Abstract
Conventional cDNA library construction often requires a minimum available amount of material (typically 1 or 2 µg of polyA+ RNA). For complex organs, such as brain, or certain species, such as humans, as well as subsets of cell types, this condition is often difficult to fulfill. Amplification by PCR can be used to circumvent this limitation because it is a powerful method to obtain working quantities of low-abundance DNAs. To effectively apply this method, known sequences need to be attached to the ends of the single-stranded cDNA (ss-cDNA). One at the 5′ end of the ss-cDNA is added during the priming of the synthesis, the other, at the 3′ end, is covalently attached by ligation using the SLIC strategy as described elsewhere (Chapter 27). With known DNA sequences attached to both ends of the synthetized cDNA, minute quantities can be amplified with sequence-specific primers to provide sufficient material to successfully generate and screen cDNA libraries. The overall scheme is illustrated in Fig 1.
Constructing cDNA libraries using the SLIC strategy. Reverse transcription is carried out with a random primer RA3′ NV. RNAs are digested and primer is removed to avoid its ligation to the A5′ NV oligonucleotide. A5′ NV bears a phosphate group at its 5′ extremity to allow ligation to the 3′ end of the ss-cDNA. To avoid formation of concatemers A5′ NV bears an amino group at its 3′ end. Two rounds of nested PCR are performed to generate a ready-to-use cDNA library.
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© 1997 Humana Press Inc.
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Ravassard, P., Icard-Liepkalns, C., Mallet, J., Edwards, J.B.D.M. (1997). cDNA Libraries from a Low Amount of Cells. In: White, B.A. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 67. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-483-6:317
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DOI: https://doi.org/10.1385/0-89603-483-6:317
Publisher Name: Humana Press, Totowa, NJ
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