Abstract
The isolation of intact messenger RNA and its conversion into cDNA copies by avian or Moloney murine reverse transcriptase, as well as subsequent amplification of gene transcripts by the PCR technique, are becoming increasingly important tools in molecular biology. At present, these techniques have been often necessary and widely used for the analysis of individual mRNA levels in cells and tissues by Northern blot analysis, nuclease protection analysis and in situ hybridization. Another important application of RNA templates is the construction of representative cDNA libraries in order to clone genes, to investigate their molecular structure and to express them in prokaryotic and/or eukaryotic cells (Belyaysky 1989).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Aviv H, Leder P (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci USA 69: 1408–1412
Belyaysky A, Vinogradova T, Rajewsky K (1989) PCR-based cDNA library construction: general cDNA libraries at the level of a few cells. Nucl Acids Res 17: 2919–2932
Cathala G, Savouret JF, Mendez B, West BL, Karin M, Martail JA, Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2: 329–335
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159
Clark JM (1988) Novel non-template nucleotide addition reactions catalyzed by prokaryotic and eucaryotic DNA polymerases. Nucl Acids Res 16: 9677–9686
Crowe JS, Cooper HJ, Smith MA, Sims MJ, Parker D, Gewert D (1991) Improved cloning efficiency of polymerase chain reaction ( PCR) products after proteinase K digestion. Nucl Acids Res 19: 184
Efstratiadis F, Kafatos FC, Maxam AM, Maniatis T (1976). Enzymatic in vitro synthesis of globin genes. Cell 7: 279–288
Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269
Jung V, Pestka SB, Pestka S (1990) Efficient cloning of PCR generated DNA containing terminal restriction endonuclease recognition sites. Nucl Acids Res 18: 6156
Kaufman DL, Evans GA (1990) Restriction endonuclease cleavage at the termini of PCR Products. Bio Techniques 9: 304–306
Kimmel AR, Berger SL (1987) Guide to molecular cloning techniques. In Berger SL, Kimmel AR (eds), Methods in Enzymology, vol 152. Academic Press, New York, pp 307–316
Okayama H, Berg P (1982) High-efficiency cloning of full-length cDNA. Mol Cell Biol 2: 161–170
Rhyner TA, Biquot NF, Berrard S, Borbely AA, Mallet J (1986) An efficient approach for the selective isolation of specific transcripts from complex brain mRNA populations. J Neurosci Res 16: 167–181
Rougen F, Mach B (1976) Cloning and amplification of rabbit α-and β-globin gene sequences into E. coli plasmids. J Biol Chem 252: 2209–2217
Sardelli AD (1991) Cloning of PCR products. Amplifications, 6: 10–11.
Stoker AW (1990) Cloning of PCR products after defined cohesive termini are created with T4 DNA polymerase. Nucl Acids Res 18: 4290–4291
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Steinbergs, J., Tsimanis, A. (1998). cDNA Synthesis and Cloning. In: Berzins, V. (eds) Basic Cloning Procedures. Springer Lab Manual. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-71965-3_1
Download citation
DOI: https://doi.org/10.1007/978-3-642-71965-3_1
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-48977-8
Online ISBN: 978-3-642-71965-3
eBook Packages: Springer Book Archive