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Minimal Length Requirement of the Single-stranded Tails for Ligation-independent Cloning (LIC) of PCR Products

Charalampos Aslanidis, ~ Pieter J. de long, 2 and Gerd Schmitz ~

1Institute for Clinical Chemistry, University of Regensburg, 93053 Regensburg, Germany; ~Human Genetics Department, Roswell Park Cancer Institute, Buffalo, New York 14263

The Iigation-independent cloning of length LIC tails revealed that using ated by Taq . (7's) In both PCR products (LIC-PCR) is a versatile 12-nucleotide cohesive ends pro- cases, it is necessary to prevent high and highly efficient cloning proce- duced four times more transfor- nonrecombinant backgrounds by treat- dure resulting in recombinant clones mants than were obtained with the ing the vector with alkaline phos- only. Recombinants are generated LIC with lO-nucleotide cohesive ends. phatase. Despite this treatment, a con- between PCR products and a PCR- When the LIC tails were 8 nucleotides siderable fraction of the clones will lack amplified vector through defined long, no transformants were ob- inserts. Recently, several alternative complementary single-stranded (ss) tained. PCR fragment purification, methods have been developed to cir- ends artificially generated with T4 T4 DNA polymerase treatment, and cumvent these problems. These meth- DNA polymerase. This procedure LIC is complete in <1 hr. ods can be divided in the ligation-depen- does not require restriction enzymes, dent and -independent approaches. alkaline phosphatase, or DNA ligase. In 1991, we were the first group to The primers used for amplification develop a highly efficient ligation-inde- contain an additional 12-nucleotide For many applications, PCR (1) has re- pendent cloning procedure for PCR frag- sequence at their $'ends that is com- placed as the method ments (LIC-PCR) that does not involve plementary in the vector- and insert- of choice for the rapid amplification and restriction enzymes, alkaline phos- specific primers. The (3' -~ 5') exonu- isolation of specific DNA sequences from phatase, T4 polynucleotide kinase, or clease activity of T4 DNA polymerase genomic DNA. Nevertheless, complex DNA ligase. (9) In essence, 12-nucleotide- is used In combination with a prede- PCR product mixtures still require clon- long single-stranded (ss) tails are created termined dNTP (dGTP for the inserts ing for the isolation of specific products, at the ends of the PCR products and the and dCTP for the vector) to specifi- for example, for use as probes. This is the PCR-amplified vector using T4 cally remove 12 nucleotides from case for PCR libraries established from DNA polymerase. The ss ends present at each 3'end of the PCR fragments. Be- microdissected chromosomes (2'3) and the PCR fragments are complementary cause of the complementarity of the for inter-Alu PCR. (4-6) In some cases, spe- to those attached to the vector. Nonco- ends that are generated, circulariza- cifically generated PCR products from valent associations between the vector tion can occur between vector and in- cDNAs of interest have to be cloned in and PCR fragments are facilitated. The sert. The recombinant molecules do specific vectors for expression studies. vector has identical, noncomplementary not require in vitro ligation for effi- To facilitate cloning, restriction endonu- tails at either end, preventing the forma- cient bacterial transformation. To clease sites may be introduced into the tion of circular forms consisting of vec- make this technique widely applica- amplification primers so that subse- tor only. These "recombinant" forms ble, we have simplified the handling quent digestion of the PCR fragments are used to transform of the PCR fragments prior to UC. with the appropriate enzymes results in very efficiently. We have not detected The PCR products do not need fur- products ready to be cloned in specific any nonrecombinant transformants ther purification following the T4 sites of vectors. Depending on the recog- since then. The procedure seems to re- DNA polymerase treatment. Incu- nition site used in the primers, the cleav- sult in 100% recombinant clones. An- bation of vector and insert PCR age may be more or less efficient. Some other LIC procedure was developed by fragments for as little as $ rain is suf- restriction endonucleases require addi- Shuldiner et al/1~ This method makes ficient for a high yield of recombi- tional nucleotides at their 5'end. Blunt use of denaturation and heterologous nants. Comparison of the transfor- end cloning is less efficient and requires annealing of the PCR product and the mation efficiencies using different- the removal of the 3' overhang gener- vector and is difficult to control. Rasht-

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chian et al. (12) have developed a LIC pro- efficient LIC of PCR products. We have reaction denatured at 95~ for 10 min, cedure that is very similar to ours. The compared the cloning efficiencies of Alu and chilled on ice. Inter-Alu sequences method is based on the addition of a 12- PCR products having 12-, 10-, and 8-nu- were amplified using 25 pM of the appro- base dUMP-containing sequence to the cleotide-long ss tails. Furthermore, we priate Alu primer, 100 ng of human ge- 5'end of PCR primers. Selective degrada- have simplified the purification steps re- nomic DNA, and 2 units of Taq poly- tion of the dUMP residues in the PCR quired for LIC. PCR fragment purifica- merase. The same cycling program was products with uracil DNA glycosylase tion, T4 DNA polymerase treatment, and used for the interoAlu PCR as described (UDG) generates the required 12-nucle- LIC is complete in <1 hr. for the vector PCR. Products from PCR otide-long ss tails. reactions were usually analyzed by elec- Other ligation-dependent cloning trophoresis in 2-3% agarose gels procedures using T4 DNA polymerase to MATERIALS AND METHODS (NuSieve GTG) in TBE buffer with ethid- generate smaller ss tails have been devel- Enzymes, Reagents, and ium bromide. (24) One 50-1~1 PCR reaction oped. (laA4) In these cases, the vector is Oligonucleotides resulted in 2-3 I~g of linearized pUC19. not amplified but is cut by specific re- striction endonucleases to generate the and buffer were from T4 DNA Polymerase Treatment compatible ends for cloning. Because Perkin-Elmer Cetus; T4 DNA polymerase the ss tails are small, a ligation step is and buffer were from Boehringer The amplified vector and inter-Alu PCR necessary prior to transformation. An- Mannheim. Competent bacteria (Max products were purified using a glass pow- other LIC procedure makes use of the Efficiency DH5a), the 1-kb ladder, and der suspension (-CleanlI from specific addition of 1 nucleotide at the the 123-bp ladder were purchased from BIOI01, San Diego) as recommended by 3'end of the PCR product by Taq poly- BRL. Oligonucleotides were synthesized the supplier. For the generation of the ss merase. Vectors have been constructed and HPLC-purified by MWG (Ebersberg, tails, the purified DNA preparations were from various groups that contain the Germany). The DNA sequences of the treated with 2 units T4 DNA polymerase complementary nucleotide at the ends individual primers are as follows: in a 50-t~1 volume [50 mM Tris-HCl (pH from the linearized vector to facilitate PDJ8312, 5'-gatggtagtaggCCACTGCACT- 8.8), 15 mM ammonium sulfate, 7 mM the cloning. (ls-17) These so called T-vec- CCAGCC-3 '; PDJ8310, 5 '-tggtagtaggCCA- magnesium chloride, 0.1 mM EDTA, 10 tors however, are confined to the use of CTGCACTCCAGCC-3'; PDJ8308, 5'-gtag- mM mercaptoethanol, and 200 i~g/ml of Taq polymerase. Other thermostable taggCCACTGCACTCCAGCC-3'; PDJ8012, bovine serum albumin] in the presence DNA , like Pfu polymerase, 5'-cctactaccatcGGATCCCCGGGT-3'; PD- of dGTP (0.5 mM) for the Alu-PCR prod- which lack the terminal transferase ac- J8010, 5'-cctactaccaGGATCCCCGGGT- ucts or dCTP (0.5 raM) for the vector. Af- tivity, cannot be used. (is) 3'; PDJ8008, 5'-cctactacGGATCCCCGG- ter incubation for 20 min at 37~ the Based on our LIC procedure, Haun GT-3'; PDJ8112, 5'-cctactaccatcGTCGA- mixtures were heated for 10 min at 65~ and Moss have constructed a plasmid CCTGCAG-3'; PDJ8110, 5'-cctactaccaG- Prior to LIC, the Alu-PCR products and vector that allows the LIC of cDNAs in TCGACCTGCAG-3'; PDJ8108, 5'-cctac- pUC19 vector were either purified using any reading frame and directs their syn- tacGTCGACCTGCAG-3'. The primers the GeneClean procedure or were used thesis in E. coli as glutathione S-trans- with the 12-nucleotide tails are identical without further purification. The vector ferase (GST)-linked fusion . (19) to PDJ81, PDJ82, and PDJ83. (9) was diluted in TE buffer to 5 ng/l~l and They confirm that the procedure is very the Alu PCR products were diluted to 10- simple, rapid, and highly efficient. In 20 ng/t~l (in TE buffer). Amplification of Vector and Human contrast with our protocol they use a DNA plasmid vector that, upon digestion with Cloning and Transformation NarI, allows the generation of the 12-nu- All PCR reactions were performed in 50- cleotide ss tails by T4 DNA poly- i~l mixtures using the following buffer A 2-t~1 aliquot of T4 DNA polymerase- merase. (2~ The LIC procedure has been conditions: 50 mM KC1, 10 mM Tris-HCl treated pUC19 (10 ng) was combined used by others for cloning and expres- (pH 8.3), 1.5 mM MgC12, 0.2 mM of each with 4 i~l of T4 DNA polymerase-treated sion of various cDNAs. (z~'z2) Now, com- dNTP, and 0.5 p.M each oligonucleotide. Alu PCR products (60 ng) in a 20-1~1 vol- mercially available cloning kits adapted Amplification was for 35 cycles in a Per- ume (TE buffer). After a 1-hr incubation from LIC are available. More recently, kin-Elmer Cetus thermal cycler (9600) as at room temperature (5 rain works just as sophisticated expression vectors to be follows: initial heating to 94~ for 5 rain; well), 5 I~l was used to transform 50 i~l of used in mammalian or baculoviral sys- then 44 sec at 92.3~ 40 sec at 60.8~ competent cells (in 1.5-ml Eppendorf tems have been constructed by S. Gruen- and 46 sec at 71.5~ for 35 cycles; and a micro test tubes) with BRL-recom- wald and S. Singh (PharMingen, San Di- final extension at 72~ for 5 min prior to mended procedures. The temperature ego). cooling to 4~ The vector was amplified for the heat shock of the cells for DNA The requirement for an additional 12 starting with 1 ng of XbaI-linearized uptake is crucial to the experiment and nucleotides at the 5'end of the PCR pUC19 plasmid, (23) 25 pM of each PCR was monitored carefully (precisely primers may prevent people from con- primer, and 2 units of Taq polymerase. 42~ SOC (24) medium (0.45 ml) was sidering this technique as the method of Prior to PCR, pUC19 was cut with XbaI as added, and the cells were incubated for choice. To keep the cost3 for the primers follows: 100 ng of pUC19 was incubated one hour at 37~ in Eppendorf thermo- low and make this procedure widely ap- with 15 units of XbaI (overkill) at 37~ mixer 5436 at 1000 rpm. From the bac- plicable, here we test the hypothesis that for 30 rain in a 20-1~1 volume. HaO was terial suspension, 100 i~l and 400 I~l ss tails shorter than 12 bases long permit added to a final volume of 100 i~l, the (upon concentration by centrifugation)

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were plated on LB agar plates (z4) supple- sequencer using fluorescence-labeled 3' ~ 5' activity of the poly- mented with 100 g.g/ml of ampicillin. universe and reverse pUC19 primers. merase. Because of the primer design, the exonuclease stops at nucleotide 13, which is dCMP. PCR of Transformants and DNA RESULTS Clonable Alu-PCR fragments were Sequencing Principle of LIC generated using Alu primer PDJ8312 and human genomic DNA as a template. In The LIC of PCR products is outlined in Transformants were checked for the addition to its 17-nucleotide Alu repeat- presence of recombinant by Figure 1. It is based on the use of short specific sequence, (2s-27) this primer con- cohesive ends (8-12 nucleotides) at the PCR using the universal and the reverse tains at its 5' end the complementary 12 clonable PCR products as well as the vec- primers flanking the multicloning site in nucleotides from the pUC19 primers. tor. The pUC19 plasmid vector is ampli- pUC19. Bacterial colonies were trans- Consequently, ss tails can be generated fied using two primers complementary ferred into the PCR mixture just by using T4 DNA polymerase and dGTP. touching the colony using disposable pi- to the rnulticloning site. The vector pette tips and pipetting up and down in primers PDJ8012 and PDJ8112 contain 12 additional nucleotides at their 5' end the PCR mixture. The buffer conditions, Cloning of PCR Products primer concentrations, and cycling pro- lacking dGMP residues. As a conse- gram were as mentioned above. Aliquots quence, the 12 nucleotides at the 3' ends Since the publication of LIC (9) we have (5 p.1) were analyzed by agarose gel elec- of the amplified vector lack dCMP. In used this procedure to clone numerous trophoresis. The DNA sequencing of the the presence of T4 DNA polymerase and PCR fragments. It is noteworthy that in clones was done on the Pharmacia ALF dCTP, the 3' ends are degraded by the the course of the last 4 years, all of the

FIGURE 1 Generation of defined ss ends on PCR products. Inter-Alu repeat sequences are amplified using one of the three Alu primers (PDJ8312, PDJ8310, and PDJ8308). These primers are partly homologous to the consensus Alu sequence (27) and contain either 12, 10, or 8 additional nucleotides at their 5' ends that are required to generate ss tails in the PCR products. XbaI-cut pUC19 is amplified with the primer pairs PDJ8012/ PDJ8112, PDJ8010/PDJ8110, or PDJ8008/PDJ8108 that are partly homologous to the multicloning site and contain 12, 10, or 8 additional nucleotides at their 5' ends. The PCR products are digested with the (3' --> 5') exonuclease associated with T4 DNA polymerase in the presence of dGTP (inter-Alu fragments) or dCTP (pUC19) to generate 12-, 10-, or 8-nucleotide-long ss ends. The 5' overhanging ends from the Alu PCR products and vector are complementary (lower left corner) and allow recombinant molecules to be formed without using DNA ligase.

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randomly picked transformants from To correlate the cloning efficiency to the vector was purified, 245 or 152 trans- different experiments proved to contain the purification status of the vector and formants were obtained, respectively. inserts (several hundred analyzed). We the Alu PCR products, experiments with These differences probably result from have simplified the cloning procedure in the 12-nucleotide ss tails were per- minor variations in the vector concen- regard to the incubation buffer, incuba- formed. The Alu PCR products (PDJ8312) tration in the individual experiments tion times, and purification of the PCR and the linear vector PCR products and are not caused by purification ef- fragments. In this study we compare LIC (PDJ8012/PDJ8112) were treated with T4 fects. efficiencies using 12-, 10-, and 8-nucle- DNA polymerase to generate the com- To determine the cloning efficiencies otide-long ss tails. plementary overhangs. The polymerase of PCR products having 10- or 8-nucle- To test this cloning procedure, hu- was heat inactivated and the PCR frag- otide-long ss tails we have used Alu PCR man genomic DNA was amplified with ments were either further purified with products and vector DNA amplified with Alu primer PDJ8312 (12-nucleotide ss GeneClean or were used without purifi- the appropriate primers in LIC. The con- tail), PDJ8310 (lO-nucleotide ss tail), and cation in LIC. Appropriate dilutions of ditions for the cloning were as given PDJ8308 (8-nucleotide ss tail) in separate both components were done in TE above for the experiment with nonpuri- reactions. With each primer complex buffer. Vector (pUC19, 10 ng) and inter- fled vector and purified Alu PCR prod- PCR mixtures have been obtained (Fig. Alu fragments (60 ng) were mixed in a ucts. Whereas fragments with 12-nucle- 2). The size of the DNA fragments is in 20-1~1 volume (TE buffer) and were incu- otide-long overhangs resulted in 170 the range of 150--3000 bp. For the gen- bated for 5 min to allow the formation of transformants per 2.5-ng vector, only 43 eration of the vector DNA (pUC19), recombinant molecules. One-fourth of transformants were obtained when 10- XbaI-cut plasmid was amplified with the the mixture was used for bacterial trans- nucleotide overhangs were used (Table primer pairs PDJ8012/PDJ8112 (12-nu- formation without ligation. The trans- 1). No colonies were detected on plates cleotide ss tails), PDJ8010/PDJ8110 (10- formed cells were plated on LB ampicil- when 8-nucleotide-long ss tails were nucleotide ss tails), and PDJ8008/ lin plates. From 2.5 ng of pUC19, 227 used for cloning. For the uptake of DNA PDJ8108 (8-nucleotide ss tails), respec- transformants were obtained when the by the DH5~ cells, a heat shock for 45 sec tively. About 2-3 tzg of linear plasmid vector and the insert were further puri- at 42~ was required. This temperature per 50-~1 PCR reaction was obtained fied. When both LIC partners were not was monitored carefully because the starting with 1 ng of XbaI-cut pUC19 purified, 163 transformants were ob- transformation efficiency dropped dra- (Fig. 2). tained. In cases where either the insert or matically at slightly higher tempera- tures. Because the noncovalent-associ- ated DNA fragments with 8- and 10- nucleotide overhangs may dissociate "0 "U more easily at 42~ compared with 12- nucleotide ss tails, we lowered the tem- perature for the heat shock. Heating the (..,) .... .i,-. : .,I-- ,,~ A cells for 5 min at 37~ did not improve & .... 6 ...... ~ aS t-,- :~: m,,- m.- ..... the results. In contrast, only 25-50~ of the transformants were obtained (data not shown). However, the transforma- tion efficiency of the DH5u cells was not affected, as could be seen when covalent closed circles of pUC19 were used. To characterize the recombinants 3,0 8~1~ from different experiments, randomly

2i0 :~- 61~5~:~ picked transformants from several clon- ing experiments were analyzed for in- serts by PCR with primers flanking the 2~ :. cloning site of the vector (universal and reverse pUC19 primers). When LIC was performed using the 12-nucleotide over- hangs, 57 of 59 transformants contained inserts. As for the LIC with the 10-nucle- otide ss tails, 38 of 39 transformants con- tained inserts. Both cloning procedures pUC 19-~R .... ALU-~R seem to result in comparable frequencies of recombinants (>96%). Considering FIGURE 2 (A) pUC19 vector PCR. XbaI-cut pUC19 (1 ng) was amplified using the primer pairs the fact that screening for recombinants PDJS012/PDJ8112 (12-nucleotide tail), PDJ8010/PDJ8110 (10-nucleotide tail), and PDJSO08/ PDJ8108 (8-nucleotide tail). Five microliters of a 50-1zl PCR reaction was analyzed by gel elec- is not used in LIC, 96% recombinant trophoresis in 0.7% agarose. The 1-kb ladder (BRL) was used as standard (size in kb). (B) Alu PCR transformants reflects the high effi- from genomic DNA. PCR products were generated with Alu primer PDJ8312 (12-nucleotide tail), ciency of this technique. The fraction of PDJ8310 (lO-nucleotide tail), and PDJ8308 (8-nucleotide tail). Ten microliters of a 50-t~1 PCR the recombinants can be raised theoret- reaction was separated in 1.2% agarose. The 123-bp ladder (BRL) was used as standard (size in bp). ically to 100% if the T4 DNA polymerase-

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TABLE 1 Transformation Efficiencies Using 12-, 10-, and 8-nucleotide ss tails The LIC procedure as described in this article is completed in 1 hr. For the T4 LIC tail Vector Insert DNA polymerase treatment of the PCR Transformants (nucleotides) (2.5 ng) (15 ng) fragments and the PCR amplified vector 3a 12 - + 170 it is necessary to purify these products. 3b 10 - + 43 This can easily be done in 20 min with 3c 8 - + 0 commercially available kits based on a glass powder matrix. The subsequent The numbers of transformants using 2.5 ng of T4 DNA polymerase-treated pUC19 vector (not purified, - ) and 15 ng of T4 DNA polymerase-treated Alu PCR products (purified, +), resulting polymerase treatment (20 min) and in- from experiments with 12-, 10-, or 8-nucleotide-long overhangs, are given. activation of the enzyme (10 min) are followed by a 5-min incubation of vector and insert prior to transformation of the bacteria. The incubation of vector and treated vector is further purified from 42~ Lowering the temperature for the insert DNA is in the presence of TE agarose gels to eliminate the minute heat shock to 37~ did not improve the buffer. No heteroduplex stabilizing amount of pUC19 that was not cut by transformation efficiency. Similar obser- agents are required. For efficient cloning XbaI.(9) vations have been made also by Rasht- the insert has to be in molar excess over To demonstrate that the recombi- chian et al. (12) These investigators used the vector (~10). Because of the high ef- nants were generated properly by virtue the uracil DNA glycosylase to generate ss ficiency of this cloning procedure, as lit- of the predetermined 12- or 10-nucle- tails at the ends of vector and clonable tle as 10 ng of vector is used for routine otide ss tails, DNA from five transfor- PCR fragments. They state that products subcloning of PCR products. It should be mants each was isolated and sequenced with 12-nucleotide ss tails cloned 5-10 kept in mind that one 50-1xl PCR reac- with pUC19-specific, fluorescence-la- times more efficiently than those with tion results in 2-3 Ixg of amplified vec- beled primers. All clones contained the 9-nucleotide ss tails, and no transfor- tor. properly ligated LIC sequences at both mants were obtained with 6-nucleotide Other investigators have designed ends of the inserts (data not shown). cohesive ends. In contrast to our proto- plasmid vectors that upon digestion with col, they used a repeated sequence for a specific restriction endonuclease, allow the annealing region [(CUA)4 ]. This re- the specific degradation of the 3' ends DISCUSSION sulted in some heterogeneity among the using T4 DNA polymerase to generate The ability of T4 DNA polymerase to de- recombinants produced because the an- 12-nucleotide-long ss tails. Haun et grade the 3' ends of PCR fragments in a nealing of ends could take place involv- al. (2~ have engineered a pBluescript vec- controlled fashion so that 5' ss tails of ing some or all of the CUA repeats. These tor that facilitates the generation of 12- predetermined DNA sequence and workers also have emphasized the high nucleotide cohesive ends upon digestion length are generated has been utilized to cloning efficiency when 12-nucleotide ss with NarI. The nonrecombinant fraction develop LIC techniques. In our original tails are used (1• s to 9x10 s transfor- of transformants is <15% in their exper- LIC protocol, we generated 12-nucle- mants/ixg vector). iments. These workers state that al- otide ss tails in the PCR fragment and in In a similar cloning procedure (in though the linearized vector was gel pu- the PCR-amplified plasmid vector. (~ The vivo cloning, IVC) Oliner et al. (28) clearly rified, the nonrecombinants resulted 12-nucleotide 5' extending vector ends demonstrated that the efficiency of LIC from plasmid DNA that had not been di- created by our procedure permit the for- is dependent on (1) the procedure used gested with NarI. The nonrecombinant mation of stable duplexes with cohesive to purify the inserts, and (2) the length fraction in our LIC is very low because tails from the PCR products, thus elimi- of the cohesive ends generated. Using the vector is amplified from small nating the need for ligation. The cloning electroporation, they obtained 100,000 amounts of XbaI-cut pUC19 (1 ng). In procedure is simple because only a single clones per microgram of vector (~90% theory, < 1 pg ofXbaI-cut plasmid is con- type of enzymatic reaction is required recombinants) when both ends were 22 tained in 10 ng of amplified vector. prior to transformation and results in a and 27 nucleotides long. With cohesive However, if functional cloning vec- high cloning efficiency (up to 5 • 10 s re- ends up to 67 nucleotides the transfor- tors are required for the cloning of combinants/txg of vector). Nonrecombi- mation efficiencies were raised to 4 • 10 s cDNAs as fusion proteins, it is advisable nant clones are practically eliminated. to 6 x 10 s. Knowing that electroporation to use nonamplified plasmid vectors to The 12-nucleotide ss tails proved to is much more efficient than the transfor- overcome possible mutations in relevant be the minimal length for efficient LIC. mation protocol used in our study, the DNA sequences introduced by Taq poly- When we used 10-nucleotide ss tails at transformation efficiency achieved by merase. Haun and Moss (10) have con- the vector and insert DNA fragments, the our procedure is very competetive. It structed plasmid vectors that facilitate transformation efficiency dropped to may be that the high efficiency observed LIC of GST fusion for expression -25%. With 8-nucleotide ss tails no with the LIC 12 primers reflects a pecu- in E. coli. Kuijper et al. (20) have designed transformants were obtained. This de- liarity of the specific DNA sequence or cloning vectors ("Prime" cloning vec- crease in transformation efficiency was the protocol used in our study. There- tors) that include phage )~ and plasmid not caused solely by the reduced stability fore, we did not consider it necessary to vectors useful for functional cloning in of 10-nucleotide versus 12-nucleotide perform experiments with longer LIC oocytes, , and mammalian cells, heteroduplexes during the heat shock of tails. Longer tails would render the prim- and their use in a "Prime" cloning sys- the transformation process (45 sec at ers too long and too expensive. tem. They also make use of the (3' --> 5')

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exonuclease activity of T4 DNA poly- 6. Nelson, D.L., S.A. Ledbetter, L. Corbo, independent cloning of glutathione merase to generate 15- and 16-nucle- M.F. Victoria, R. Ramirez-Solis, T.D. Web- S-transferase fusion genes for expression otide-long ss tails for cloning. Based on ster, D.H. Ledbetter, and C.T. Caskey. in Escherichia coil Gene 112: 37-43. our LIC (9) and the procedure by Haun et 1989. Alu polymerase chain reaction: A 20. Haun, R.S., I.M. Servent, and J. Moss. al. ~19'2~ a panel of LIC vectors for vari- method for rapid isolation of human-spe- 1992. Rapid, reliable ligation-indepen- cific sequences from complex DNA dent cloning of PCR products using mod- ous applications, including cloning and sources. Proc. NatL Acad. Sci. 86: 6686- ified plasmid vectors. BioTechniques mammalian and baculoviral expression, 6690. 13: 515-518. has been engineered by S. Gruenwald 7. Clark, J.M. 1988. Novel non-templated 21. Hamel, C.P., E. Tsilou, B.A. Pfeffer, J.J. and S. Singh (PharMingen, San Diego, nucleotide addition reactions catalyzed Hooks, B. Detrick, and T.M. Redmont. CA). by procaryotic and eucaryotic DNA poly- 1993. Molecular cloning and expression The results described in this report merases. Nucleic Acids Res. 16: 9677-9686. of RPE65, a novel retinal pigment epithe- demonstrate the ease and applicability 8. Hemsley, A., N. Arnheim, M.D. Toney, G. lium-specific microsomal that is of the LIC technique to a variety of PCR Cortopassi, and D.J. Galas. 1989. A simple post-transcriptionally regulated in vitro. J. products. From several independent ex- method for site-directed mutagenesis us- Biol. Chem. 268: 15751-15757. ing the polymerase chain reaction. Nu- periments a 100% recombinant fraction 22. Serventi, I.M., E. Cavanaugh, J. Moss, cleic Acids Res. 17: 6545-6551. and M. Vaughan. 1993. Characterization of clones was achieved (>400 indepen- 9. Aslanidis, C. and P.J. de Jong. 1990. Liga- of the gene for ADP-ribosylation factor dently picked transformants) when the tion-independent cloning of PCR prod- (ARF) 2, a developmentally regulated, se- amplified vector was gel purified. ~9~ ucts (LIC-PCR). Nucleic Acids Res. 18: lectively expressed member of the ARF Here, we have shown that if the vector is 6069-6074. family of -20-kDa guanine nucleotide- not gel purified the recombination frac- 10. Shuldiner, A.R., L.A. Scott, and J. Roth. binding proteins. J. Biol. Chem. 268: tion of the clones is still very high 1990. PCR-induced (ligase-free) subclon- 4863-4872. (>96%). The manipulations of the vec- ing: A rapid reliable method to subclone 23. Viera, J. and J. Messing. 1987. Production tor and the insert and the incubation polymerase chain reaction (PCR) prod- of single-stranded plasmid DNA. Methods times prior to cloning have been mini- ucts. Nucleic Acids Res. 18: 1920. Enzymol. 153: 3-11. 11. Shuldiner, A.R., K. Tanner, L.A. Scott, C.A. mized. These properties make LIC a gen- 24. Sambrook, J., E.F. Fritsch, and T. Maniatis. Moore, and J. Roth. 1991. Ligase-free sub- 1989. Molecular cloning: A laboratory man- eral and versatile method for cloning of cloning: A versatile method to subclone ual. Cold Spring Harbor Laboratory, Cold PCR products. polymerase chain reaction (PCR) prod- Spring Harbor, New York. ucts in a single day. AnaL Biochem. 25. Jelinek, W.R. and C.W. Schmid. 1982. Re- 194: 9-15. petitive sequences in eucaryotic DNA and ACKNOWLEDGMENTS 12. Rashtchian, A., G.W. Buchman, D.M. their expression. Annu. Rev. Biochem. The excellent technical assistance of U1- Schuster, and M.S. Berninger. 1992. Uracil 51: 813-844. rike St6ckl is acknowledged. DNA glycosylase-mediated cloning of 26. Jurka, J. and T. Smith. 1988. A fundamen- polymerase chain reaction-amplified tal division in the Alu family of repeated DNA: Application to genomic and cDNA sequences. Proc. Natl. Acad. Sci. 85: 4775- cloning. Anal. Biochem. 206: 91-97. 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PCR Methods and Applications 177 Downloaded from genome.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press

Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products.

C Aslanidis, P J de Jong and G Schmitz

Genome Res. 1994 4: 172-177

References This article cites 28 articles, 5 of which can be accessed free at: http://genome.cshlp.org/content/4/3/172.full.html#ref-list-1

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