Emulsion PCR for Large Nucleic Acid Linkage Analysis
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Solution Overview
Problem
Existing emulsion PCR techniques are unsuitable for analyzing large nucleic acids, such as whole chromosomes, due to their reliance on small DNA fragments, which limits their application in genotyping and sequencing of certain genotypes.
Innovation Solution
The method involves preparing large nucleic acid fragments or whole chromosomes through molecular combing to create uniformly stretched DNA molecules, which are then encapsulated in emulsion droplets for PCR, allowing for the analysis of genotypes characterized by allelic linkage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If small DNA fragments are used in emulsion PCR, then amplification efficiency is improved, but the ability to analyze large nucleic acid fragments and maintain allelic linkage is lost
Solution Approach 1:
The patent changes the physical parameters of the nucleic acid template by using large fragment shearing conditions that preserve fragments greater than 1000 base pairs, rather than standard fragmentation. This parameter change enables maintenance of allelic linkage while still allowing emulsion PCR amplification to proceed effectively.
2Loss of information
If large nucleic acid fragments are used in emulsion PCR, then allelic linkage analysis is improved, but amplification efficiency and sequencing suitability deteriorate
Solution Approach 1:
The patent segments the large nucleic acid fragments into smaller sub-fragments through controlled shearing, creating a distribution of fragment sizes. This segmentation preserves some fragments large enough to maintain allelic linkage (greater than 1000 base pairs) while creating smaller fragments that are more suitable for efficient amplification and sequencing, thus resolving the contradiction between maintaining linkage information and achieving amplification efficiency.
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 enables efficient sequencing and genotyping of large nucleic acids, facilitating the detection of genotypes like the 2+0 genotype, which cannot be distinguished by dosage analysis alone, and improving sequencing efficiency and accuracy.
Implementation Method 1
isolation of individual DNA molecules along with primer-coated beads in aqueous droplets within an oil phase
Implementation Method 2
creating an emulsion in which each drop of the emulsion contains an average of between about 0-2, 0-1.75, 0-1.5, 0-1.0, 0-0.75, 0-0.5, or fewer chromosomes or fragments
Implementation Method 3
A PCR step coats each bead with clonal copies of the DNA molecule which are then immobilized for later sequencing
Data Source
AI summary
The present invention provides methods for analyzing large nucleic acids including chromosomes and chromosomal fragments. In one aspect, the present invention provides a method of nucleic acid analysis comprising the steps of (a) obtaining a sample of nucleic acid comprising at least one chromosome or fragment greater than about 1,000 base pairs in length and containing a target region; (b) creating an emulsion in which each drop of the emulsion contains an average of between about 0-2, 0-1.75, 0-1.5, 0-1.0, 0-0.75, 0-0.5, or fewer chromosomes or fragments of step (a), (c) performing emulsion PCR, (d) quantifying the number of emulsion droplets containing amplified nucleic acid from the target region; (e) calculating the ratio of droplets containing amplified nucleic acid from the target region to total droplets; and (f) comparing the ratio of step (e) to a reference ratio representing a known genotype.