SMART M-Seq Immunoglobulin Sequencing
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Solution Overview
Problem
Current methods for sequencing the variable region of immunoglobulin heavy and light chains in biological samples are hindered by the lack of a priori knowledge of gene fragments used and the presence of multiple B lymphocytes/plasma cells producing different immunoglobulins, leading to inaccuracies and artifactual sequences due to the use of DNA polymerases without proof-reading activity and error-prone bacterial amplification.
Innovation Solution
The SMART M-Seq method employs two-step reverse PCR with high-fidelity DNA polymerase and real-time sequencing of single DNA molecules to accurately amplify and sequence the variable region of immunoglobulins, allowing for the identification of whole sequences and classification based on relative abundance, using high-fidelity enzymes like Q5, Phusion, or Platinum Taq for precise amplification and Pacific Biosciences' SMRT technology for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If DNA polymerase without proof-reading activity is used for amplification, then cloning with TOPO TA system is enabled, but sequencing accuracy deteriorates due to increased risk of nucleotide misincorporation
Solution Approach 1:
The method separates the amplification and sequencing steps, using high-fidelity DNA polymerase for amplification to ensure accuracy, then using the 3' A-overhang feature for subsequent TOPO TA cloning. This segmentation allows optimization of each step for its specific purpose without compromise.
Solution Approach 2:
The method performs preliminary amplification with high-fidelity DNA polymerase that incorporates a 3' A-overhang, which is then used for cloning. This preliminary action ensures both high sequencing accuracy and compatibility with the cloning system before the actual sequencing occurs.
2Productivity
If bacterial transformation and amplification are used, then DNA replication occurs, but sequencing errors increase due to error-prone bacterial replicative apparatus
Solution Approach 1:
The method replaces the bacterial transformation and amplification system with direct Sanger sequencing of the PCR amplicon. This substitution eliminates the error-prone bacterial replicative apparatus while maintaining DNA amplification capability through high-fidelity PCR, thereby improving sequence fidelity.
3Adaptability or versatility
If multiple B lymphocytes/plasma cells producing different immunoglobulins are present, then biological sample complexity increases, but sequence identification accuracy decreases due to lack of a priori knowledge of gene fragments
Solution Approach 1:
The method employs universal primers that can amplify immunoglobulin variable regions from multiple different B lymphocytes and plasma cells simultaneously. This universality allows the method to handle complex samples with diverse immunoglobulin sequences while still enabling accurate identification of monoclonal sequences through their characteristic patterns.
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
SMART M-Seq provides accurate and reproducible identification of monoclonal immunoglobulin sequences with high fidelity, reducing errors and artifactual sequences, enabling the detection of clonal sequences in patients with monoclonal gammapathy, even in samples with low clone presence, and allowing parallel analysis of multiple samples.
Implementation Method 1
two-step reverse PCR with high-fidelity DNA polymerase for accurate amplification
Implementation Method 2
real-time sequencing of single DNA molecules, which allows the complete sequence of the variable region of one or more isotypes of the heavy and/or light chains of the immunoglobulins present in the biological sample, to be obtained
Data Source
AI summary
The present invention relates to a method for the identification of the whole nucleotide sequence of the variable region of the heavy and or light chains of immunoglobulins in a biological sample and the quantification of their relative frequency. The invention is particularly used for the identification of monoclonal heavy and light chains, i.e. tumours, in biological samples from patients suffering from a monoclonal gammapathy.