Multiplex TRFLP Analysis Using Fluorescently Labeled Primers
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Solution Overview
Problem
Current nucleic acid analysis techniques, such as TRFLP, are limited in their ability to simultaneously analyze multiple taxa, requiring multiple procedures and being time-consuming and expensive, with low resolution and inability to detect complex microbial communities effectively.
Innovation Solution
A method involving the use of multiple pairs of labelled primers complementary to marker sequences, followed by PCR amplification and restriction enzyme digestion, allowing for the simultaneous analysis of multiple taxa in a single reaction, using a DNA sequencer to detect terminal restriction fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional TRFLP method is used to analyze multiple taxa, then each taxon can be analyzed with good resolution, but multiple separate procedures are required which increases time and cost
Solution Approach 1:
The patent combines multiple TRFLP analyses for different taxa into a single multiplex PCR reaction by using primer pairs with different fluorescent labels. Each primer pair targets a specific taxon and is labeled with a unique fluorophore, allowing simultaneous amplification and detection of multiple taxa in one reaction tube, thereby eliminating the need for multiple separate procedures
Solution Approach 2:
The invention creates a universal analysis platform that can detect multiple taxa simultaneously using a single reaction system. The method uses a common set of reaction conditions and a single sequencing run to analyze diverse microbial communities, making the technique broadly applicable to various taxa without requiring taxon-specific optimization of entire protocols
2Measurement precision
If conventional TRFLP method is used to analyze multiple taxa, then each taxon can be analyzed with good resolution, but multiple separate procedures are required which increases cost
Solution Approach 1:
The patent combines multiple TRFLP analyses for different taxa into a single multiplex PCR reaction by using primer pairs with different fluorescent labels. Each primer pair targets a specific taxon and is labeled with a unique fluorophore, allowing simultaneous amplification and detection of multiple taxa in one reaction tube, thereby eliminating the need for multiple separate procedures
Solution Approach 2:
The invention employs different fluorescent labels (colors) on primer pairs to distinguish between different taxa. Each taxon-specific primer pair is tagged with a unique fluorophore that emits at a distinct wavelength, allowing the detection system to differentiate and quantify multiple taxa simultaneously based on their color signatures during a single analysis run
3Adaptability or versatility
If multiplex-PCR is used for detection of specific microbial strains, then specific strains can be detected, but prior knowledge of likely contaminants is required and resolution is limited
Solution Approach 1:
The patent creates a dynamic and flexible primer design system where primer pairs can be selectively added or removed from the multiplex reaction based on the specific analysis needs. The method allows adaptation to different taxa and research questions by simply changing which primer pairs are included, without requiring redesign of the entire system or prior specification of all possible targets
Solution Approach 2:
The invention uses fluorescently labeled primers as intermediaries that bridge the gap between specific taxon targets and the universal detection system. These labeled primers serve as mediators that enable specific binding to taxon-specific sequences while simultaneously providing a universal fluorescent signal that can be detected by the sequencing system, thus simplifying the overall detection architecture
4Measurement precision
If conventional TRFLP is used with multiple primer sets and enzymes for higher resolution, then resolution improves, but the approach becomes time consuming and expensive
Solution Approach 1:
The patent combines multiple TRFLP analyses for different taxa into a single multiplex PCR reaction by using primer pairs with different fluorescent labels. Each primer pair targets a specific taxon and is labeled with a unique fluorophore, allowing simultaneous amplification and detection of multiple taxa in one reaction tube, thereby eliminating the need for multiple separate procedures
Solution Approach 2:
The invention adds the dimension of fluorescent labeling to the traditional TRFLP approach. Instead of increasing resolution by adding more physical separation steps (more enzymes, more gels), the method uses spectral differentiation through fluorescent labels to achieve multiplexing, thus improving resolution and throughput without proportionally increasing procedural complexity
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
Enables rapid, sensitive, and cost-effective analysis of multiple microbial communities or target genes in one reaction, providing high reproducibility and reducing labor and time, while maintaining the ability to identify pathogens and their antibiotic resistance.
Implementation Method 1
the target gene is amplified using the polymerase chain reaction (PCR) using a 5' fluorescently tagged primer
Implementation Method 2
The amplification products are then digested with at least one restriction enzyme to give different length fragments
Implementation Method 3
The sequencer is able to detect and measure the fluorescently labelled fragments
Implementation Method 4
providing at least two pairs of labelled primers, wherein each said primer pair is complementary to a marker sequence in a nucleic acid of at least one member
Data Source
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AI summary
There is provided a method of nucleic acid analysis which allows analysis of genetic diversity in multiple populations to be performed rapidly and simultaneously. The method comprises a) isolating nucleic acid from said sample; b) providing at least two pairs of labelled primers, wherein each said primer pair is complementary to a marker sequence in a nucleic acid of at least one member; c) amplifying the nucleic acid; d) digesting the labelled amplified nucleic acid with at least one restriction enzyme to produce restriction fragments, and size sorting said fragments to produce a restriction fragment length profile, and e) analysing said restriction fragment length profile so obtained, wherein the primer pairs provided for each marker have a different sequence to the sequence of the primer pairs for each other marker, and wherein each said primer pair is uniquely labelled relative to the other primer pair(s). In one embodiment each primer pair is uniquely labelled at the 5' end with a fluorophore. The restriction fragments can be conveniently analysed by a DNA sequencer. The method of the invention has the advantage that it allows nucleic acid amplified using multiple marker sequences to be simultaneously analysed.