Haplotype Analysis via Enzymatic Cleavage and Digital PCR
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Solution Overview
Problem
Conventional genotyping technologies cannot accurately determine the combination of alleles at linked loci on the same chromosome, leading to underestimation of copy number variations and haplotype information, especially when multiple copies of a target nucleic acid are present on the same polynucleotide in digital polymerase chain reaction (dPCR) assays.
Innovation Solution
A method involving contacting a sample with an agent that cleaves specifically between target nucleic acids on the same polynucleotide, separating the sample into spatially isolated partitions, and enumerating the number of partitions containing the target nucleic acids to accurately determine copy number and haplotype information, using techniques such as digital PCR and restriction enzymes to physically separate and amplify target sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If digital PCR is used to determine copy number of target nucleic acid, then copy number can be quantified, but multiple copies on the same polynucleotide are underestimated as single copies
Solution Approach 1:
The method segments the polynucleotide by using site-specific endonuclease cleavage at a target site located between two target nucleic acid sequences. This physical separation converts linked copies into independent fragments that can be individually enumerated in digital PCR partitions, resolving the underestimation problem where multiple copies on the same polynucleotide were counted as a single copy.
2Measurement precision
If conventional genotyping technologies are used, then allele presence at individual loci can be detected, but haplotype information and allele combinations on the same chromosome cannot be determined
Solution Approach 1:
The method introduces a site-specific endonuclease as an intermediary that selectively cleaves polynucleotides at a specific target site between two target sequences. By comparing cleaved and uncleaved samples, the method infers haplotype information and determines whether alleles are in cis or trans configuration, thereby recovering phase information that conventional genotyping technologies cannot provide.
3Measurement precision
If target nucleic acids are physically separated by cleavage, then accurate copy number can be determined, but the process requires additional steps and reagents
Solution Approach 1:
The method changes the chemical state of the polynucleotide by introducing a site-specific endonuclease that cleaves at a specific recognition site between target sequences. This parameter change (from intact to cleaved) creates distinguishable populations that can be enumerated separately, enabling accurate copy number determination while maintaining a relatively simple digital PCR workflow.
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 effectively addresses the underestimation of copy number variations and improves haplotype determination by accurately separating and counting target nucleic acids, even when they are present in multiple copies on the same polynucleotide, enhancing the precision of genetic analysis.
Implementation Method 1
contacting the first subsample with an agent capable of physically separating the first target nucleic acid from the second target nucleic acid if they are present on the same polynucleotide
Implementation Method 2
subjecting the polynucleotides to an amplification reaction
Data Source
AI summary
Method of haplotype analysis. In an exemplary method, an aqueous phase containing nucleic acid may be partitioned into a plurality of discrete volumes. At least one allele sequence may be amplified in the volumes from each of a first polymorphic locus and a second polymorphic locus that exhibit sequence variation in the nucleic acid. At least one measure of co-amplification of allele sequences from both loci in the same volumes may be determined. A haplotype of the first and second loci may be selected based on the at least one measure of co-amplification.