Strand-Specific RNA Library Preparation via 5'-Modified Primers
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Solution Overview
Problem
Current methods for generating strand-specific RNA libraries are cumbersome, prone to biases, and require additional enzymatic steps, such as the dUTP method, which is not 100% efficient, and the use of complex tagged primers and T4 PNK, which exhibits base bias and requires precise optimization.
Innovation Solution
A method using 5'-modified, preferably 5'-phosphorylated random oligonucleotides for cDNA synthesis, allowing direct ligation of adaptors to the cDNA strand, eliminating the need for additional enzymatic reactions and reducing bias, with the first strand allowing no adapter ligation at its 5' terminus and the second strand allowing ligation, ensuring strand specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If dUTP method is used for strand-specific RNA library preparation, then strand specificity is achieved, but the process becomes more complicated and less efficient
Solution Approach 1:
The patent applies preliminary action by incorporating a phosphate group at the 5' end of the adaptor during the adaptor ligation step itself, rather than requiring a separate phosphorylation step afterward. This pre-preparation of the phosphate group eliminates the need for additional enzymatic reactions and resolves the technical contradiction by maintaining strand specificity while simplifying the overall library construction process
2Productivity
If T4 PNK is used for adaptor phosphorylation, then ligation efficiency is improved, but base bias and optimization complexity increase
Solution Approach 1:
The patent extracts the phosphate group addition function from the T4 PNK enzyme system and implements it directly through chemical modification of the adaptor during synthesis. This removes the need for T4 PNK enzyme and its associated optimization complexities, while maintaining the essential phosphorylation function needed for efficient ligation
Solution Approach 2:
The patent uses a simple, chemically modified adaptor with a pre-incorporated phosphate group that can be directly ligated without requiring expensive enzyme systems like T4 PNK. This disposable, pre-modified adaptor approach eliminates the need for complex enzymatic phosphorylation and reduces optimization requirements
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 method enhances the efficiency and accuracy of RNA library preparation, achieving a higher percentage of uniquely mapped reads and reducing the complexity of the library construction process, with no additional enzymatic steps required post-adaptor ligation, resulting in better data quality and strand specificity.
Implementation Method 1
T4 Polynucleotide Kinase (T4 PNK) plays a key role; its kinase activity adds phosphate groups at 5' fragment ends
Implementation Method 2
its phosphatase activity removes phosphates from at 3' fragment ends and leaves hydroxyl groups
Implementation Method 3
ligating adapters to the 3' and 5' termini of the double-stranded nucleic acid
Data Source
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AI summary
The invention relates to a method for preparing a strand-specific library from an nucleic acid or preferably RNA sample, for RNA comprising the steps of: (i) optionally fragmenting said RNA sample, (ii) generating a plurality of first cDNA strands by subjecting said fragmented RNA to reverse transcription by using a reverse transcriptase and first oligonucleotide primers, (iii) generating a plurality of second cDNA strands by using a DNA polymerase, second oligonucleotide primers, and the plurality of first cDNA strands, and (iv) ligating adapters to the 3' and 5' termini of the of double- stranded cDNA, (v) wherein the first cDNA strand allows no adapter ligation at its 5' terminus and said second cDNA strand allows adapter ligation at its 5' terminus, or vice versa, and, (v) optionally cloning, sequencing or otherwise using the strand-specific library. The invention also relates to a kit for preparing a strand-specific library from an RNA sample, wherein said kit comprises, (i) random oligonucleotide primers comprising a 5' terminus nucleotide which allows no adaptor ligation, (ii) random oligonucleotide primers comprising a 5' terminus nucleotide which allows adaptor ligation, (iii) optionally a reverse transcriptase for synthesizing a first cDNA strand complementary to the template RNA, (iv) optionally dNTPs, and (v) optionally a DNA polymerase