Amplifying Short Non-PolyA RNAs via PolyA Tailing
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Solution Overview
Problem
Current methods face challenges in amplifying and detecting short non-polyA tailed RNAs, such as miRNA, snoRNA, and piRNA, due to their short length and lack of polyA tails, which limits their amplification and quantification.
Innovation Solution
The methods involve reverse transcribing the short RNA using a primer with a 5' overhang that is not complementary to the RNA, followed by extension with a DNA polymerase-DNA binding domain fusion, and subsequent amplification using a primer complementary to the 3' region of the cDNA, allowing for the generation of longer cDNA sequences that can be quantified and sequenced.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PCR amplification is used on short non-polyA tailed RNAs, then amplification can be performed, but the short length and lack of polyA tails prevent effective primer binding and amplification
Solution Approach 1:
The patent applies preliminary action by adding a polyA tail to the 3' end of short non-polyA tailed RNAs before amplification. This pre-modification enables subsequent primer binding and PCR amplification to proceed effectively, resolving the contradiction between maintaining short RNA length and achieving amplification capability.
Solution Approach 2:
The patent introduces a polyA tail as an intermediary element that mediates between the short RNA template and the PCR primers. This intermediary structure provides the necessary binding site for oligo-dT or polyA-specific primers, enabling amplification without altering the original short RNA sequence or requiring the RNA to be naturally polyA-tailed.
2Stability of the object's composition
If the RNA is kept short to maintain its natural state, then the native structure is preserved, but amplification and detection become difficult
Solution Approach 1:
The patent performs preliminary polyA tailing on the short RNA molecules before detection. This preliminary modification preserves the native short RNA structure and sequence while adding the necessary polyA tail for subsequent amplification and detection, thereby resolving the contradiction between maintaining native structure and enabling detection.
Solution Approach 2:
The patent applies local quality modification by adding the polyA tail only at the 3' end of the short RNA without altering the rest of the native sequence. This localized modification maintains the native structure and composition of the short RNA while providing the necessary feature for amplification and detection.
3Productivity
If polyA tailing is performed on short RNAs, then amplification becomes possible, but the added tail increases the total length beyond the original short RNA
Solution Approach 1:
The patent applies preliminary polyA tailing in a controlled manner, adding only the minimum necessary polyA residues (typically 5-20 nucleotides) to enable primer binding. This limited preliminary modification provides amplification capability while minimizing the increase in total length, thus resolving the contradiction between enabling amplification and maintaining short length.
Solution Approach 2:
The patent applies partial action by adding only a short polyA tail segment rather than a full polyA tail. This partial modification is sufficient to enable primer binding and amplification while keeping the total length increase minimal, thereby resolving the contradiction between achieving amplification capability and maintaining the short RNA characteristic.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient amplification and detection of short non-polyA tailed RNAs, overcoming the limitations of existing methods by producing longer cDNA sequences that can be accurately quantified and sequenced, facilitating the analysis of these small RNA molecules.
Implementation Method 1
reverse transcribing the RNA by extending a first primer having at least 6 contiguous nucleotides complementary to the RNA
Implementation Method 2
amplifying the first strand cDNA with a DNA polymerase-DNA binding domain fusion to extend a second primer
Implementation Method 3
adding random poly-W (A/T) or poly-S (G/C) nucleotide sequences to the 3' end of target non-polyA tailed RNA with a terminal transferase
Implementation Method 4
submitting the RNA molecules comprising the random poly-W or poly-S nucleotide sequences to conditions such that the RNA molecules comprising the random poly-W or poly-S nucleotide sequences anneal to form a duplex
Implementation Method 5
the adding further comprises contacting the non-polyA tailed RNA with a poly A polymerase (e.g. a yeast poly A polymerase)
Data Source
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AI summary
Methods of detecting and amplifying short RNAs are provided.