SDL-PCR Gene Analysis Using Universal Primers
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Solution Overview
Problem
Current gene analysis methods, such as multiplex PCR, face limitations in amplifying multiple gene regions simultaneously due to cross-reactions and require complex experimental procedures, leading to contamination and inefficiencies in sensitivity and specificity.
Innovation Solution
The SDL-PCR method involves ligating probes complementary to a gene of interest with ligase, followed by an extension reaction to create a template probe, which is then separated and amplified using universal primers, allowing for accurate and rapid amplification of multiple genes in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex polymerase chain reaction is used to amplify multiple gene regions simultaneously, then the number of gene regions analyzed at the same time increases, but cross-reactions between primers occur and the number of amplifiable gene regions is limited
Solution Approach 1:
The method divides the gene analysis process into two distinct stages: (1) probe ligation stage where gene-specific probes are ligated to genomic DNA to create template probes, and (2) amplification stage where universal primers amplify all template probes simultaneously. This segmentation allows multiple gene regions to be analyzed without cross-reactions because each gene has its own ligation-specific template probe that is subsequently amplified by universal primers.
Solution Approach 2:
The patent introduces template probes as an intermediary between the gene-specific recognition step and the universal amplification step. These template probes are created by ligating gene-specific probes to genomic DNA, and then serve as templates for PCR amplification using universal primers. This intermediary structure enables specific gene regions to be selectively amplified without direct interaction between multiple gene-specific primers.
2Measurement precision
If conventional gene analysis methods are used, then amplification of a single gene region is accurate, but the same operation must be repeatedly performed for multiple genes
Solution Approach 1:
The method combines multiple gene analysis operations into a single reaction tube by using universal primers that can simultaneously amplify multiple different template probes. Each template probe contains a unique ligation junction specific to a particular gene region, allowing specific amplification of multiple genes in parallel without requiring separate reactions for each gene.
Solution Approach 2:
The patent employs universal primers that serve multiple functions: they can bind to and amplify template probes for different gene regions simultaneously. These universal primers are designed to recognize common sequences in the probe structures while the ligation-specific template probes provide gene-region specificity, enabling one set of primers to perform what would traditionally require multiple gene-specific primer sets.
3Ease of operation
If probe ligation and extension reactions are performed without separation, then the procedure is simpler, but non-specific amplification occurs reducing sensitivity and specificity
Solution Approach 1:
The method extracts and removes unligated probes and non-specific products from the reaction mixture using magnetic beads before the amplification step. This separation ensures that only the specifically ligated template probes remain for amplification, eliminating non-specific amplification and improving the sensitivity and specificity of the detection while maintaining procedural simplicity through magnetic bead-based separation.
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 method enables accurate and rapid amplification of multiple genes with reduced non-specific amplification and contamination, improving sensitivity and specificity compared to conventional methods.
Implementation Method 1
adding ligase thereto to ligate probe 1 and probe 2 with each other
Implementation Method 2
adding probe 3, DNA polymerase and dNTP to the mixture resulting from step (b) to hybridize probe 3 to probe 2, and extending probe 3
Implementation Method 3
followed by separation of the template probe
Implementation Method 4
amplifying the separated template probe of step (c) by a polymerase chain reaction (PCR) using the universal forward primer and the universal reverse primer
Data Source
AI summary
The present invention relates to a method for analyzing genes using SDL-PCR (separation of displaced ligation probe-based PCR), and more particularly to a method for analyzing genes using SDL-PCR, in which probes comprising a nucleotide sequence complementary to the gene of interest are ligated with each other by ligase, and another probe capable of hybridizing to the probes is hybridized and extended, thereby preparing a template probe, and the template probe for the gene of interest is amplified using universal primers.According to the SDL-PCR method of the present invention, non-specific amplification can be minimized by removing non-ligated probes or genomic DNA using a tag, and separation can be achieved within a shorter time compared to a separation method that is performed using exonuclease. In addition, ligation, separation and polymerase chain reaction processes can be performed in a single solution in a single tube, and thus a plurality of genes can be amplified at the same time in an accurate and rapid manner.