Nucleic Acid Barcoding via Cleavable Blocking Groups
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Solution Overview
Problem
Current methods for molecular barcoding of nucleic acid targets, particularly at the 5′ and 3′ ends, are limited in their ability to efficiently quantify gene expression and obtain full-length sequences of nucleic acid targets.
Innovation Solution
The method involves contacting nucleic acid targets with a first plurality of oligonucleotide barcodes, which include a universal sequence, a molecular label, and a target-binding region. These barcodes are extended to generate barcoded nucleic acid molecules, which are then hybridized with a second plurality of oligonucleotide barcodes containing a cleavage domain and a blocking group. The blocking group is removed using a cleaving enzyme, allowing for further extension of the barcoded nucleic acid molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If molecular barcoding is performed at both 5' and 3' ends of nucleic acid targets, then quantitative analysis of gene expression and full-length sequence acquisition are improved, but the complexity of the barcoding process increases
Solution Approach 1:
The barcoding process is divided into two distinct stages: first barcoding at the 5' end using first oligonucleotide barcodes, then barcoding at the 3' end using second oligonucleotide barcodes. This segmentation allows each end to be processed independently with specialized reagents and conditions, improving overall measurement precision while managing complexity through modular design
Solution Approach 2:
The 5' end barcoding is performed as a preliminary action before 3' end barcoding. The first oligonucleotide barcodes are hybridized and extended to create intermediate barcoded molecules that serve as substrates for the second barcoding step. This preliminary action establishes a foundation that enables subsequent full-length sequencing and quantitative analysis
2Manufacturing precision
If blocking groups are used to prevent premature extension of oligonucleotide barcodes, then manufacturing precision of barcoded molecules is improved, but the number of processing steps increases
Solution Approach 1:
Blocking groups are pre-installed on the oligonucleotide barcodes before hybridization to the nucleic acid targets. This preliminary action prevents premature extension during the hybridization and washing steps, ensuring that extension only occurs when and where intended, thereby improving manufacturing precision of the barcoded molecules
Solution Approach 2:
The blocking groups are temporarily introduced during barcode synthesis and are subsequently removed after the barcoding process is complete. This extraction of the blocking groups after they have served their protective function allows recovery of the fully functional barcoded molecules without the blocking interference, maintaining precision while enabling downstream applications
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 enables efficient quantitative analysis of gene expression and the acquisition of full-length sequences of nucleic acid targets, improving the accuracy and completeness of molecular barcoding.
Implementation Method 1
a first target-binding region capable of hybridizing to the nucleic acid target
Implementation Method 2
a cleaving enzyme is capable of cleaving the oligonucleotide barcode at a point within or adjacent to the cleavage domain when the cleavage domain is hybridized to a barcoded nucleic acid molecule
Implementation Method 3
extending the 3′ ends of oligonucleotide barcodes of the second plurality of oligonucleotide barcodes hybridized to the barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for full-length whole transcriptome analysis (WTA). Some embodiments comprise 5′-based, 3′-based, and internal-based gene expression profiling. Immune repertoire profiling methods are also provided in some embodiments.