Nested primer sets for amplifying mouse immunoglobulin variable gene segments
Inventors
Devinder, Sehgal • Soma, Rohatgi
Assignees
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Publication Number
US-8143007-B2
Publication Date
2012-03-27
Expiration Date
Abstract
The present invention provides oligonucleotides for detection of rearrangement of immunoglobulin genes for identifying clonality of cells, cancer cells, hypermutation in immunoglobulin gene, antibody isotype producing cell and/or assaying B cell repertoire in a sample. The oligonucleotides disclosed in the present invention are very specific to the immunoglobulin genes.
Core Innovation
The invention is a nested (RT-)PCR primer system defined by a set of oligonucleotides (SEQ ID NO: 1-112) for amplifying rearranged and expressed mouse immunoglobulin variable gene families. The system targets immunoglobulin variable gene families including V_H, V_k, and V_l, and amplifies from genomic DNA or total RNA using (RT-)PCR. The oligonucleotides are designed to provide low degeneracy and strong binding characteristics while minimizing mismatches.
The design provides pooled-primers compatibility to support use as a nested (RT-)PCR system and reports low cross-family priming. Constant-region pooled primers enable isotype-specific amplification for antibody isotypes including IgM, IgG, and IgA and k/l. The design is described across mouse strains, including C57BL/6 and BALB/c, and is presented with sequencing-based confirmation of functional V-family targeting.
The system is experimentally validated for amplification from pooled hybridomas and microdissected B220+ cells, including single-cell and low-cell sensitivity described in the partial content. The reported use includes clonality detection and hypermutation analysis, as well as B-cell repertoire and isotype analysis, including mention of a B-cell lymphoma context. The overall problem addressed is assaying rearrangement of immunoglobulin genes to identify clonality and related features in samples.
Claims Coverage
The independent claims cover four aspects of the nested (RT-)PCR immunoglobulin gene assay: a defined oligonucleotide set, processes for assaying rearrangement, processes for constructing an immunoglobulin gene polynucleotide library via cloning and transforming, and a corresponding assay kit. Across these independent claims, the inventive features are grounded in explicitly enumerated SEQ ID NO oligonucleotide sets used for PCR workflows, amplification detection, and downstream library construction or kit composition.
Defined oligonucleotide set for assaying immunoglobulin gene rearrangement
A set of oligonucleotides comprising 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104; and 3′ oligonucleotides as set forth in SEQ ID NO: 35-37, SEQ ID NO: 94-95 and SEQ ID NO: 105-108; or 3′ oligonucleotides as set forth in SEQ ID NO: 38-54, SEQ ID NO: 96-98 and SEQ ID NO: 109-112, for identifying clonality of cells, cancer cells, hypermutation in immunoglobulin gene, antibody isotype producing cell and/or assaying B cell repertoire in a sample.
Process for assaying immunoglobulin gene rearrangement using the SEQ ID oligonucleotide sets with PCR and detection
A process of assaying rearrangement of immunoglobulin genes for identifying clonality of cells, cancer cells, hypermutation in immunoglobulin gene, antibody isotype producing cell and/or assaying B cell repertoire in a sample, comprising providing a sample; providing 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104; providing 3′ oligonucleotides as set forth in SEQ ID NO: 35-37, SEQ ID NO: 94-95 and SEQ ID NO: 105-108; or 3′ oligonucleotides as set forth in SEQ ID NO: 38-54, SEQ ID NO: 96-98 and SEQ ID NO: 109-112; performing a polymerase chain reaction; and detecting presence of an amplified product.
Two-round PCR and RT-based variant using enumerated SEQ ID oligonucleotides with amplified-product detection
A process comprising providing a sample; providing a first set of oligonucleotides as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105 and SEQ ID NO: 106; performing a first round of polymerase chain reaction to obtain first product; performing second round of polymerase chain reaction using said first product and oligonucleotides as set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 107 and SEQ ID NO: 108; and detecting presence of an amplified product; or comprising an RT reaction using oligonucleotides as set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 96 and SEQ ID NO: 109, followed by providing a first set of oligonucleotides and performing first and second round polymerase chain reaction using the specified oligonucleotides and detecting presence of an amplified product.
Assay kit comprising the SEQ ID oligonucleotide sets for immunoglobulin gene rearrangement clonality analysis
A kit for assaying rearrangement of immunoglobulin genes for identifying clonality of cells, cancer cells, hypermutation in immunoglobulin gene, antibody isotype producing cell and/or assaying B cell repertoire in a sample, wherein the kit comprises a set of oligonucleotides comprising 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104 and 3′ oligonucleotides as set forth in SEQ ID NO: 35-37, SEQ ID NO: 94-95 and SEQ ID NO: 105-108; or 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104 and 3′ oligonucleotides as set forth in SEQ ID NO: 38-54, SEQ ID NO: 96-98 and SEQ ID NO: 109-112.
Library construction from amplified immunoglobulin genes using SEQ ID oligonucleotide sets with cloning and transformation
A process for constructing a library of polynucleotides encoding immunoglobulin genes, comprising amplifying immunoglobulin genes using a set of oligonucleotides comprising 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104 and 3′ oligonucleotides as set forth in SEQ ID NO: 35-37, SEQ ID NO: 94-95 and SEQ ID NO: 105-108; or 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104 and 3′ oligonucleotides as set forth in SEQ ID NO: 38-54, SEQ ID NO: 96-98 and SEQ ID NO: 109-112 to obtain amplified product; cloning said amplified product in an expression vector to obtain a recombinant expression vector; and transforming said recombinant expression vector in a host cell.
Overall, the claim coverage is centered on a specifically defined oligonucleotide set (SEQ ID NO ranges) for assaying immunoglobulin gene rearrangement, PCR-based workflows that detect amplified products, including a two-round PCR format and an RT-based variant, and downstream library construction via cloning into an expression vector and transforming into a host cell, plus a corresponding kit composition containing the SEQ ID oligonucleotides.
Stated Advantages
Low degeneracy.
High binding with ≤2 mismatches.
Majority ≤4-fold degeneracy.
Low cross-family priming.
Isotype-specific amplification enabled by constant-region pooled primers for IgM/IgG/IgA and κ/λ.
Compatibility across mouse strains including C57BL/6 and BALB/c.
Functional V-family targeting confirmed by sequencing-based confirmation.
Support for clonality detection, hypermutation analysis, and B-cell repertoire and isotype analysis.
Single-cell/low-cell sensitivity described for amplification from microdissected B220+ cells.
Documented Applications
Assaying rearrangement of immunoglobulin genes for identifying clonality of cells.
Assaying immunoglobulin gene hypermutation.
Detecting antibody isotype producing cell and analyzing antibody isotypes (IgM/IgG/IgA and κ/λ).
Assaying B cell repertoire in a sample.
Amplification from pooled hybridomas.
Amplification from microdissected B220+ cells, including single-cell/low-cell sensitivity.
Application context includes B-cell lymphoma.
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