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Communication Mol. Celis, Vol. 4, pp. 377-380 A Highly Efficient Method for the Construction of a Plasmid-Based cDNA Library C hae Oh lim l, M oo J e C hou and lnhwan Hwang 1* IPlant Molecular Biology and Biotechnology Research Center and 2Department of Biochem istry, Gyeongsang National Univel:~ity. Ch inju 660- 70/ , Korea (Received on June 16, 1994) We have developed a highly efficient method for the construction of a plasmid-based cDNA library. The new method was based on the addition of complementary single stranded oligomers to cDNA and vector as 5' overhangs and annealing of the 5' overhangs before ligation to increase ligation efficiency. To generate the long, complementary 5' overhangs to cDNA and vector DNA a common un phosphorylated adaptor composed of 8 bases (Ad-B) and 12 bases (Ad-T) of two complementary oligomers was ligated to the cDNA and vector. Only one of the two adaptor stands was ligated to either cDNA or vector. The annealing of the 12 bases of 5' overhangs prior to ligation greatly simplified the ligation step and increased the efficiency of ligation reaction. The ligated DNA was then transformed into E. coli by the electroporation method. By this method we have constructed two plasmid-basmid cDNA libraries of approximately 5 X 106 and 5 X 107 cfu from 100 ng cDNA made from the flower bud and leaf tissues of chinese cabbage (Brassica campestris L. ssp. pekinensis) using pBluescript KS( + ) as a cloning vector, respectively. To isolate genes in molecular biology, one of most important steps is the construction of a good cDNA library. M any methods have been developed (Burrell, 1984; Gubler a nd Hoffma n, 1983; H eideker and Messing, 1983; O kayama and Berg, 1982); however, it is still a challenge to construct a good cDNA library. In most cases, la mbda DNA has been used as a cloning vector (Frisch auf et af. , 1983; G ubler and H offman, 1983; Young and Davis, 1983), and it is often sufficient to screen a lam bda cDNA library to isolate a cDNA clone, Recently there has, however, been an increasing demand for a plasmid-based cDNA library fo r random cDNA sequenci ng projects (Adams et al., 1991; Adams et al., 1992; Park et aI., 1993), funcitona1 screenings of genes (Fields and Song, 1989; Zevros et aI. , 1993), and so on. Especially, to do functional screening of a pa rticula r gene in yeast it is necesary to construct a cDNA library using a yeast expression plasmid a~ a cloning vector. The same is true in the construction of a double stranded cDNA in the case of the lambda and plasmid libraries. One major difference is the ligation step where a double . cDNA is inserted into the vector. In the case of the lambda cDNA lib rary, vector a rms and inserts are concatemerized and then packaged into lambda phages in vitro (Hohn and Murray, 1977; Hyunh et al., 1985). Therefore it is rather simple to ligate the inserts to vector arms. H owever, in the case of the p lasmid library, the inserts have to be ligated to the vector one by one to give a circular molecule. It is often difficult to get a right molar ratio of the vector to insert for the ligation. We have developed a highly efficient method for the construction of a plasmid-based cDNA lib rary. Th is method is based on the addition of single stranded complementary oligomers to vector and cDNA to make a long 5' overhang, and annealing of the complementary long 5' overhangs of the vector and cDNA before ligation. The ligated molecules are then transformed into E. coli by electroporation. Materials and Methods Preparation of double stranded cDNA Total RNA was prepared from the flower bud and leaf tissues of B. campestris L. ssp. pekinenesis (from Seoul Seed Co., Korea) according to the protocol described previously (Ausubel et at., 1992; Shirley et al., 1993). Poly(At RNA was isolated from the total RNA using an mRNA purification kit (Ph armacia, USA) according to the manufacture's protocol (Aviv and Leder, 1972). Double stranded cDNA was prepared using a commercially available cDNA construction ki t (Stratagene, USA) according to the manufacture's protocol. The double stranded cDNA was polished using three units of T4 DNA polymerase (NE B, USA) and 4 ).ll of 2.5 mM dNTPs in a 50 ).ll reaction volume at 37 °C for 30 min (1 X reaction buffe r: 50 mM Tris ' H Cl, pH 7.9, 10 mM MgCb l.0 mM OTT). A ddition of long 5' overhangs to double stranded cDNA and vector An adaptor is composed of two oligomers, Ad-T © 1994 The Korean Society for Molecular Biology 378 A Highly Efficient Method for cDNA Construction (5' AATTCGGATCCC 3': 12 bases) a nd Ad-B (5' GGGATCCG 3': 8 bases), which were obtained from th e Korea n Biotech Co. a nd used to generate long 5' overhangs of cDNA and vector DNA. The two oligomers were a nnealed at a I : I molar ratio by slowly cooling down to room temperature for a period of 30 min afte r heating at 65 t for 5 min. 10 Ilg of the a nnea led adaptor was ligated to 5 Ilg of Eco Rldigested vector [p Bluescript KS( + )J in a 30 III reaction volume with 2 III of T4 DNA ligase (approximately 7 units/ Ill, NEB, USA) (1 X . reaction buffer: 50 mM Tris 'HCI pH 7.8, 10 mM MgCh, 10 mM DTT, 1 mM ATP) at 15 t overnight. For cDNA, 5 Ilg of the annealed adaptor was ligated to the double stranded cDNA with 1.5 III of ligase (7 unitsl!ll) in a 20 III reaction volume. Size fractionation of double stranded cDNA and preparation of vector DNA The adaptor-ligated double stranded cDNA was fractionated on a 1.0% low melting point agarose gel (a mini gel) in 1 X TAE at 80 V for 50 min, a nd the agarose block containing the desired size (500 bp to 4 kb) of cDNA based on a size marker was cut out fro m the gel. The agarose block containing the cDNA was returned to the place at the reverse OIientation a nd sealed with molten low melting point aga rose. After the sealing was solidified th e gel was placed back in the electrophoresis box and run at exactly the same co ndition (80 V for 50 min) to concentrate the spread cDNA. After the second electrophoresis the co nce ntrated DNA band was excised under a lo ng wavelength UV. Th e cDNA was extracted from the gel by the phenol extraction method (Maniatis et al. , 1982). The aqueous ph ase was reduced to approximately 50 III by the seco ndary butanol concentration method (Ma niatis et al., 1982). The DNA was then extracted sequentially with equal volumes of phenol, phenol/CH Ch, a nd CHCh followed by eth anol precipitation with three volumes of eth a nol at - 20 t overnight. After addition of th e adaptor to the vector, the vector DNA was also gel-purified from agarose gel by the electroelution method (Ma niatis et at., 1982). The vector was phosphorylated with 10 units of T4 polynucleotide kinase (NEB, USA) in a 20 III reaction volume containing I mM ATP at 37 t for 30 min and subsequently digested with 50 units of XhoI in a 50 III reaction volume. The vector DNA was gel purified again. Ligation of the cDNA to vector and electroporation 200 ng of vector a nd 100 ng of cDNA were mixed in a 15 III volume co ntaining 2 III of lO X T4 DNA ligase buffe r (-ATP and -DTT) and then heated at 65 t for 5 min followed by a slow c.ooling down to room temperature over a period of 30 min. 2 III of 10 mM ATP, 2 III of 0.1 M DTT, 1.0 III of T4 DNA ligase (7.0 units/ Ill) were added to the annealed cDNA a nd vector mixture. The mix was then incuba- Mol. Cells ted at 15 °C overnight. After phenol/CHCJ 3 a nd CHC13 extraction the ligated DNA was eth a nol precipitated at - 20 °C. A fraction of the ligated DNA was transformed into E. coli cells (MC I06J) by electroporation (Dower et at., 1988) to estimate a titer. The rest of th e ligated DNA was then transfo rmed into MC 106 l host cells by electroporation a nd plated on 24 X 24 cm bioassay plates. Results and Discussion The strategy for the construction of the cDNA library using a plasmid as vecto r is shown in Figure 1. One major difference of this method from other cDNA library construction methods is the addition of long complementalY single strand 5' overhangs, AdT and Ad-B of cDNA a nd vector, respectively. which can be used to anneal the cDNA and vector molecules before ligation reaction. The lo ng 5' overhangs were generated by ligating an unphosphorylated adaptor to cDNA and vector DNA. Since the annealed adaptor oligomers did not have 5' phosphate groups a t both ends, only one strand of the adaptor, eith er Ad-T or Ad-B, was ligated to eith er cDNA or vector DNA, respectively. Therefore the 5' overhangs added to the cDNA and vector DNA were complementary to each other. The co mplementary 5' overhangs were then a nn ea led befo re ligation reaction. Since the cDNA a nd vector DNA were annealed to a one to one mola r ratio there was no need to test for an optimal condition for the ligation step. The ligation reaction of the a nnea led moleculer was then almost the same as a self-ligatio n of linea r DNA molecules. Previously there were ma ny different methods to utili ze complementary homopolymelic tails for annealing of cDNA a nd vector DNA (Heideker and Messing, 1983; Okayama and Berg, 1982). The homopolymelic tail was added using ternlinal deoxynucleotidyl tra nsferase with a specific deoxynucleotide triphosph ate. H owever, it is difficult to co ntrol th e size of the homopolymeric tails. To make li bra ries for exp ression screening or for random sequencing projects it is desirable to have a short and defin ed 5' end of cDNA. To solve this problem we used sho rt and defined oligomers to generate co mpl ementary si ngle strand overh a ngs to cDNA and vector DNA. Another difference of the new method is the size fractio natio n step. This method utilized low melting point aga rose gel electrophoresis instead of Sepharose 4B column chromatography to isolate desired sizes of cDNA. The gel electrophoresis method ca n give much better size fractionation compared to the column chromatograp hy. However one problem is the large agarose gel volume containing the desired cDNA fractions since the cDNA is sp read to a large area due to heteroge neity of the cDNA population. To solve this problem we devised a simple co ncentration method of the spread cDNA in the gel. After gel electrophoresis, the desired area was cut out from the gel and returned to the sa me gel at the reve- Vol. 4 (1 994) 379 C hae O h Lim el al. A. eDNA preplntiOll M 1 2 3 4 5 6 7 · 8 9 10 11 12 13 14 15 16 (Kb) X)'o I (,:)18=== AmeaJed odapor p===ds<=DN=A=== A Ug.tion wi'" T4 DNA tipoe )(hoI I I + B. Vector preparation I t 1. U gatiojn with T4 DN A li gase 2. ElcclIoclution on agarose gel k " j c. 1. PhosphoryraLi on with T4 kinase 2. Xho I digesti on 3. Elcct roc lution I, Anneali ng and ligalio n Vector eDNA ~ "" = 2.0 0.56 1.)(holcl~OII p Xhol 23 2. Size i'naicnoIioa OIl _1'1 )(hoI hoI 23.1 9.4 6.6 4.4 Annealing I, ~ Lis" ion "ilh T4 DNA ligm o Figure I. A schemati c prese ntati o n of th e constructio n of a pl as mid-based cDNA library. A) eDNA preparatio n. An a nnealed ada pto r was ligated to do uble stra nded eDN A a nd the liga ted eDNA was d igested with Xho i. The resul ti ng e DNA was size fractio nated o n a low melting poi nt agarose gel as desc ribed in the Materi als a nd Methods secti o n. (AfT) 18 ind icates eightee n base pairs of A!f used for the primi ng o f eDNA. 8) Vecto r DNA preparatio n. T he vector DNA was ligated with a nnealed ada pto r afte r Eco RJ d igestio n. T he liga ted vecto r DNA was gel-purified after electropho resis. The vecto r DNA was th en phospho ly lated with T4 kinase followed by digesti o n with Xhoi. After digestio n, vector DNA was gel purified aga in. C) Liga tio n o f eD NA a nd vecto r. eDNA a nd th e vector we re a nnealed at a I: I mola r ratio a nd th en ligated with T4 DNA Ligase. Figure 2. eDNA clones isolated from the libraly. Sixteen cDNA clones were randomly selected and plasmid DNA was prepared by the alka li ne/SDS lysis method. cDNAs were digested with BamHI and X/lUI and ekctrophoresed on a 1.0% agarose gel. M de notes size marker. lambda DNA digested with Hilld ll l. The size of the DNA fragments is indicated on the left. rse orien tation. The cut was sealed with molten low melting poin t agarose gel. After the sealing was solidified , the gel was ru n at the exactly same condition. Afte r the second gel electrophoresis, the band containing concentrated cDNA was excised from the gel and cD NA was purified from the low melting agarose as described previously (Maniatis ef af., 1982). By this method we co ncen trated cDNA and minimized the loss o f th e cDNA dU li ng gel plllification. To test this method we have constructed cDNA libra ri es fro m the flower bud and leaf tissues of chinese cabbage using pB luescripts KS( + ) as a cloning vector. Approx im ately o ne /lg of adaptor ligated double-stranded cDNA was prepa red from 5 /lg of poly(A) + RNA. Also vector DNA was prepared as described in the Ma teria ls a nd Methods section. One to one molar ratio of cDNA (assuming that the average size of th e cDNA is 1.5 kb) and vector DNA (100 ng of cDNA and 200 ng of vector) were ligated as described above a nd then transformed into E. coli Me 1061 host cells by electroporation (Dower el af., 1988). Approximately 5 X 106 and 5 X 10 7 cfu were obtained from 100 ng of the leaf and flower bud cDNAs, respectively. In order to examine the size of the cDNA insert 16 clones were randomly selected from plate containing flower bud cDNA library, and plasmid DNA was prepared by the alkaline lysis method (Birnboim, 1988). T he plasmid DNA was analyzed on a gel after restriction d igest with BamHI and XhoI. As shown in Figure 2, more than 90% of the clones have inserts ranging from approximately 500 bases to 2.0 kb in size. As demonstrated by the construction of the two cDNA li bralies using pBluscript KS( + ) as a vector, th is meth od was highly efficient. There was no need to test for the ligation condition between the cDNA and vector DNA. Simple calculation of the molar ratio of th e vector and cDNA (based on the average size) was eno ugh to give a good ligation as shown by the titer of cDNA libraries. Based on the titer of the two cDN A libraries, more than 5 X 107 cfu can be easily obtai ned from I /lg of cDNA which was prepared a 380 A Highly Efficient Method for cDNA Construction from th e 5 Ilg of poly(At RNA This efficiency is a t least five to ten times higher than other commerciall y ava ilable cDNA construction kits based on the lambda vector (The manufacture of the commercial cDNA construction kit claims to give 2 X 106 pfu/5 Ilg poly(At RNA, Stratagene, USA). This easy method for the construction of the plasmid-based cDNA libraIY will contlibute greatly to studies such as the functional screening of genes in yeast and random cDNA seq uencing projects where the lambda cDNA library can not be used. Acknowledgments The authors wish to thank Dr. Do Hyun Lee for supplying Brassica flower bud and seeds. This work was supported by a grant from the Plant Molecular Biology and Biotechnology Research Center of the Korean Science and Engineeling Foundation. References Adams, M. 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