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Nucleic Acids Research Pages 3451-3452  

Solid-phase cDNA library construction,a versatile approach
Acknowledgements
References

Solid-phase cDNA library construction,a versatile approach

Solid-phase cDNA library construction,a versatile approach

Thomas Roeder*

University of Hamburg, Zoological Institute, Department of Neurophysiology, Martin-Luther-King-Platz 3,D-20146 Hamburg, Germany

Received April 3, 1998; Revised and Accepted June 2, 1998

ABSTRACT

A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for most purposes.

The construction of cDNA libraries is a basic step in most molecular biological techniques. Several procedures for the construction of cDNA libraries are available which meet the needs of different applications (1). The synthesis of cDNA libraries is a chain of enzymatic reactions, each requiring specific buffers, substances and enzymes. All these protocols share common steps (2,3) such as first strand synthesis by a reverse transcriptase followed by second-strand synthesis, blunting, adaptor ligation, kinasing and ligation into the vector of choice.

A major problem of the multi-step cDNA-synthesis is to ensure optimal conditions during each step. To overcome problems encountered with DNA purification, most enzymatic steps are performed without complete buffer exchange. This approach, although straightforward and quick, is accompanied by problems such as suboptimal buffer composition and the persistence of potentially interfering compounds throughout the whole cDNA synthesis process. To take advantage of the benefits of the classical cDNA synthesis approach and eliminate its drawbacks, I modified the protocol towards optimal conditions in each step of the cDNA construction process. The only difference in my approach is that the cDNA is synthesised on a solid support, which enables complete buffer exchange without loss of material in less than a minute. This procedure allows library construction from the RNA isolation to transformation of competent bacteria in a single day. Solid-phase cDNA synthesis is a well known technique, introduced and almost exclusively used to obtain reusable pools of first-strand synthesis. This restriction to very few applications might be due to the use of oligodT-coupled beads, which irreversibly fixes the first-strand cDNA to the beads. This excludes approaches where the cDNA has to be removed, such as the synthesis of conventional, double-stranded cDNA libraries (4).

To demonstrate the usefulness of this approach, I prepared cDNA-libraries from different parts of the locust brain (Schistocerca gregaria). After RNA isolation (Trizol, Life Technologies, Eggenstein, Germany), the mRNA was isolated using a modified 5[prime]-biotinylated oligodT(25)-primer with an internal recognition sequence for NotI [5[prime]-biotin-GAGAGAGAGAGAGCGGCCGCT(25)G/A/C-3[prime]] bound to streptavidin coated magnetic beads (Dynal, Hamburg, Germany) (see Fig. 1). The mRNA (starting with 50-100 µg total RNA which includes ~1-2 µg mRNA) is bound to the solid phase via the oligodT-primer. If the cDNA-synthesis should be primed with this oligonucleotide, the mRNA is not eluted with low salt buffer. Directly following the wash steps in the mRNA purification protocol, first-strand cDNA synthesis succeeded for 1 h at 48°C (42 mM Tris-HCl pH 8.3; 48°C, 8 mM MgCl2, 8 mM DTT, 1 mM methyl-dNTPs, 50 U RNase inhibitor and 200 U Superscript+; LifeTechnologies, Eggenstein, Germany). If the cDNA is primed with a modified random-oligonucleotide, an alternative strategy is required. The mRNA is eluted from the beads and the random-oligonucleotide is added at a molar ratio of 2:1. The random-primed cDNA (5[prime]-biotin-GAGAGAGAGAGAGCGGCCGCNNNNNNNN-3[prime]) requires an alternative temperature regime to ensure effective cDNA synthesis. Before addition of the enzyme, the entire mixture is heated to 68°C (10 min) and immediately chilled on ice. After addition of RNase inhibitor and reverse transcriptase, incubation proceeded for 10 min at 20°C, 10 min at 37°C and 45 min at 48°C. The random-primed first-strand cDNA has to be immobilised to streptavidin-Dynabeads. Therefore, beads (20 pmol) are added in 100 µl binding buffer (10 mM Tris-HCl pH 8, 1 M NaCl) and the medium is incubated under constant shaking for 30 min at 43°C. Alternatively, the Dynabeads kilobaseBinder system (Dynal, Hamburg, Germany) could be used for immobilisation. From now on, both alternative approaches are treated equally. All following experimental steps are preceded by aspiration of the supernatant followed by two washes with the buffer required for the corresponding enzymatic reactions. The reactions are performed under constant shaking. Second-strand synthesis (5) proceeded for 2 h at 16°C with gentle shaking (50 mM Tris-HCl pH 7.6, 100 mM KCl, 5 mM MgCl2, 50 µg/ml BSA, 5 mM DTT 8 U/ml RNase H and 230 U/ml Escherichia coli DNA polymerase). After completion of the reaction, the cDNA is blunted (5 min, 37°C with 5 U T4-DNA-polymerase, 100 µM dNTPs, 0.1 mg/ml BSA, 33 mM Tris acetate pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate and 0.5 mM DTT). The cDNA is now ready for adaptor ligation (15 pmol EcoRI adaptors, 10 U T4-DNA-ligase and 30 min under constant shaking; Fast-Link, Epicentre Technologies, Biozym, Hessisch-Oldendorf, Germany). Directly following adaptor ligation, the cDNA is phosphorylated (10 U T4-polynucleotide kinase at 37°C for 30 min, 70 mM Tris-HCl pH 7.6, 10 mM MgCl2 and 5 mM DTT). Kinasing is the last enzymatic step that is performed on the solid support. To remove the completed cDNA from its support and to enable the cloning of the corresponding cDNA fragments, the cDNA is restricted with NotI. Restriction is carried out for ~30 min and the supernatant, containing the completed cDNA fragments, is aspirated and the DNA purified using PCR purification columns (Qiagen, Hilden, Germany).


Figure 1. Outline of the solid-phase cDNA-synthesis approach. The mRNA of interest is isolated using an oligodT-primer coupled to magnetic beads via a 5[prime]-attached biotin residue. First-strand synthesis is performed either on the solid support primed by the oligodT-primer used to capture the mRNA, or in solution primed by a modified biotinylated oligonucleotide carrying an eight base long random sequence at its 3[prime]-end. After capture of the random primed cDNA, the procedure for either random- or oligodT-primed cDNA is the same. Each of the following steps (3-7) is accomplished after aspiration of the supernatant and washes with the buffer required for the subsequently following enzymatic reactions. These steps are second-strand synthesis accomplished by RNase H and DNA-Polymerase (3), blunting catalysed by T4-DNA-Polymerase (4), EcoRI adaptor ligation (5), kinasing of the cDNA (6) and restriction with NotI to liberate the cDNA from the beads (7). The whole procedure and the transformation of competent bacteria could be performed in a single day.

The cDNA is now ready for ligation into the appropriate vector (e.g. pSportI). Alternatively, the cDNA could be size fractionated prior to ligation. Ligation has to be performed with insert/vector molar ratios ranging from 1:1 to 5:1 (see adaptor ligation). Directly following the ligation, competent bacteria (Xl10Gold, Stratagene, Heidelberg, Germany) are transformed with 1 µl aliquots of the ligation mixtures and plated on appropriate plates. A total number of 500 000-2 000 000 independent clones were routinely achieved.

We used this method to make high quality cDNA libraries from different parts of the nervous system of the desert locust S.gregaria. To analyse the cDNA size distribution, an aliquot of the second-strand synthesis reaction was supplemented with digoxygenin (DIG) labelled nucleotides. After electrophoretic separation (0.7% agarose in 1× TBE), the DNA was blotted onto nylon membrane, probed with [alpha]-DIG coupled alkaline phosphatase antibodies and visualised with CDP-star. The DNA smear ranged from ~300 bp to 7 kb, including highly abundant transcripts indicated by strong extra bands in this smear. The random-primed cDNA has a different appearance. Its mean size is smaller, beginning at <100 bp, but it approaches the length of the oligodT-primed DNA. Extra bands are missing. In comparison to cDNA libraries made from the same source, these newly produced libraries exhibit a higher quality, as seen in the mean length of inserts. This should result from the optimised buffer compositions, together with careful handling of the cDNA. Although some reports are known that address the problem of capture of large cDNAs using magnetic beads, I could not reproduce this problem as even large cDNAs (>5 kb) are present in the produced library. To show the usefulness of this approach, I randomly chose 10 clones from both pSportI libraries (90% of insert containing clones with insert length ranging from 800 bp to 4.8 kb) and sequenced them from their 5[prime]-end. Eight out of 10 for the oligodT-primed and seven out of 10 for the random-primed cDNA revealed a start codon followed by an uninterrupted open reading frame of >400 bp. As only very few Schistocerca sequences are known, sequence comparison could not reveal identities of deposited sequences. Nevertheless, homologies to other proteins such as actin or choline acetyl transferase are obvious. To test whether rare messages are still present in the libraries, I performed test hybridisations. Two cDNAs were used, the TA20 cDNA coding for a transcription factor of the zinc-finger family, which is exclusively expressed in the thoracic ganglia of locusts and the olA12 cDNA coding for the insect centrosomin gene. Both are rare transcripts as the transcription factor is exclusively expressed in only a few neurons of the thoracic ganglia and centrosomin is only expressed in mitotic cells. Neurogenesis is an extremely rare event in the brain of adult insects, thus centrosomin is expressed at very low levels. Test hybridisations gave frequencies of ~15 and 5 positives per 100 000 clones, respectively, which shows that even low copy transcripts are present in the solid-phase synthesised libraries.

The main advantage of my cDNA synthesis solid-phase approach is the easy manipulation of the synthesised cDNA. Buffer exchanges without loss of cDNA and the risk of contamination are easy to perform. In addition, truly representative cDNA libraries, which include small cDNAs, could be made this way because a size fractionation prior to cloning is not necessary. Taken together, the solid-phase approach combines the advantages of conventional cDNA-synthesis protocols while eliminating most of their drawbacks. The method is simple to perform, reliable, inexpensive and gives cDNA-libraries of superior quality. It could therefore replace most currently used cDNA-library protocols.

ACKNOWLEDGEMENTS

I would like to thank Professor Gewecke for continuous support. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ge 249).

REFERENCES

1. Kimmel,A.R. and Berger,S.L. (1987) Methods Enzymol., 152, 307-316. MEDLINE Abstract

2. Gubler,U. and Hoffman,B.J. (1983) Gene, 25, 263-269. MEDLINE Abstract

3. Okayama,H. and Berg,P. (1982) Mol. Cell. Biol., 2, 161-170. MEDLINE Abstract

4. DYNAL Technical Handbook (1995) Biomagnetic Techniques in Molecular Biology (2nd ed.), pp. 61-70.

5. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

*Tel: +49 40 4123 3941; Fax: +49 40 4123 3937; Email: roeder@zoologie.uni-hamburg.de

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