Sequencing Template Adaptor and Anchor Probe Hybridization
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Solution Overview
Problem
Conventional sequencing methods are limited by signal degradation and low signal-to-noise ratios, restricting sequencing efficiency and making them unsuitable for single-molecule sequencing, particularly in determining long nucleotide sequences effectively.
Innovation Solution
The method involves using a sequencing template with adaptors and anchor probes, where degenerate bases are used for hybridization and sequencing probes are labeled to detect specific bases, allowing for the determination of nucleotide sequences through ligation and detection processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used, then sequencing can be performed with standard protocols, but signal degradation limits the determination to only a few tens of nucleotides
Solution Approach 1:
The sequencing process is divided into multiple cycles, each determining a specific position in the sequence. At each cycle, probes specific to that position are hybridized and detected, allowing the sequence to be read one nucleotide at a time across multiple steps rather than attempting to read the entire sequence in a single step.
Solution Approach 2:
Adaptor sequences are ligated to the ends of the template DNA before sequencing begins. These adaptors contain known sequences that serve as binding sites for sequencing probes, preparing the template in advance for systematic sequencing by providing anchor points for probe hybridization at each position.
2Reliability
If conventional sequencing methods are used, then standard sequencing protocols can be applied, but signal-to-noise ratios are too low for single-molecule sequencing
Solution Approach 1:
Different nucleotide incorporations are detected using fluorescently labeled probes that emit different colors or intensity levels. When a probe hybridizes to its complementary sequence and is ligated, the fluorescent label produces a detectable signal that indicates which nucleotide was incorporated, enabling optical detection of single-molecule events.
Solution Approach 2:
Sequencing probes with fluorescent labels serve as intermediaries between the nucleotide incorporation event and the detection system. These probes bind to the template and amplify the signal of nucleotide incorporation through their fluorescent tags, making single-molecule events detectable above the noise background.
3Measurement precision
If shorter read lengths are used, then sequencing reactions can be performed with higher accuracy, but more reads are needed to assemble complete sequences
Solution Approach 1:
The sequencing process continues through multiple cycles, with each cycle determining the next nucleotide position in the sequence. By maintaining the same template and systematically advancing through positions one by one, the method accumulates sequence information continuously over many steps, achieving long reads through sustained sequencing activity rather than relying on long exposure times that would degrade signal quality.
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 enhances sequencing efficiency by enabling the determination of longer sequences and improving the accuracy of nucleotide identification, overcoming the limitations of conventional methods.
Implementation Method 1
hybridizing an anchor probe to the anchor site
Implementation Method 2
hybridizing a pool of sequencing probes for determination of the sequence
Implementation Method 3
ligating the anchor probe and the sequencing probe
Implementation Method 4
the sequencing probe is detectably labeled to identify the presence of a particular base
Data Source
AI summary
The present invention is directed to compositions and methods for nucleic acid identification and detection. Compositions and methods of the present invention include extracting and fragmenting target nucleic acids from a sample, using the fragmented target nucleic acids to produce target nucleic acid templates and subjecting those target nucleic acid templates to amplification methods to form nucleic acid nanoballs. The invention also includes methods of detecting and identifying sequences using various sequencing applications, including sequencing by ligation methods.