DNA Ligation and Repeat Element Amplification for ctDNA Sequencing
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Solution Overview
Problem
Current methods for analyzing circulating tumor DNA (ctDNA) face challenges due to limited sample volume and high fragmentation, leading to restricted diagnostic information and potential false positives from amplification errors during sequencing.
Innovation Solution
The method involves random ligation of fragmented DNA to form concatemers, enabling amplification between repeat elements and incorporating unique molecular identifiers to correct sequencing errors, thereby enhancing the amplification of genomic regions and reducing false positives.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to amplify ctDNA, then the amount of DNA is increased, but false positive mutations are introduced due to polymerase mis-incorporation errors
Solution Approach 1:
The method segments the amplification process into multiple independent PCR reactions, each targeting a specific genomic region. By dividing the genome into smaller amplicons and amplifying them separately, the method reduces the cumulative error rate while maintaining sufficient DNA quantity for detection
Solution Approach 2:
The patent introduces unique molecular identifiers (UMIs) as intermediary tags that are ligated to DNA fragments before amplification. These UMIs serve as mediators to track original DNA molecules through the PCR process, enabling error correction by comparing sequences from multiple amplifications of the same original molecule
2Quantity of substance
If standard blood draw is used to obtain ctDNA, then sample volume is limited, but diagnostic information is restricted due to insufficient DNA quantity
Solution Approach 1:
The method performs preliminary enrichment of ctDNA by capturing circulating DNA fragments containing tumor-derived mutations before amplification. This preliminary action concentrates the rare ctDNA molecules among vast excess of normal DNA, ensuring sufficient diagnostic information is preserved even from limited sample volumes
Solution Approach 2:
The patent combines multiple small DNA fragments containing the same genomic region into larger concatemers through ligation. This merging process increases the effective concentration of target sequences, enabling sufficient diagnostic information to be obtained from minimal starting material
3Ease of operation
If ctDNA is highly fragmented into 120-200 bp segments, then sample handling is complicated, but amplification between repeat elements is prevented
Solution Approach 1:
The patent uses repeat element sequences as intermediary anchors to bridge fragmented DNA segments. By designing primers that target conserved repeat elements (such as Alu elements) present in multiple copies throughout the genome, the method enables amplification across fragmented regions without requiring the fragments to be naturally contiguous
Solution Approach 2:
The method changes the amplification parameters by using primers with higher melting temperatures and optimized annealing conditions that allow specific binding to repeat elements even in the presence of fragmentation. This parameter optimization enables successful amplification despite the fragmented state of the DNA
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 increases the amount of amplifiable DNA, improves the accuracy of sequencing, and allows for the detection of clinically relevant mutations from small blood samples, facilitating minimally invasive testing and monitoring of tumor status.
Implementation Method 1
applying random ligation conditions to the sample to form a plurality of double-stranded concatemers
Implementation Method 2
performing DNA amplification using a pair of primers, wherein a first primer of the pair of primers is complementary to a sequence in the first strand within the first repeat element and the second primer of the pair of primers is complementary to a sequence in the second strand within the second repeat element
Implementation Method 3
a first primer of the pair of primers is complementary to a sequence in the first strand within the first repeat element and the second primer of the pair of primers is complementary to a sequence in the second strand within the second repeat element
Data Source
AI summary
Presently described are methods for enriching regions of a genome in a sample using ligation of fragmented genomic nucleic acid and amplification using repeat elements. The methods can be used for a number of applications, including genome-wide homopolymer indel detection, and enable increasing the amount of information obtained from a limited sample of genomic nucleic acid.