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Mint cDNA synthesis

Method overview

Mint cDNA synthesis is based on a novel technology (patent pending) utilizing the specific features of MMLV-based reverse transcriptase (RT). First strand cDNA synthesis starts from the 3'-primer comprising an oligo(dT) sequence to anneal to the polyA+ stretch of RNA. When RT reaches the 5' end of the mRNA, it adds several non-template nucleotides, primarily deoxycytidines, to the 3' end of the newly synthesized first-strand cDNA (Schmidt and Mueller, 1999). This oligo(dC) stretch base pairs to the complementary oligo(dG) sequence located at the 3' end of a special 30-mer deoxyribooligonucleotide called PlugOligo. RT identifies PlugOligo as an extra part of the RNA-template and continues first strand cDNA synthesis to the end of the oligonucleotide, thus incorporating PlugOligo sequence into the 5' end of cDNA.

The last 3'-dG residue of the PlugOligo is a terminator nucleotide comprising 3'-phosphate group. This blocking group prevents unwanted annealing and extension of the PlugOligo. Under standard conditions RT can hardly use PlugOligo as a template, however our special IP-solution (solution for Incorporation of PlugOligo sequence) dramatically increases the efficiency of this process. Synthesis of ds cDNA is then performed using PCR amplification.

Schematic outline of Mint cDNA synthesis.
Enlarge scheme

Mint-amplified cDNA from different sources.

1 - mouse liver; 2 - mouse skeletal muscle; 3 - mouse brain; 4 - human leucocytes; 5 - human lung; 6 - human skeletal muscle; 7 - mosquito grub; 8 - copepod Pontella sp.; 9 - tomato Lycopersicon esculentum. M - 1 kb DNA size marker, SibEnzyme, Russia.

References:

  • Schmidt WM, Mueller MW. CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs. Nucleic Acids Res. 1999; 27 (21):e31. / pmid: 10518626
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