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5' protection dependent amplification

Through double-stranded specific labeling technology with a protective group at the 5' end of RNA, it solves the problems of information loss and degradation caused by low RNA selectivity and multi-step solutions in the existing technology, and achieves efficient and accurate RNA labeling. Labeling and purification.

Active Publication Date: 2015-03-18
LEXOGEN GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Also, enzyme preparations are not really pure and even small amounts of nucleases can increase the chances of RNA degradation
Finally, each additional reaction step also increases the chance of introducing RNase contamination that degrades RNA
[0019] A bias towards shorter RNA molecules is also introduced due to the increased risk of degradation of longer RNA molecules compared to shorter RNA molecules

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0109] Example 1A: Study of double-strand-specific junctions using artificial RNA molecules with defined sequences

[0110] Since the structure represented by the cap is guanosine in nature, the N7 of its purine ring is methylated, and its 5' end is connected to the 5' end of the RNA by a triphosphate bridge. The cDNA in hybrids with the capped RNA is ligated in a strand-specific manner. Therefore, in a manual assay, capped T7 transcripts and oligonucleotides with different 3' overhangs or 3' deleted nucleotides were set up in the ligation reaction to investigate whether this duplex specificity could be achieved . Of particular interest is whether additional nucleotides are required on the cDNA that can pair or stack with the cap.

[0111] connect:

[0112] Representatives of different oligonucleotides with different 3' extensions (Seq ID: 22-26) were ordered from Microsynth AG (Balgach, CH) with RNA (Seq ID: 4,5 ) reverse complement. Whereas Seq ID:22 has no 3' extension...

Embodiment 1B

[0115] Example 1B: Investigation of duplex-specific ligation of adapters with 5' overhangs to capped or uncapped RNA::cDNA hybrids

[0116] As mentioned above, the cap provides a one nucleotide overhang if during reverse transcription of the capped RNA (mRNA), the reverse transcriptase preferably does not add additional nucleotides (C) to the cDNA. However, this cap overhang will consist of inverted nucleotides, which are further attached to the 5' end of the RNA by a triphosphate bridge. We therefore investigated whether this cap overhang could interact with the 5' overhang of the adapter in a manner that enables specific ligation of the adapter oligonucleotide to the 3' duplex of the cDNA (e.g., base stacking or base pairing). ).

[0117] In contrast to capped RNA, if reverse transcriptase accesses the 5' end of uncapped RNA, it will blunt-end or add extra nucleotides to the cDNA in a non-templated manner. Therefore, if a control reaction is included, the uncapped RNA hybr...

Embodiment 2

[0123] Example 2: T7 exonuclease digestion of artificial RNAs with different 5' ends

[0124] To demonstrate the specificity of exonuclease digestion for different 5' ends of RNA when hybridized to different cDNA molecules, experiments were performed using artificial oligonucleotides.

[0125] hybridization:

[0126] Templates for T7 exonuclease digestion were performed on PAGE purified hybrids of RNA (Seq ID: 4, 7, 8) with different oligonucleotides mimicking cDNA (Seq ID: 22, 23). Different RNAs (Seq ID: 7, 8) were ordered from Eurogentec (Seraing, Belgium). Hybridization was performed by heating RNA and oligonucleotides to 95°C in 10 mM Tris pH 7.0 for 30 seconds, cooling to 45°C with a 2% ramp rate (ABI9700 thermal cycler), adding an equal volume of 2x Hybridization Buffer (100mM Tris pH 7.9, 6mM MgCl 2 ). After maintaining for 15 minutes, the temperature was further lowered to 4°C with a 2% ramp speed limit. Hybrids were subsequently synthesized: Seq ID:4 / Seq ID:22, ...

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Abstract

The present invention relates to methods for generating a labelled nucleic acid from an RNA comprising a 5' protecting group, said method comprises the steps of obtaining a mixture of template strands of nucleic acids, said mixture comprising said RNA and further potentially other nucleic acids without a 5' protecting group, annealing at least one oligonucleotide primer to the template strand of said RNA and potentially other nucleic acids, and template sequence dependent extending said primer, thereby obtaining a complementary nucleic acid strand annealed to its template strand, or providing the RNA in duplex with a complementary nucleic acid strand annealed to its template strand, and modifying the extension product of said nucleic acids without 5' protecting group either on the 5' end of the template strand or on the 3' end of the complementary strand, or both, and labelling a complementary nucleic acid of a double stranded nucleic acid not modified in step c), thereby specifically generating a labelled nucleic acid complementary to an RNA at least originally comprising a 5' protecting group.

Description

technical field [0001] The present invention relates to the field of analysis of complex nucleic acid mixtures, in particular the analysis and selection of nucleic acids providing different nucleotides in the 5' region of the nucleic acid, in particular the purification of RNA, especially capped RNA such as messenger RNA (mRNA), and enrichment for cDNA copies derived from the RNA. Background technique [0002] Nucleic acids, such as total RNA extracts, can provide different chemical structures at their 5' ends. For example, 28S and 18S ribosomal RNA (rRNA) as well as microRNA (miRNA) and 5' degraded RNA have a 5' monophosphate. Other RNAs have a 5' diphosphate such as 5S rRNA intermediates or a 5'OH such as 18S rRNA intermediates and transfer RNA (tRNA) intermediates. Prokaryotic mRNA has a 5' triphosphate, and eukaryotic mRNA has a cap structure. The cap structure is essentially guanosine, the N7 of the purine ring is methylated, and its 5' position is connected to the 5...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12P19/34C12Q1/68
CPCC12N15/1096C12P19/34C12Q1/6844C12Q1/6858C12Q1/686C12Q2521/107C12Q2533/107
Owner LEXOGEN GMBH