Selective Adaptor Ligation for Genomic Complexity Reduction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genome analysis methods face challenges in reducing the complexity of genomic samples in a predictable and reproducible manner, particularly in identifying and genotyping single nucleotide polymorphisms (SNPs) for whole-genome association studies, which require analyzing hundreds of thousands of polymorphisms.
Innovation Solution
The use of selective adaptor ligation and amplification techniques involving restriction enzymes with degenerate recognition sites and complementary adaptors to fragment and amplify specific subsets of genomic DNA, allowing for targeted analysis of SNPs using allele-specific probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If whole-genome association studies analyze hundreds of thousands of polymorphisms, then the comprehensiveness of genetic analysis is improved, but the complexity of genomic samples becomes unmanageable
Solution Approach 1:
The patent segments the complex genomic sample into manageable subsets by using restriction enzymes with degenerate recognition sites to create fragments with specific overhang sequences. Selective adaptors are then ligated to these fragments, allowing segmentation of the genome into analyzable portions that can be processed systematically through amplification and array hybridization.
Solution Approach 2:
The patent extracts specific subsets of genomic DNA fragments that contain informative markers by using selective adaptors complementary to specific overhang sequences. This extraction process isolates the relevant fragments containing SNPs and other polymorphisms from the complex genomic background, enabling focused analysis of hundreds of thousands of polymorphisms.
2Device complexity
If selective adaptor ligation is used to reduce sample complexity, then the manageability of genomic samples is improved, but the predictability and reproducibility of complexity reduction becomes challenging
Solution Approach 1:
The patent changes the parameter of restriction enzyme recognition site degeneracy to control the complexity reduction process. By selecting enzymes with specific degenerate patterns (e.g., CCWWGG where W=A or T), the patent creates predictable overhang sequences that determine which adaptors will ligate, making the complexity reduction process reproducible and statistically predictable.
Solution Approach 2:
The patent incorporates feedback through the use of adaptors with known sequences that complement specific overhangs. The ligation process provides feedback about which fragments are successfully captured, allowing for standardized protocols that can be replicated and whose outcomes can be predicted based on the enzyme-adaptor pairing.
3Measurement precision
If restriction enzymes with degenerate recognition sites are used to fragment genomic DNA, then the specificity of marker selection is improved, but the difficulty of controlling fragment characteristics increases
Solution Approach 1:
The patent applies local quality by making the adaptor sequences specific to particular overhang types while keeping the restriction enzyme recognition sites degenerate. This creates a system where the adaptors provide local specificity to capture particular fragment types, while the degenerate enzyme sites provide broad coverage across the genome.
Solution Approach 2:
The patent introduces dynamics through the degenerate recognition sites that can accommodate multiple base pairs (e.g., CCWWGG where W=A or T). This dynamic recognition allows the same enzyme to generate multiple overhang types, providing flexibility in fragment generation while maintaining controllable specificity through adaptor selection.
4Reliability
If selective adaptors are ligated to fragments with variable overhangs, then the reproducibility of amplification is improved, but the complexity of adaptor selection and amplification protocols increases
Solution Approach 1:
The patent applies universality by designing adaptor sets that can be systematically applied across different restriction enzymes and overhang types. The adaptors follow a standardized structure that allows them to function with multiple enzyme-adaptor combinations, reducing the need for entirely new protocols for each application while maintaining reproducibility.
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 effectively reduces sample complexity, improves signal-to-noise ratios, and enables the genotyping of hundreds of thousands of SNPs, enhancing the specificity and reliability of detection while maintaining informative markers for association studies.
Implementation Method 1
a restriction enzyme that has a variable sequence in the recognition site is used to cleave the genomic DNA. The variable position is positioned so that when the enzyme cleaves the variable position is within the single stranded overhang that is generated
Implementation Method 2
Selective adaptors that are complementary to one or some of the overhangs but not complementary to all of the overhangs may be ligated to the fragments
Implementation Method 3
The adaptor ligated fragments can be amplified using primers complementary to the selective adaptor
Implementation Method 4
hybridized to an array of allele specific probes that are perfectly complementary to one allele of a known sequence variant
Data Source
AI summary
The present invention provides methods for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences. Complexity reduction can be accomplished by fragmenting the nucleic acid sample with a restriction enzyme that has at least one variable position in the recognition sequence. In some aspects adaptors that ligate to some but not all possible overhangs generated by digestion are ligated to the fragments. This selective adaptor ligation allows for selective amplification of a subset of the fragments using primers complementary to the adaptor sequence. In another aspect primers that are complementary to a subset of the fragments after adaptor ligation are used for amplification. Amplified fragments may be analyzed to genotype polymorphisms by hybridization to an array of probes that are complementary to target sequences that will be amplified.