Telomere Adaptor Ligation for High-Resolution Strand Sequencing
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Solution Overview
Problem
Existing methods for sequencing telomeres are inefficient due to their highly repetitive and non-linear nature, leading to difficulties in characterizing their sequence, and the use of polymerases to copy long stretches of repetitive sequences is problematic.
Innovation Solution
A method involving ligation of a polynucleotide telomere adaptor to the 5′ end of the non-overhanging strand at the telomere, allowing specific characterization of the ligated strand in the 5′ to 3′ direction, without the need for restriction digestion or PCR, and enabling characterization of both strands with increased resolution and identification of modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR is used to copy long stretches of repetitive sequences, then sequencing can be performed, but polymerases cannot effectively copy long stretches of repetitive sequences as found in telomeres
Solution Approach 1:
The patent extracts the problematic repetitive telomeric sequences from the genomic DNA and isolates them as separate entities. By using adaptors that specifically bind to telomeric overhangs and ligation to attach sequencing adapters, the method separates the telomere sequencing task from general genomic sequencing, allowing specialized handling of these difficult repetitive regions without relying on problematic PCR amplification.
Solution Approach 2:
The patent introduces adaptor molecules as intermediaries between the telomeric DNA and the sequencing system. These adaptors specifically hybridize to telomeric overhangs and provide a platform for ligation-based adapter attachment, serving as a mediator that enables sequencing of repetitive telomeric sequences without requiring direct PCR amplification of the telomeres themselves.
2Measurement precision
If restriction digestion and Southern blot analysis are used, then telomere characterization is possible, but the sequence has to be reconstructed from shorter sequences
Solution Approach 1:
The patent performs preliminary actions by first attaching adaptors to the telomeric overhangs and then ligating sequencing adapters directly to these adaptors. This preliminary preparation creates continuous reading frames that extend through the telomeric repeats, enabling direct sequencing of long stretches of telomeric DNA without requiring subsequent reconstruction from fragmented data.
3Productivity
If nanopores are used to sequence telomeres, then direct sequencing is possible, but many other 3′ overhangs in genomic DNA were extended non-specifically
Solution Approach 1:
The patent applies local quality by designing adaptors with specific binding characteristics tailored to telomeric overhangs. The adaptor sequences and binding mechanisms are optimized to specifically recognize and bind to telomeric 3' overhangs while excluding other genomic DNA overhangs, thereby achieving both direct sequencing capability and telomere-specificity simultaneously.
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
The method provides specific and high-resolution sequencing of telomeres, allowing for the identification of modifications and characterizing beyond the telomere into the chromosome, without the need for restriction digestion or PCR, and can identify methylated nucleotides and telomere-binding proteins.
Implementation Method 1
the 3′ end of the adaptor specifically hybridises to the first part of the overhanging strand
Data Source
AI summary
The invention relates to methods for characterising, such as sequencing, at least part of a telomere and adaptors for use in such methods.