Reverse transcriptase reaction

To produce scFv molecules, you will need to do two first strand cDNA n-thesis reactions one for the heavy chain and one for the light chain. Since the spleen mRNA comes from an hyperimmunized mouse, we are only interested in the preparation of IgG DNA and not IgM DNA. 

Set up the ft)llowing reverse transcription mix in duplicate in RNase-free micro-centrifuge tubes. Label one tube as light chain and the second one as heavy chain  

Place the mRNA sample in a 500 pi RNase-free microcentrifuge tube and add RNase-Free Water (component of the Kit), if necessary, to bring the mRNA to 20 pi final volume. 

Heat the mRNA at 65 °C for 10 min and cool immediately on ice. Start the first strand cDNA reaction within 2 min after placing on ice. 

Mouse heavy chain constant r on primers MulgGl/2For 5 CTG GAC AGG GAT CCA GAG TTC CA 3  

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This step removes any residual proteins. It is important to avoid contamination of phenol in the extracted phase because this will affect the reverse transcriptase reaction. It is also important to avoid the interphase, which is enriched with DNA. 

Figure4.4 Reverse transcriptase reaction scheme (Lottspeich, 1998). Tth = Thermits thermophilus.

Daunomycin and adriamycin at 10 jug/reaction mixture (0.25 ml) inhibit the reverse-transcriptase reaction by 60—70%. The dihydro derivative is also quite effective whereas the N-guanidine derivative has moderate activity. The N-acetyl derivative was completely ineffective in the MSV (M) and FLV systems but moderately inhibited the RSV system. 

Mature mRNA transcripts (sense strand) from eukaryotic cells can be purified and then reverse transcribed, with the assistance of a reverse transcriptase enzyme (from Moloney murine leukemia virus, MMLV), into complementary DNAs (cDNAs) that will anneal with the mRNA transcripts by Watson-Crick base pairing to give anti-parallel DNA/RNA duplexes or double helices. The poly(A) tail in each mature mRNA transcript is actually a usefiil handle for each reverse transcriptase reaction. Thereafter, DNA/RNA duplexes must be broken down with the assistance ofRNAse enzymes (specific for the hydrolysis of RNA phospho diester links) and a sense strand of DNA constructed instead on each cDNA single strand so that equivalent, more stable antiparallel DNA/DNA duplexes are generated instead, with the assistance of a DNA polymerase enzyme. In this instance, the poly(T) tail in each cDNA molecule turns out to be important for the DNA polymerase reaction ... 

Single-stranded cDNA is synthesized from mRNAs from individuals using oligo-dT priming and Thermoscript reverse transcriptase reactions (Invitrogen, Carlsbad, CA). 

Superscript II reverse transcriptase (Life Technologies, Paisley, UK) This enzyme is a recombinant RNase H" reverse transcriptase from Moloney Murine Leukaemia Virus (MMLV) and is supplied with tubes of 0.1 M dithiothreitol (DTT) and 5X reverse transcriptase reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl and 15 mM MgClj)... 

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