Restriction Site Tag Microarrays for SNP Discovery Without Genome Sequencing
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Solution Overview
Problem
Current methods for detecting large numbers of polymorphisms, especially single nucleotide polymorphisms (SNPs), are inefficient and often require prior genomic and cDNA sequence information, limiting their application to organisms with well-developed genomics infrastructure, and are not routine for detecting polymorphisms between individuals lacking thorough sequence data.
Innovation Solution
The use of restriction site tags isolated from genomic DNA, which are hybridized to microarrays or bead libraries, allows for the detection of polymorphisms through differential hybridization, enabling the identification of SNPs and other variants without the need for prior knowledge of the genome sequence, using techniques such as restriction site associated DNA (RAD) and primer extension methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high-throughput approaches using high-density oligonucleotide arrays are employed for SNP discovery, then the number of SNPs identified increases, but the requirement for prior genomic and cDNA sequence information and significant cost increases
Solution Approach 1:
The invention segments the genome into restriction fragments that can be individually analyzed. By using restriction enzymes to cut genomic DNA into manageable fragments and analyzing these segments separately through hybridization, the method enables SNP discovery without requiring complete genome sequencing or complex high-density arrays, thus reducing the genomics infrastructure requirement while maintaining SNP detection capability
Solution Approach 2:
The invention introduces restriction site tags as intermediary elements that bridge the gap between simple genomic DNA and complex genome-wide analysis. These tags serve as mediators that can be hybridized to detect SNPs without requiring direct whole-genome sequencing or complex array infrastructure, thereby reducing the required genomics infrastructure while preserving SNP detection accuracy
2Measurement precision
If comparative genome sequencing of lab populations is used to generate high-resolution SNP maps, then SNP mapping resolution improves, but the effort and cost make it unlikely that additional lines of interest will have SNPs discovered at high density
Solution Approach 1:
The invention creates a universal restriction fragment hybridization platform that can be applied to multiple different populations and organisms without requiring population-specific genome sequencing. The same basic methodology and probe sets can be used across different lab populations, enabling high-resolution SNP mapping in many more populations than traditional comparative sequencing approaches, thus increasing productivity while maintaining mapping resolution
Solution Approach 2:
The invention uses relatively simple and inexpensive restriction fragment probes instead of expensive whole-genome sequencing for each population. This cost-effective approach allows many more populations to be analyzed within the same resource constraints, increasing the number of populations that can be studied while maintaining adequate SNP mapping resolution through the hybridization detection method
3Measurement precision
If methods requiring prior genomic and cDNA sequence information are used, then SNP detection accuracy improves, but applicability to organisms without well-developed genomics infrastructure is limited
Solution Approach 1:
The invention enables organisms to essentially analyze themselves by using their own genomic DNA directly as the source of restriction fragments. The method requires no external reference genome or cDNA sequence information - the organism's own DNA is digested, tagged, and hybridized to detect SNPs. This self-service approach maintains SNP detection accuracy while dramatically expanding applicability to any organism regardless of whether it has a well-developed genomics infrastructure
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 routine detection of a large number of SNPs in any population of interest, using only genomic DNA and a few restriction endonucleases, allowing for high-resolution mapping and SNP discovery without the need for pre-existing SNP maps or sequenced genomes.
Implementation Method 1
contacting a preparation of genomic or other DNA with a restriction endonuclease; subjecting the preparation of genomic DNA shearing force to fragment the DNA
Implementation Method 2
hybridizing the tags from two individuals or samples to a nucleic acid array (or other detection platform, such as a collection of beads loaded with target nucleic acid molecules); hybridizing the tags from two individuals or samples to a collection of nucleic acid elements
Data Source
AI summary
Described are methods for the high-throughput discovery and genotyping of nucleotide polymorphisms in DNA, including single nucleotide polymorphism (SNPs) and short deletions and insertions. These methods take advantage of the fact that differences in DNA sequence result in the differential presence of restriction endonuclease digestion sites. Approaches involve isolation of short DNA fragments (“tags”) near restriction endonuclease sites. The presence of one (or two) of these tags indicates that a site was present. Regions of DNA with a restriction site in only one individual create an opportunity for primer extension to produce labeled material, which can be assayed on a platform that employs a collection of nucleic acids. Efficient variant detection microarrays and bead libraries are provided that contain genomic tags with different representations between two populations, so that most elements in the collection of nucleic acids contain a SNP between populations of interest.