Nucleic Acids Research, Vol. 18, No. 4
1990 Oxford University Press 1071
An improved method for the construction of high efficiency cDNA library in plasmid or lambda vector Kripamoy Aguan, Tomonobu Kusano, Nobuhiro Suzuki and Yoshichika Kitagawa Laboratory of Plant Genetic Engineering, Biotechnology Institute, Akita Prefectural College of Agriculture, Akita, Ohgata 010-04, Japan Submitted January 24, 1990
Although there are several methods available for cDNA library construction in plasmid or phage vector, all suffer from a drawback that a higher proportion of non-recombinant clones appear in the ligation step even in the case of lambda gtlO which reduces these types of clones by a way of genetic selection. In addition, the cloning efficiency of such library decreases since self-ligation of individual cDNA-adaptor complex species results in long cDNA molecule which are beyond the cloning capacity of the vectors. A method has been developed using special adaptor which can generate 106 p.f.u. or clones per 100 ng of poly(A+) mRNA with reduced background. The method requires fewer steps in cloning, namely; complementary oligodeoxynucleotides of the adaptor are kinased together prior to its ligation to blunt ended cDNA, obviating the need for two steps of kination in most protocol. The cloned insert could easily be isolated either by EcoRI or by NotI enzyme digestion. The ds cDNA was prepared as described previously (1). The two oligodeoxynucleotides (P1 and P2) complementary except for the 5' overhang of the sticky ends were synthesized, mixed in equi-molar amount (0.1 tg/jl) and phosphorylated by T4 polynucleotide kinase. The kinased adaptor was ligated to blunt ended cDNA in a 100-fold molar excess ratio. The reaction conditions were as mentioned previously (2) except for the reaction temperature which was 12°C. The unincorporated mono and dimer form of the adaptors were removed by gel electrophoresis. The vectors (plasmid/lambda) were digested with EcoRI lambda/plasmid vectors +AATTC-----------------G G------------------CTTAA dATP,
-----------GAA -----------CTTAA
Klenow
+AATTC--------
enzyme and the recessed 3' ends were filled with dATP in the presence of Klenow fragment of DNA polymerase I. The free dATPs were removed by spin column chromatography. This fill-
in step inhibits the formation of parental non-recombinant plasmid phage and thus obviates the need for dephosphorylation (CIP/BAP treatment) of the vector arms (3). Ligation was carried out in a volume of 10 1ul containing 0.5 jig of vector and 0.3 ,ug of cDNA-adaptor complex at 4°C for 12-16 hrs. The ligated product was packaged in vitro using Gigapack Gold (Stratagene) and plated on E. coli strain C600hfl-. In the case of lambda gtlO, 83 plaques were found to contain inserts out of 100 random plaques. In the case of plasmid vector, transformation of ligated product resulted in 5 % blue colonies, and 90% white colonies (45 out of 50) contained inserts. The presence of Notl site in the adaptor allowed cloned cDNAs to be recovered intact and hence would facilitate sequencing steps. Using this method we have successfully constructed 5 cDNA libraries (3 on plasmid and 2 on lambda gtlO). The cloning or
efficiency was 106-107 independent p.f.u. (A +) RNA.
or
colonies/4g poly
REFERENCES 1. Gubler,U. (1988) Nucl. Acids Res. 16, 2726. 2. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 3. van Doorn,L.-J., Belkum,A. and Kos,T. (1989) Nucl. Acids Res. 17, 9496.
adaptors TTCGCGGCCGC: Pl GCGCCGGCG: P2
I ligntion to cDNA TTCGCGGCCGC**********GCGGCCGCG
fragment
GCGCCGGCG**********CGCCGGCGCTT ligation -----------GGAATTCGCGGCCGC***A** ******GCGGCCGCGMTTC----------AAG--------
CTTAAGCGCCGGCG************CGCCGGCGCTTAAG------------
1990 Oxford University Press 1071
An improved method for the construction of high efficiency cDNA library in plasmid or lambda vector Kripamoy Aguan, Tomonobu Kusano, Nobuhiro Suzuki and Yoshichika Kitagawa Laboratory of Plant Genetic Engineering, Biotechnology Institute, Akita Prefectural College of Agriculture, Akita, Ohgata 010-04, Japan Submitted January 24, 1990
Although there are several methods available for cDNA library construction in plasmid or phage vector, all suffer from a drawback that a higher proportion of non-recombinant clones appear in the ligation step even in the case of lambda gtlO which reduces these types of clones by a way of genetic selection. In addition, the cloning efficiency of such library decreases since self-ligation of individual cDNA-adaptor complex species results in long cDNA molecule which are beyond the cloning capacity of the vectors. A method has been developed using special adaptor which can generate 106 p.f.u. or clones per 100 ng of poly(A+) mRNA with reduced background. The method requires fewer steps in cloning, namely; complementary oligodeoxynucleotides of the adaptor are kinased together prior to its ligation to blunt ended cDNA, obviating the need for two steps of kination in most protocol. The cloned insert could easily be isolated either by EcoRI or by NotI enzyme digestion. The ds cDNA was prepared as described previously (1). The two oligodeoxynucleotides (P1 and P2) complementary except for the 5' overhang of the sticky ends were synthesized, mixed in equi-molar amount (0.1 tg/jl) and phosphorylated by T4 polynucleotide kinase. The kinased adaptor was ligated to blunt ended cDNA in a 100-fold molar excess ratio. The reaction conditions were as mentioned previously (2) except for the reaction temperature which was 12°C. The unincorporated mono and dimer form of the adaptors were removed by gel electrophoresis. The vectors (plasmid/lambda) were digested with EcoRI lambda/plasmid vectors +AATTC-----------------G G------------------CTTAA dATP,
-----------GAA -----------CTTAA
Klenow
+AATTC--------
enzyme and the recessed 3' ends were filled with dATP in the presence of Klenow fragment of DNA polymerase I. The free dATPs were removed by spin column chromatography. This fill-
in step inhibits the formation of parental non-recombinant plasmid phage and thus obviates the need for dephosphorylation (CIP/BAP treatment) of the vector arms (3). Ligation was carried out in a volume of 10 1ul containing 0.5 jig of vector and 0.3 ,ug of cDNA-adaptor complex at 4°C for 12-16 hrs. The ligated product was packaged in vitro using Gigapack Gold (Stratagene) and plated on E. coli strain C600hfl-. In the case of lambda gtlO, 83 plaques were found to contain inserts out of 100 random plaques. In the case of plasmid vector, transformation of ligated product resulted in 5 % blue colonies, and 90% white colonies (45 out of 50) contained inserts. The presence of Notl site in the adaptor allowed cloned cDNAs to be recovered intact and hence would facilitate sequencing steps. Using this method we have successfully constructed 5 cDNA libraries (3 on plasmid and 2 on lambda gtlO). The cloning or
efficiency was 106-107 independent p.f.u. (A +) RNA.
or
colonies/4g poly
REFERENCES 1. Gubler,U. (1988) Nucl. Acids Res. 16, 2726. 2. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York. 3. van Doorn,L.-J., Belkum,A. and Kos,T. (1989) Nucl. Acids Res. 17, 9496.
adaptors TTCGCGGCCGC: Pl GCGCCGGCG: P2
I ligntion to cDNA TTCGCGGCCGC**********GCGGCCGCG
fragment
GCGCCGGCG**********CGCCGGCGCTT ligation -----------GGAATTCGCGGCCGC***A** ******GCGGCCGCGMTTC----------AAG--------
CTTAAGCGCCGGCG************CGCCGGCGCTTAAG------------
