any segment region of amplified DNA to allow its ligation into a recipient vector (Mullis and Faloona, 1987; Chapter 15) in any configuration. The incorporation of additional nucleotides at the 5' ends of the oligonucleotide primers permits the creation of novel restriction sites or changes in reading frame and coding sequence. The oligonucleotide primers can also be designed to contain mismatches, deletions, or insertions in the region of homology (UNIT 8.5). However, it is not necessary to always incorporate a new restriction site in the primer. Amplified PCR fragments can also be subcloned by blunt-end or sticky-end ligation using preexisting restriction sites within the amplified DNA. For example, PCR might be used to amplify a target DNA that already contains appropriate restriction sites, but is available in limited quantities. Finally, sequential polymerase chain reactions can be used to generate more complex recombinant PCR products which can subsequently be subcloned into a recipient vector.
The most obvious
disadvantage of PCR cloning is the need to verify that the subcloned PCR product
does not contain mutations generated during the polymerase chain reaction. In
cases where longer DNA segments (i.e., >1 kb) are being amplified, it may
be more advantageous to use a polymerase which has a 3'5'
exonuclease activity (e.g., Pfu polymerase, Stratagene) to reduce the chances
of generating mutations. After subcloning, several independent PCR products
should be analyzed by DNA sequencing to be sure that the recombinant DNA molecule
is not mutated. Sequencing can be laborious when a large fragment of DNA is
subcloned. However, subcloned PCR products can be prescreened by either biological
or biochemical functional assays if they are available. In some instances, it
may be more desirable to break down the cloning into several steps that might
involve the introduction of a needed restriction site within a short piece of
DNA first.
In general, the DNA preparation, purification, and ligation guidelines outlined in UNIT 3.16 should be applied to PCR cloning to ensure recovery of the desired ligation products. However, the following points deserve special consideration.
Design of oligonucleotide primers. Primers should only hybridize to the sequence of interest. This can be predicted in instances where sequence information is available. In general, primers with homology of 16 to 20 nucleotides to the target DNA and a GC content of ~50% should be chosen. A longer oligonucleotide of ~25 nucleotides should be used for AT-rich regions. In instances where genomic DNA is the source of the target DNA, the oligonucleotide primers should contain at least 20 nucleotides of homology to the target DNA to ensure that they anneal specifically (Arnheim and Erlich, 1992).
When using primers to introduce a specific restriction site, a sequence within the target DNA should be selected that requires the addition of the fewest noncomplementary nucleotides to create the new site, if possible. Special consideration should be given to the choice of site itself, as restriction endonucleases vary in their ability to cleave recognition sequences within ten nucleotides of the end of a DNA duplex (consult Table 8.5.1 for the efficacies of different restriction enzymes in cleaving terminal recognition sequences). It is also recommended that four to five additional nucleotides be added on the 5' side of the restriction site in the primer. Because DNA duplexes "breathe" at termini, potentially interfering with the ability of a restriction enzyme to cleave (Innis et al., 1990), it is useful to use the GCGC "clamp" sequence that is most thermostable (Sheffield et al., 1989).
Finally, the sequence of the primer should be checked for internal complementarity to avoid secondary structure formation that will interfere with hybridization of the primer to the target DNA. The 3' ends of the two primers being used must not be complementary, so that the formation of primer-dimers that will compete with the synthesis of the desired PCR product will be avoided.
Additional details on primer design are discussed in UNIT 15.1.
DNA polymerase.
Commercially available Taq DNA polymerase (Perkin-Elmer Cetus) lacks
the 3'5'
proofreading exonuclease activity used by DNA polymerase I Klenow fragment and
T4 DNA polymerase to reduce error frequency (Kornberg, 1992). This absence of
proofreading activity in Taq DNA polymerase is thought to result in a
heightened error frequency. Old estimates indicate that the average rate of
misincorporation is 8.5 
10-6
nucleotides per cycle (Goodenow et al., 1989; Fucharoen et al., 1989). Two other
thermostable DNA polymerases possessing proofreading 3'5'
exonuclease activity have recently become commercially available: Pfu DNA polymerase,
purified from Pyrococcus furiosus (Stratagene) and Vent DNA polymerase,
purified from Thermococcus litoralis (New England Biolabs and Promega).
Both are more thermostable than Taq DNA polymerase. Pfu DNA polymerase
is 12-fold more accurate than Taq DNA polymerase, as assayed by the method
of Kohler et al. (1991). Vent DNA polymerase is 4-fold more accurate than Taq
DNA polymerase (Cariello et al., 1991). Although it is difficult to compare
the relative error frequencies of three enzymes because they were assayed by
different methods, the use of either Vent or Pfu DNA polymerases may reduce
the amount of misincorporation.
Removal of unincorporated nucleotide triphosphates. It is recommended that the amplified PCR fragment be purified from unincorporated nucleotides and primers. Any method of purification that involves electrophoresis can also separate the desired PCR product from any undesired DNA species produced during amplification. Typical methods of DNA purification include electrophoresis through low-gelling/melting temperature agarose or electrophoresis through agarose followed by DNA purification by electroelution or adsorption to glass beads (UNIT 2.6). However, amplified DNA can be more rapidly purified from unincorporated nucleotide triphosphates and primers using a Centricon microconcentration unit (Amicon). The disadvantage of using the microconcentrator is that undesirable PCR products and the starting template DNA will copurify with the amplified PCR fragment.
PCR amplification. The use of appropriately designed primers should allow the amplification of the DNA segment of interest. Occasionally, however, primers may not be specific, leading to the amplification of undesired DNA segments. The specificity of primer to template hybridization will depend upon temperature and salt (see UNITS 15.1 & 6.4 for a thorough discussion). The highest annealing temperature possible should be used to reduce nonspecific associations. Some nonspecific amplifications can be avoided by employing a "hot-start" technique--i.e., adding the DNA polymerase to a prewarmed sample (D'Aquila et al., 1991). In addition, purifying the PCR product by gel electrophoresis will help ensure that the proper DNA fragment is subcloned. If the synthesized primer does not bind with specificity, it may be simplest to have another one synthesized.
In setting up the amplification cycle, keep in mind that for cloning, fidelity is more important than yield, so it is better to keep a low cycle number and not to raise the MgCl2 concentration too much. 1.5 mM MgCl2 in the amplification buffer should be sufficient for most primers.
Cloning of the amplified fragment. "Sticky-end" ligation of the amplified DNA can sometimes be difficult, due to poor cutting of the terminal restriction site by the desired restriction endonuclease. Careful choice of restriction sites and the addition of extra nucleotides to the 5' end of the primer (see critical parameters) will facilitate digestion.
Other common explanations for poor cutting by restriction endonucleases include:
(1) Blockage of the duplex
terminus by bound Taq DNA polymerase. If this is the case, the amplified
DNA can be treated with proteinase K to remove associated protein. This is done
by adding 50 µg/ml proteinase K in 10 mM Tris·Cl (pH 7.8)/5 mM
EDTA/0.5% (v/v) SDS to the sample and incubating 30 min at 37°C. The sample
must be extracted with phenol/chloroform (UNIT 2.1) to remove the proteinase
K.
(2) Inefficient extension by Taq DNA polymerase. The nonduplex ends generated
in this fashion can be repaired by filling in with Klenow fragment (UNIT
3.5).
(3) An insufficient number of extra nucleotides 5' to the restriction site in
the primer. In this case, the primer should be resynthesized. Prolonged (typically
overnight) DNA digestion should also be tried.
(4) 5' terminal breathing of duplex DNA. Duplex formation can be stabilized
by the inclusion of 0.1 mM spermidine in the digestion reaction.
An alternative approach is to internalize the restriction site by concatemerizing the PCR products prior to restriction endonuclease digestion (Jung et al., 1990). To do this, 5' phosphorylated oligonucleotide primers are used in the PCR amplification, or following amplification, the DNA fragment is phosphorylated using T4 polynucleotide kinase (UNIT 3.10). The amplified fragments are then concatemerized by ligation using T4 DNA ligase (UNIT 3.16) prior to restriction endonuclease treatment. Aliquots of the amplified DNA before and after concatemerization, and after digestion, can be compared on an agarose gel to confirm that the procedure worked.
Blunt-end
ligations are often inhibited by nonflushed ends in the PCR fragment, due
to the presence of a nontemplate-directed nucleotide (usually dATP) added
by Taq DNA polymerase (Clark, 1988). Treatment of the PCR fragment
with Klenow polymerase in the presence of dNTPs (UNIT 3.5) to make
the ends flush should circumvent this problem. In the event that this simple
approach does not work, the PCR fragment can be subcloned into a MstII
(or Bsu36 I) site (CC
TNAGG)
which leaves a 5' dT overhang. This approach requires a recipient vector with
a unique MstII site.
All of the cloning approaches outlined are reliable and should result in efficient recovery of the desired recombinant molecules.
Once the oligonucleotides have been synthesized, the PCR amplification, purification, ligation, and transformation steps can all be done within 2 days. The appropriate subclones can then be sequenced or tested in a functional assay immediately thereafter.
PCR cloning is particularly useful for creating in-frame fusions between two open reading frames, as is often done for synthesizing fusion proteins with E. coli expression vectors (Chapter 16). The essence of this type of subcloning involves incorporating additional noncomplementary nucleotides within the oligonucleotide primer that will encode the junction sequences of the amplified PCR fragment. Consider the introduction of a unique EcoRI site into a piece of target DNA that is to be fused with an open reading frame in the recipient vector.
For this experiment, the primers should be designed as indicated in panel A. Each primer is designed to contain a unique restriction site not present within the target DNA. The primer carrying the EcoRI site contains an additional nucleotide (shown in bold) to allow the ATG of the amplified target DNA to be in-frame with the open reading frame in the vector (bold bracket). The second primer contains a unique BamHI site. Both oligonucleotide primers are designed to be homologous to and anneal with ~20 nucleotides of DNA flanking the target DNA. They are oriented such that their 3' hydroxyl ends point toward the target DNA. These unique restriction sites are 5' to the region of the primer that is homologous to the target DNA. Each new restriction site is separated from the 5' end of the oligonucleotide by four additional nucleotides to facilitate enzymatic digestion of the amplified DNA. Shown in this example is a GC clamp (Myers et al., 1985) which favors duplex formation at the ends of the amplified fragment.
Panel B depicts the sequence of events in this experiment. Following primer annealing and PCR amplification, the amplified DNA is first digested with restriction endonucleases that cleave at the new restriction sites, then purified by gel electrophoresis. The recipient vector DNA is digested with either the same or compatible restriction endonucleases and is purified before being ligated to the recipient vector.
In this example, one oligonucleotide contains additional bases to create an in-frame fusion with the plasmid-borne open reading frame. More elaborate primers can be designed to include additional restriction sites in different reading frames to allow subcloning of the same PCR fragment into multiple recipient fusion vectors that may have nonidentical cloning sites. This is efficient both in terms of labor and the cost of having to synthesize a new oligonucleotide primer. Note that an ATG can also be incorporated into the 5' oligonucleotide primer to create an open reading frame with a new translational start (e.g., in the construction of a promoter-exon fusion).
Consider creating a chimeric DNA molecule by sequential polymerase chain reactions rather than by ligation. This technique is useful for complex cloning schemes that involve fusing together more than two pieces of DNA, as depicted in the creation of the gene fusion. In this example, two PCR products are made from noncontiguous regions of DNA (that are also nonhomologous) in separate reactions.
Two of the first-round amplification primers are designed to contain 5' extensions that are homologous to a portion of the other target gene (see primers 1b and 1c). In this example, the primers for target gene 1 are labeled as 1a and 1b. Primer 1a contains a unique EcoRI site; primer 1b contains a 5' extension that is homologous to a region in target gene 2 that will be amplified (thin line of arrow). Primers 1c and 1d are for amplifying target gene 2: primer 1c contains a 5' extension that is homologous to a portion of target gene 1 that will be amplified (bold line of arrow), and primer 1d contains a unique BamHI restriction site.
Because primers 1b and 1c contain complementary 5' extensions, two PCR products containing a region of overlapping homology are generated. The two PCR fragments are purified away from the primers, then mixed together and annealed by denaturation and renaturation. Four DNA species are generated in this reaction: two heteroduplexes associating at the region of overlapping homology and two parental homoduplexes. The recessed 3' ends of the heteroduplexes are extended by Taq DNA polymerase to produce a single fragment that is equal in length to the sum of the two overlapping fragments.
In a second round of amplification, the combined heteroduplex DNA species is amplified by adding the outside set of primers (1b and 1d) to the PCR assay. These primers will now have complete homology to the amplified heteroduplex DNA species. (Note that the parental homoduplexes will not be amplified because only one of each outside primers will anneal to each parental homoduplex.)
The complementary primers used in the first polymerase chain reaction step can be designed to either insert a restriction site at the junction between the joined PCR products, or alter a reading frame.
Consider deleting a segment of DNA from a plasmid by inserting a unique restriction site by inverse PCR. Divergent primers containing the same novel restriction site (R) are annealed to two portions of the plasmid. (Note that R can be any restriction site not found in the plasmid.) The primers are oriented with their 3' ends facing away from each other so that sequences flanking the region to be deleted will be amplified. The full-length PCR product is purified and digested at the new restriction site and the ends are ligated in a unimolecular reaction. The amount of spacing between the two primers will determine whether the final product contains all of the original sequence or a deletion. Insertions can also be made at specific sites by including 5' extensions in the primers. Mismatches within the body of the primer can also be used to introduce mutations.
This method is useful for rapid introduction of desired restriction sites, and is limited only by the size of the plasmid and the ability of DNA polymerase to synthesize complete products. This methodology allows the amplification of DNA flanking a region of known sequence. It is also useful for cloning DNA that has not yet been sequenced and for making hybridization probes (Ochman et al., 1990).
Arnheim, N. and Erlich, H. 1992. Polymerase chain reaction strategy. Annu. Rev. Biochem. 61:131-156.
Cariello, N.F., Swenberg, J.A., and Skopek, T.R. 1991. Fidelity of Thermococcus litoralis DNA polymerase (Vent) in PCR determined by denaturing gradient gel electrophoresis. Nucl. Acids Res. 19:4193-4198.
Clark, J.M. 1988. Novel nontemplated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucl. Acids Res. 16:9677-9689.
D'Aquila, R.T., Bechtel, L.J., Videler, J.A., Eron, J.J., Gorczyca, P., and Kaplan, J.C. 1991. Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucl. Acids Res. 19:3749.
Fucharoen, S., Fucharoen, G., Fucharoen, P., and Fukamaki, Y. 1989. A novel ochre mutation in the beta-thalassemia gene of a Thai identified by direct cloning of the entire beta-globin gene amplified using polymerase chain reactions. J. Biol. Chem. 264:7780-7783.
Goodenow, M., Huet, T., Saurin, W., Kwok, S., Sninsky, J., and Wain-Hobson, S. 1989. HIV-1 isolates are rapidly evolving quasi-species: Evidence for viral mixtures and preferred nucleotide substitutions. J. Acquired Immunol. Defic. Syndr. 2:344-352.
Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. (eds.) 1990. PCR Protocols. Academic Press, San Diego.
Jung, V., Pestka, S.B., and Pestka, S. 1990. Efficient cloning of PCR-generated DNA containing terminal restriction endonuclease sites. Nucl. Acids Res. 18:6156.
Kornberg, A. and Baker, T.A. 1992. DNA Replication, 2nd ed. W.H. Freeman, New York.
Mullis, K.B. and Faloona, F.A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155:335-350.
Ochman, H., Medhora, M.M., Garza, D., and Hartl, D.L. 1990. Amplification of flanking sequences by inverse PCR. In PCR Protocols. (M.A. Innis, D.H. Gelfand, J.J Sninsky, and T.J. White, eds.) pp. 219-227. Academic Press, San Diego, Calif.
Sheffield, V.C., Cox, D.R., Lerman, L.S., and Myers, R.M. 1989. Attachment of a 40 base pair G+C-rich sequence (GC clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc. Natl. Acad. Sci. U.S.A. 86:232-236.
Innis et al., 1990. See above.
Provides an in-depth analysis of PCR methods and techniques.