ITE-PCR Primers for Human DNA Isolation from Microbial Contamination

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Solution Overview

Problem

Current methods for analyzing human DNA sequences in samples contaminated with microbial DNA, such as sputum and feces, face challenges due to interference from bacterial and viral DNA, which complicates the detection of cancer cells and genetic variations.

Innovation Solution

The use of Alu-, MIR-, and SVA-consensus sequence-based PCR primers for amplifying inter-transposable element (ITE) genomic segments through PCR, followed by massively parallel sequencing (MPS) on the AlmivaScan platform, allows for the isolation of human DNA sequences with minimal interference from microbial DNA.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional whole genome sequencing or whole exome sequencing is used to analyze human DNA, then comprehensive genomic information can be obtained, but the analysis is rendered difficult by the presence of bacterial and viral DNA in contaminated samples

Engineering Contradiction:

Improvedetection accuracy of human genomic variationsVSAvoidinterference from microbial DNA

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The invention extracts and amplifies only human-specific genomic regions containing transposable elements (Alu, LINE-1, SVA) from contaminated DNA samples. By using primers that specifically bind to human transposable element sequences, the method isolates human DNA signals from the background of microbial DNA, enabling accurate detection of human genomic variations despite sample contamination

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention uses transposable element sequences as intermediary markers to detect human DNA. These conserved human-specific sequences serve as mediators that allow selective amplification and detection of human genomic material in the presence of microbial DNA, bridging the gap between contaminated sample input and clean human DNA analysis

Inventive Principle:

Principle #24Intermediary (Mediator)

2Quantity of substance

If Alu-, MIR- and SVA-consensus sequence-based PCR primers are used to amplify human genomic DNA, then human-specific amplicons can be obtained even in the presence of bacterial and viral DNAs, but the primers must be designed to avoid amplification of microbial DNA to an extent that interferes significantly with sequence analysis

Engineering Contradiction:

Improveyield of human-specific ampliconsVSAvoidspecificity of amplification

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The invention designs primers with local specificity to human transposable element consensus sequences. Each primer is tailored to bind specifically to human Alu, LINE-1, or SVA sequences while avoiding microbial DNA sequences. This localized optimization of primer binding specificity ensures high-yield amplification of human amplicons without significant microbial DNA interference

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The invention optimizes PCR amplification parameters including primer concentration, annealing temperature, and cycle number to maximize human-specific amplicon yield while minimizing non-specific amplification. By adjusting these parameters, the method achieves high sensitivity for human DNA detection while maintaining reliability in the presence of microbial DNA

Inventive Principle:

Principle #35Parameter changes

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 the efficient analysis of human DNA in contaminated samples by producing high-yield, diverse amplicons that can identify genomic variations associated with traits and diseases, facilitating early cancer detection and monitoring.

Implementation Method 1

amplification of inter-transposable element (ITE) genomic segments using polynucleotide chain reaction (PCR) with Alu-, MIR- and SVA-consensus sequence-based PCR primers

Methodology Applied

Scientific EffectPolymerase chain reaction (PCR):

Implementation Method 2

sequence analysis of the resultant amplicons by means of massively parallel sequencing (MPS) in an 'AlmivaScan' sequencing platform

Methodology Applied

Scientific EffectMassively parallel sequencing (MPS):

Data Source

PatentUS11634774B2Genome-wide capture of inter-transposable element segments for genomic sequence analysis of human DNA samples with microbial contamination

Publication Date: 2023.04.25 PHARMACOGENETICS

AI summary

A method for identifying one or more genomic variations in human genomic DNA, comprising employment of Alu, MIR and SVA sequence-based PCR primers and performing an inter-transposable-element (ITE) polymerase chain reaction on the assay mixture to produce an array of amplicons comprising the ITE genomic segments. Also provided the use of the method for identifying one or more genomic variants associated with a trait or a disease.