# Patent

## Title
Nested primer sets for amplifying mouse immunoglobulin variable gene segments

## 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.

## Background
CLAIM OF PRIORITY
The present patent application claims the priority benefit of the filing date of Indian Application No. 634/DEL/2008 filed Mar. 13, 2008, the entire content of which is incorporated herein by reference in its entirety.
FIELD OF INVENTION
The present invention concerns the fields of immunology. More specifically, the present invention relates to oligonucleotides for detection of rearrangement of immunoglobulin genes.
BACKGROUND OF THE INVENTION
Humoral immunity relies on the specific recognition and elimination of foreign antigen (Ag), brought about by immunoglobulin (Ig) Ag receptors present on B cells. The humoral immune system needs a vast and diverse array of antibody (Ab) molecules to specifically recognize the extensive and varied world of potential Ags. It achieves this in part by encoding a large array of germline variable (V), diversity (D) and joining (J) gene segments. The germline, junctional and combinatorial diversity, together with Ag dependent changes such as somatic hypermutation and receptor editing, collectively produce a potentially vast diversity of V gene sequences.
The specialized microenvironments present in secondary lymphoid organs like the spleen and lymph nodes, where T-cell dependent Ab responses occur, are referred to as germinal centers. In germinal centers, Ag specific B cells undergo clonal expansion, somatic hypermutation, isotype class switching, affinity maturation, Ag-driven selection and differentiation into memory B cells or plasma cells.
PCR technology has been used for studying various aspects of B cell biology such as B cell repertoire analysis, expression of Ab using phage display systems and detection of B cell clonality in diagnostic haematopathology. Using PCR technology, numerous investigators have tried to devise a universal primer or set of primers for amplifying all possible mouse V gene rearrangements (Chiang et al., 1989; LeBoeuf et al., 1989; Orlandi et al., 1989; Larrick et al., 1990; Coloma et al., 1991; Kettleborough et al., 1993; Nicholls et al., 1993; Zhou et al., 1994; Dattamajumdar et al., 1996; Lan et al., 1996; Krebber et al., 1997). These primers, degenerate or otherwise, were however not successful in amplifying all the V gene segments (Owens and Young, 1994; Ruberti et al., 1994; Wang et al., 2006). Several workers have tried to overcome the shortcomings of the earlier efforts by designing a few, highly degenerate (1- to 128-fold or more degeneracy) primers (Jones and Bendig, 1991; Wang et al., 2000; Essono et al., 2003). In general, highly degenerate primers have higher propensity to lead to cross-family amplification. An alternative approach involves designing a panel of primers with minimum degeneracy (Chardes et al., 1999; Seijen et al., 2001). These studies (Chiang et al., 1989; LeBoeuf et al., 1989; Orlandi et al., 1989; Larrick et al., 1990; Coloma et al., 1991; Jones and Bendig, 1991; Kettleborough et al., 1993; Nicholls et al., 1993; Owens and Young, 1994; Ruberti et al., 1994; Zhou et al., 1994; Dattamajumdar et al., 1996; Lan et al., 1996; Krebber et al., 1997; Chardes et al., 1999; Wang et al., 2000; Seijen et al., 2001; Essono et al., 2003; Wang et al., 2006) designed primers for a standard PCR. Further, they do not report the PCR detection limit achieved using their primer set which is unlikely to be sensitive enough to be able to amplify the rearranged VH and VL genes from a single or a small number of B cells. To enhance the sensitivity and specificity of the PCR for analysis of the rearranged VH and VL genes from single B cells, researchers working on the human and rabbit systems have resorted to a nested PCR strategy (Brezinschek et al., 1995; Foster et al., 1997; Sehgal et al., 1998; Farner et al., 1999; Dohmen et al., 2005).
The major challenge in using PCR technology for studying mouse Ab genes, whether expressed in homogeneous hybridoma cell lines or a heterogeneous splenic B cell population, is to ensure broad and highly specific amplification. This need arises due to the fact that the mouse V genes show intra- and inter-family sequence variability, and somatically rearrange to generate nearly limitless Ab diversity. Additionally, somatic hypermutation events that coincide with the primer binding sites can potentially adversely affect the efficiency of amplification.
In order to study V gene usage, somatic hypermutation patterns, clonal expansion and selection in Ag-specific splenic B cells in experimental system, one would require a PCR primer set and an amplification strategy that would potentially amplify all possible rearranged V genes from a single or a small number of B cells in a highly sensitive and specific manner. A nested primer set that can potentially amplify the rearranged VH and VL genes belonging to any V gene family from single or small number of mouse B cells in a representative and highly specific manner has not been reported.
The present invention discloses a novel nested PCR primer set that amplifies all possible mouse rearranged/expressed VH, Vκ and Vλ genes from C57BL/6 and BALB/c mice in an isotype-independent manner with minimum cross reactivity, low degeneracy, high specificity and sensitivity, using genomic DNA or total RNA from small number of B cells.
SUMMARY OF THE INVENTION
The present invention relates to 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 provided in the present invention amplify all possible mouse rearranged/expressed VH, Vκ and Vλ genes from C57BL/6 and BALB/c mice in an isotype-independent manner with minimum cross reactivity, low degeneracy, high specificity and sensitivity, using genomic DNA or total RNA from small number of B cells.
One aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 112.
Another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 1: SEQ ID NO: 1 to 37, SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108; and set 44: SEQ ID NO: 1 to 34, SEQ ID NO: 38 to 93, SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112.
Another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 2: SEQ ID NO: 1 to 37, set 3: SEQ ID NO: 1 to 4 and SEQ ID NO: 35-37, set 4: SEQ ID NO: 5 to 6 and SEQ ID NO: 35 to 37, set 5: SEQ ID NO: 7 to 8 and SEQ ID NO: 35-37, set 6: SEQ ID NO: 9 to 10 and SEQ ID NO: 35-37, set 7: SEQ ID NO: 11 to 12 and SEQ ID NO: 35-37; set 8: SEQ ID NO: 13 to 14 and SEQ ID NO: 35-37; set 9: SEQ ID NO: 15 to 16 and SEQ ID NO: 35-37; set 10: SEQ ID NO: 17 to 18 and SEQ ID NO: 35-37; set 11: SEQ ID NO: 19 to 20 and SEQ ID NO: 35-37; set 12: SEQ ID NO: 21-22 and SEQ ID NO: 35-37; set 13: SEQ ID NO: 23 to 24 and SEQ ID NO: 35-37; set 14: SEQ ID NO: 25 to 26 and SEQ ID NO: 35-37; set 15: SEQ ID NO: 27 to 28 and SEQ ID NO: 29 to 30; set 16: SEQ ID NO: 29 to 30 and SEQ ID NO: 35-37; set 17: SEQ ID NO: 31 to 32 and SEQ ID NO: 35-37; and set 18: SEQ ID NO: 33-37.
Still another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides are as set forth in set 19: SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108.
Still another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 20: SEQ ID NO: 55 to 95; set 21: SEQ ID NO: 55 to 57 and SEQ ID NO: 94 to 95; set 22: SEQ ID NO: 58 to 59 and SEQ ID NO: 94 to 95; set 23: SEQ ID NO: 60 to 61 and SEQ ID NO: 94 to 95; set 24: SEQ ID NO: 62 to 63 and SEQ ID NO: 94 to 95; set 25: SEQ ID NO: 64 to 65 and SEQ ID NO: 94 to 95; set 26: SEQ ID NO: 66 to 67 and SEQ ID NO: 94 to 95; set 27: SEQ ID NO: 68 to 69 and SEQ ID NO: 94 to 95; set 28: SEQ ID NO: 70 to 71 and SEQ ID NO: 94 to 95; set 29: SEQ ID NO: 72 to 73 and SEQ ID NO: 94 to 95; set 30: SEQ ID NO: 74 to 75 and SEQ ID NO: 94 to 95; set 31: SEQ ID NO: 76 to 77 and SEQ ID NO: 94 to 95; set 32: SEQ ID NO: 78 to 79 and SEQ ID NO: 94 to 95; set 33: SEQ ID NO: 80 to 81 and SEQ ID NO: 94 to 95; set 34: SEQ ID NO: 82 to 83 and SEQ ID NO: 94 to 95; set 35: SEQ ID NO: 84 to 85 and SEQ ID NO: 94 to 95; set 36: SEQ ID NO: 86 to 87 and SEQ ID NO: 94 to 95; set 37: SEQ ID NO: 88 to 89 and SEQ ID NO: 94 to 95; set 38: SEQ ID NO: 90 to 91 and SEQ ID NO: 94 to 95; and set 39: SEQ ID NO: 92 to 95.
Still another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 40: SEQ ID NO: 99 to 108; set 41: SEQ ID NO: 99 to 100 and SEQ ID NO: 105 to 108; set 42: SEQ ID NO: 101 to 102 and SEQ ID NO: 105 to 108; and set 43: SEQ ID NO: 103 to 108.
Further aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 45: SEQ ID NO: 1 to 34, SEQ ID NO: 38-54; set 46: SEQ ID NO: 1 to 34, SEQ ID NO: 38, SEQ ID NO: 43 to 44; set 47: SEQ ID NO: 1 to 34, SEQ ID NO: 39, SEQ ID NO: 45 to 48; set 48: SEQ ID NO: 1 to 34, SEQ ID NO: 40, SEQ ID NO: 49 to 50; set 49: SEQ ID NO: 1 to 34, SEQ ID NO: 41, SEQ ID NO: 51 to 52; set 50: SEQ ID NO: 1 to 34, SEQ ID NO: 42, SEQ ID NO: 53 to 54; set 51: SEQ ID NO: 1 to 4 and SEQ ID NO: 38 to 54; set 52: SEQ ID NO: 5 to 6 and SEQ ID NO: 38 to 54; set 53: SEQ ID NO: 7 to 8 and SEQ ID NO: 38 to 54; set 54: SEQ ID NO: 9 to 10 and SEQ ID NO: 38 to 54; set 55: SEQ ID NO: 11 to 12 and SEQ ID NO: 38 to 54; set 56: SEQ ID NO: 13 to 14 and SEQ ID NO: 38 to 54; set 57: SEQ ID NO: 15 to 16 and SEQ ID NO: 38 to 54; set 58: SEQ ID NO: 17 to 18 and SEQ ID NO: 38 to 54; set 59: SEQ ID NO: 19 to 20 and SEQ ID NO: 38 to 54; set 60: SEQ ID NO: 21 to 22 and SEQ ID NO: 38 to 54; set 61: SEQ ID NO: 23 to 24 and SEQ ID NO: 38 to 54; set 62: SEQ ID NO: 25 to 26 and SEQ ID NO: 38 to 54; set 63: SEQ ID NO: 27 to 28 and SEQ ID NO: 38 to 54; set 64: SEQ ID NO: 29 to 30 and SEQ ID NO: 38 to 54; set 65: SEQ ID NO: 31 to 32 and SEQ ID NO: 38 to 54; and set 66: SEQ ID NO: 33 to 34 and SEQ ID NO: 38 to 54.
Another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 67: SEQ ID NO: 55 to 93; SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112; set 68: SEQ ID NO: 55 to 93 and SEQ ID NO: 96 to 98, set 69: SEQ ID NO: 55 to 57 and SEQ ID NO: 96 to 98, set 70: SEQ ID NO: 58 to 59 and SEQ ID NO: 96 to 98, set 71: SEQ ID NO: 60 to 61 and SEQ ID NO: 96 to 98, set 72: SEQ ID NO: 62 to 63 and SEQ ID NO: 96 to 98, set 73: SEQ ID NO: 64 to 65 and SEQ ID NO: 96 to 98, set 74: SEQ ID NO: 66 to 67 and SEQ ID NO: 96 to 98, set 75: SEQ ID NO: 68 to 69 and SEQ ID NO: 96 to 98, set 76: SEQ ID NO: 70 to 71 and SEQ ID NO: 96 to 98, set 77: SEQ ID NO: 72 to 73 and SEQ ID NO: 96 to 98, set 78: SEQ ID NO: 74 to 75 and SEQ ID NO: 96 to 98, set 79: SEQ ID NO: 76 to 77 and SEQ ID NO: 96 to 98, set 80: SEQ ID NO: 78 to 79 and SEQ ID NO: 96 to 98; set 81: SEQ ID NO: 80 to 81 and SEQ ID NO: 96 to 98; set 82: SEQ ID NO: 82 to 83 and SEQ ID NO: 96 to 98; set 83: SEQ ID NO: 84 to 85 and SEQ ID NO: 96 to 98; set 84: SEQ ID NO: 86 to 87 and SEQ ID NO: 96 to 98; set 85: SEQ ID NO: 88 to 89 and SEQ ID NO: 96 to 98; set 86: SEQ ID NO: 90 to 91 and SEQ ID NO: 96 to 98; set 87: SEQ ID NO: 92 to 93 and SEQ ID NO: 96 to 98; set 88: SEQ ID NO: 99 to 104 and SEQ ID NO: 109 to 112; set 89: SEQ ID NO: 99 to 100 and SEQ ID NO: 109 to 112; set 90: SEQ ID NO: 101 to 102 and SEQ ID NO: 109 to 112; and set 91: SEQ ID NO: 103 to 104 and SEQ ID NO: 109 to 112.
Another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 54.
Yet another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 55 to 98.
Still another aspect of the present invention relates to a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 98 to 112.
Still another aspect of the present invention relates to 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 using the set of oligonucleotides as set forth in SEQ ID NO: 1-112.
Still another aspect of the present invention relates to 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; the process comprises 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: 1, 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 oligonucleotide 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.
Still another aspect of the present invention relates to 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; the process comprises 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 oligonucleotide 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: SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO: 54; detecting presence of an amplified product.
Still another aspect of the present invention relates to a process for constructing library of polynucleotides encoding immunoglobulin genes, said process comprises amplifying immunoglobulin genes using the oligonucleotide sequences as set forth in SEQ ID NO: 1-112 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.
Still another aspect of the present invention relates to 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 said kit comprises nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 112.
Still another aspect of the present invention relates to 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 1: SEQ ID NO: 1 to 37, SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108; and set 44: SEQ ID NO: 1 to 34, SEQ ID NO: 38 to 93, SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112.
Still another aspect of the present invention relates to 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 2: SEQ ID NO: 1 to 37, set 3: SEQ ID NO: 1 to 4 and SEQ ID NO: 35-37, set 4: SEQ ID NO: 5 to 6 and SEQ ID NO: 35 to 37, set 5: SEQ ID NO: 7 to 8 and SEQ ID NO: 35-37, set 6: SEQ ID NO: 9 to 10 and SEQ ID NO: 35-37, set 7: SEQ ID NO: 11 to 12 and SEQ ID NO: 35-37; set 8: SEQ ID NO: 13 to 14 and SEQ ID NO: 35-37; set 9: SEQ ID NO: 15 to 16 and SEQ ID NO: 35-37; set 10: SEQ ID NO: 17 to 18 and SEQ ID NO: 35-37; set 11: SEQ ID NO: 19 to 20 and SEQ ID NO: 35-37; set 12: SEQ ID NO: 21-22 and SEQ ID NO: 35-37; set 13: SEQ ID NO: 23 to 24 and SEQ ID NO: 35-37; set 14: SEQ ID NO: 25 to 26 and SEQ ID NO: 35-37; set 15: SEQ ID NO: 27 to 28 and SEQ ID NO: 29 to 30; set 16: SEQ ID NO: 29 to 30 and SEQ ID NO: 35-37; set 17: SEQ ID NO: 31 to 32 and SEQ ID NO: 35-37; and set 18: SEQ ID NO: 33-37.
Still another aspect of the present invention relates to 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 said kit comprises set 19: SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108.
Still another aspect of the present invention relates to 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 20: SEQ ID NO: 55 to 95; set 21: SEQ ID NO: 55 to 57 and SEQ ID NO: 94 to 95; set 22: SEQ ID NO: 58 to 59 and SEQ ID NO: 94 to 95; set 23: SEQ ID NO: 60 to 61 and SEQ ID NO: 94 to 95; set 24: SEQ ID NO: 62 to 63 and SEQ ID NO: 94 to 95; set 25: SEQ ID NO: 64 to 65 and SEQ ID NO: 94 to 95; set 26: SEQ ID NO: 66 to 67 and SEQ ID NO: 94 to 95; set 27: SEQ ID NO: 68 to 69 and SEQ ID NO: 94 to 95; set 28: SEQ ID NO: 70 to 71 and SEQ ID NO: 94 to 95; set 29: SEQ ID NO: 72 to 73 and SEQ ID NO: 94 to 95; set 30: SEQ ID NO: 74 to 75 and SEQ ID NO: 94 to 95; set 31: SEQ ID NO: 76 to 77 and SEQ ID NO: 94 to 95; set 32: SEQ ID NO: 78 to 79 and SEQ ID NO: 94 to 95; set 33: SEQ ID NO: 80 to 81 and SEQ ID NO: 94 to 95; set 34: SEQ ID NO: 82 to 83 and SEQ ID NO: 94 to 95; set 35: SEQ ID NO: 84 to 85 and SEQ ID NO: 94 to 95; set 36: SEQ ID NO: 86 to 87 and SEQ ID NO: 94 to 95; set 37: SEQ ID NO: 88 to 89 and SEQ ID NO: 94 to 95; set 38: SEQ ID NO: 90 to 91 and SEQ ID NO: 94 to 95; and set 39: SEQ ID NO: 92 to 95.
Still another aspect of the present invention relates to 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 40: SEQ ID NO: 99 to 108; set 41: SEQ ID NO: 99 to 100 and SEQ ID NO: 105 to 108; set 42: SEQ ID NO: 101 to 102 and SEQ ID NO:105 to 108; and set 43: SEQ ID NO: 103 to 108.
Still another aspect of the present invention relates to 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 45: SEQ ID NO: 1 to 34, SEQ ID NO: 38-54; set 46: SEQ ID NO: 1 to 34, SEQ ID NO: 38, SEQ ID NO: 43 to 44; set 47: SEQ ID NO: 1 to 34, SEQ ID NO: 39, SEQ ID NO: 45 to 48; set 48: SEQ ID NO: 1 to 34, SEQ ID NO: 40, SEQ ID NO: 49 to 50; set 49: SEQ ID NO: 1 to 34, SEQ ID NO: 41, SEQ ID NO: 51 to 52; set 50: SEQ ID NO: 1 to 34, SEQ ID NO: 42, SEQ ID NO: 53 to 54; set 51: SEQ ID NO: 1 to 4 and SEQ ID NO: 38 to 54; set 52: SEQ ID NO: 5 to 6 and SEQ ID NO: 38 to 54; set 53: SEQ ID NO: 7 to 8 and SEQ ID NO: 38 to 54; set 54: SEQ ID NO: 9 to 10 and SEQ ID NO: 38 to 54; set 55: SEQ ID NO: 11 to 12 and SEQ ID NO: 38 to 54; set 56: SEQ ID NO: 13 to 14 and SEQ ID NO: 38 to 54; set 57: SEQ ID NO: 15 to 16 and SEQ ID NO: 38 to 54; set 58: SEQ ID NO: 17 to 18 and SEQ ID NO: 38 to 54; set 59: SEQ ID NO: 19 to 20 and SEQ ID NO: 38 to 54; set 60: SEQ ID NO: 21 to 22 and SEQ ID NO: 38 to 54; set 61: SEQ ID NO: 23 to 24 and SEQ ID NO: 38 to 54; set 62: SEQ ID NO: 25 to 26 and SEQ ID NO: 38 to 54; set 63: SEQ ID NO: 27 to 28 and SEQ ID NO: 38 to 54; set 64: SEQ ID NO: 29 to 30 and SEQ ID NO: 38 to 54; set 65: SEQ ID NO: 31 to 32 and SEQ ID NO: 38 to 54; and set 66: SEQ ID NO: 33 to 34 and SEQ ID NO: 38 to 54.
Still another aspect of the present invention relates to 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 67: SEQ ID NO: 55 to 93; SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112; set 68: SEQ ID NO: 55 to 93 and SEQ ID NO: 96 to 98, set 69: SEQ ID NO: 55 to 57 and SEQ ID NO: 96 to 98, set 70: SEQ ID NO: 58 to 59 and SEQ ID NO: 96 to 98, set 71: SEQ ID NO: 60 to 61 and SEQ ID NO: 96 to 98, set 72: SEQ ID NO: 62 to 63 and SEQ ID NO: 96 to 98, set 73: SEQ ID NO: 64 to 65 and SEQ ID NO: 96 to 98, set 74: SEQ ID NO: 66 to 67 and SEQ ID NO: 96 to 98, set 75: SEQ ID NO: 68 to 69 and SEQ ID NO: 96 to 98, set 76: SEQ ID NO: 70 to 71 and SEQ ID NO: 96 to 98, set 77: SEQ ID NO: 72 to 73 and SEQ ID NO: 96 to 98, set 78: SEQ ID NO: 74 to 75 and SEQ ID NO: 96 to 98, set 79: SEQ ID NO: 76 to 77 and SEQ ID NO: 96 to 98, set 80: SEQ ID NO: 78 to 79 and SEQ ID NO: 96 to 98; set 81: SEQ ID NO: 80 to 81 and SEQ ID NO: 96 to 98; set 82: SEQ ID NO: 82 to 83 and SEQ ID NO: 96 to 98; set 83: SEQ ID NO: 84 to 85 and SEQ ID NO: 96 to 98; set 84: SEQ ID NO: 86 to 87 and SEQ ID NO: 96 to 98; set 85: SEQ ID NO: 88 to 89 and SEQ ID NO: 96 to 98; set 86: SEQ ID NO: 90 to 91 and SEQ ID NO: 96 to 98; set 87: SEQ ID NO: 92 to 93 and SEQ ID NO: 96 to 98; set 88: SEQ ID NO: 99 to 104 and SEQ ID NO: 109 to 112; set 89: SEQ ID NO:99 to 100 and SEQ ID NO: 109 to 112; set 90: SEQ ID NO: 101 to 102 and SEQ ID NO: 109 to 112; and set 91: SEQ ID NO: 103 to 104 and SEQ ID NO: 109 to 112.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows schematic diagram outlining the nested (RT-) PCR based strategy used. The (RT-) PCR strategy for amplifying the mouse rearranged/expressed VH genes using genomic DNA from splenocytes (A) and total RNA from splenocytes or hybridomas (B) as starting template is shown.
FIG. 2 shows PCR amplification of V gene families from mouse splenocyte genomic DNA. PCR products obtained for individual VH (A), Vκ (B) and Vλ (C) gene families (or genes where the family has only one member) using primers having nucleotide sequence as set in SEQ ID NO: 1-112. The name of the VH, Vκ and Vλ gene family is indicated. ‘1⁄2’ in (C) denotes Vλ1 and Vλ2 genes; M, molecular weight marker (766, 500, 300, 150 and 50 bp).
FIG. 3 shows amplification of the rearranged V genes using pooled primers. Nested PCR was performed to amplify the rearranged V genes using genomic DNA from mouse splenocytes and 3 separate cocktails comprising of primers targeting all VH (lane 1), Vκ (lane 3) and Vλ (lane 5) gene families. The corresponding negative controls (water only) are in lanes 2, 4 and 6, respectively; M, molecular weight marker (766, 500, 300, 150 and 50 bp).
FIG. 4 shows determination of detection limit of the designed primers using RT-PCR. The expressed Vκ gene was amplified using Vκ pooled primer mix, Cκ primer and decreasing numbers of hybridoma cells (100 to 1 cell) (A). The corresponding primers used for the expressed VH gene were pooled VH and Cμ primers (B). ‘No cell’ and ‘no reverse transcriptase’ negative controls are shown; M, molecular weight marker (766, 500, 300, 150 and 50 bp).
FIG. 5 shows PCR amplification of rearranged VH and Vκ genes from immunohistochemically stained B cells microdissected from splenic sections from naïve BALB/c mice.



DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a nested PCR targeted to mouse rearranged/expressed immunoglobulin V gene family comprising VH, Vκ, Vλ, JH, Jκ, Jλ, CH, Cκ and Cλ genes assay B cell repertoire and/or isotyping antibody producing cells from a sample. The nested PCR disclosed in the present invention is a specific and sensitive tool for amplification of mouse rearranged/expressed immunoglobulin V gene family.
The present invention provides a novel highly specific and sensitive universal nested PCR primer set for (RT-) PCR capable of potentially amplifying the rearranged/expressed VH and VL gene belonging to any mouse immunoglobulin V gene family from a single or a small number of B cells.
A database of germline immunoglobulin sequences was used to design 112 primers (SEQ ID NO:1-12) for a nested (RT-) PCR based strategy to cover all VH, VL, JH, JL, CH and CL gene families/genes from C57BL/6 and BALB/c mice. 93.7% of the primers had 4-fold or less, while 71.4% had no degeneracy. The proportions of germline V genes to which the primers bind with no, up to 1 and up to 2 mismatches are 59.7%, 84.1% and 94.9%, respectively.
We have validated the broad V gene family coverage provided by this set of 112 (SEQ ID NO: 1-112) primers by amplifying the rearranged/expressed V genes from splenocytes and a panel of 38 hybridomas under conditions where pooled primers and genomic DNA or total RNA was used as starting template.
We experimentally confirmed the V gene family-specific nature of the designed primers for 6 randomly selected VH, 6 Vκ and 2 Vλ families. Cocktail of constant region specific primers allowed efficient RT-PCR amplification of IgM, IgG, IgA, Igκ and Igλ isotypes.
The primers disclosed in the present invention permitted RT-PCR amplification of IgM, IgG, IgA, Igκ and Igλ isotypes. The broad V gene family coverage and single cell level sensitivity offered by our primer set can be taken advantage of to study B cell repertoire by single cell RT-PCR. Analysis at the single cell level avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells (Wang and Stollar, 2000).
The primers disclosed in the present invention cover all V genes, with the exception of a few pseudogenes. The V gene primers are compatible with all the J- and C region primers. As a consequence of the high sensitivity of the assay, RT-PCR or genomic PCR can be done directly with small number of hybridoma or B cells thus bypassing the need and effort to isolate total RNA or genomic DNA. The amount of RT-PCR amplified product obtained from hybridoma cells is sufficient for sequencing with the CH or CL internal primer thus doing away with the cloning step. Due to the high sensitivity and specificity, and broad V gene family coverage, the designed primers have the potential to be used for studying various mouse B cell subsets, to follow progress of B cell lymphomas and test the heterogeneity of B cell populations at the single cell level (Jacob et al., 1991a; Jacob et al., 1991b; Kim and Berek, 2007; Tiller et al., 2007). It would now be possible to amplify and sequence V genes from small numbers of B cells recovered from lesions or during the course of infection. This will facilitate the study of anti-infectious disease Ab repertoire.
The present invention discloses a functional set of nested primers having nucleotide sequence as set forth in SEQ ID NO: 1-112 that cover all mouse VH, Vκ, Vλ, JH, Jκ, Jλ, CH, Cκ, and Cλ genes. The primer set and the PCR strategy permits amplification of essentially all rearranged VH and VL gene families from C57BL/6 and BALB/c mice from either genomic DNA or transcript in a highly sensitive and V gene specific manner. The primers exhibit low degeneracy and cross-family priming, and amplify the rearranged V genes in an isotype independent fashion. The design principle outlined here can be extended to Ig gene systems from other species, other members of the Ig super family such as the T cell receptor and in designing primers for complex multigene families.
The present invention describes that amplification of the rearranged VH and VL genes from a mixture of hybridomas occurred in a representative manner. We successfully amplified the expressed/rearranged VH and VL gene from a single hybridoma cell by RT-PCR, and 10-15 microdissected B cells from immunohistochemically stained splenic sections by genomic PCR.
The present invention discloses novel universal nested primers for (RT-) PCR amplification of the mouse rearranged/expressed VH and VL genes. The versatile and comprehensive set of nested primers disclosed in the present invention are indispensable for applications where sample or template quantity is limiting (as in the case of microdissected or biopsy material) and where broad V gene family coverage is required. These primer sets facilitate study of B cell subsets, ontogeny and B cell lymphomas.
We demonstrated that the amplification of the rearranged VH and VL genes from a mixture of hybridomas occurred in a representative manner. We successfully amplified the expressed/rearranged VH and VL gene from a single hybridoma cell by RT-PCR, and 10-15 microdissected B cells from immunohistochemically stained splenic sections by genomic PCR.
V gene family-specific nature of the primers (SEQ ID NO: 1-112) disclosed in the present invention is experimentally confirmed for 6 randomly selected VH, 6 Vκ and 2 Vλ families. Cocktail of constant region specific primers allowed efficient RT-PCR amplification of IgM, IgG, IgA, Igκ and Igλ isotypes.
Most but not all V gene family specific primers designed allow amplification of full-length V genes. These nested primers permit PCR amplification of rearranged V genes belonging to all VH and VL gene families from splenocyte genomic DNA. The V gene family-specific nature of the primers was experimentally confirmed for randomly selected 6 VH and 6 Vκ families, and all Vλ genes. The broad V gene family coverage of the primer set was experimentally validated by amplifying the rearranged/expressed VH and VL genes from splenocytes and a panel of 38 hybridomas under conditions where primer mixes and genomic DNA or total RNA was used as starting template. We observed no or low-level cross-family priming. Pooled constant region specific primers allowed efficient RT-PCR amplification of H and L chain isotypes. The expressed VH and VL genes belonging to different V gene families RT-PCR amplified from a mixture of hybridomas in a representative manner.
This, first of its kind, comprehensive set of highly sensitive and specific nested primers that provide broad V gene family coverage will open up new avenues and opportunities to study various aspects of mouse B cell biology.
Designing Universal Nested PCR Primers for Amplification of Mouse Rearranged/Expressed VH and VL Genes
In order to develop highly specific and sensitive PCR that can potentially amplify mouse rearranged/expressed VH and VL gene belonging to any V gene family we adopted a nested strategy. The primers used for first and second (nested) round of PCR are referred to as external and internal primers, respectively. The leader (LH), variable (VH), diversity (DH), joining (JH), constant (CH) and complementary determining region 3 (HCDR3) regions of the Ig H chain are indicated. In most cases, the binding site for the 5′ external and internal primer is located in the leader and framework region 1, respectively. The instances were both 5′ external and internal primer hybridizes to the framework region 1 are indicated in Tables 1 and 2. The arrows pointing to the right and left indicate the orientation of the sense and antisense primers, respectively. The antisense primer (RT) used for first strand cDNA synthesis binds in the CH1 region. The strategy (not shown) used for (RT-) PCR amplification of the rearranged/expressed Vκ and Vλ genes was essentially identical to the one used for the H chain (FIG. 1).
Mouse germline Ig sequences were downloaded from international ImMunoGeneTics information system and IgBLAST databases for designing the primers. We focused our primer designing effort on the two most commonly used inbred strains of mice, namely C57BL/6 and BALB/c. The sequences were grouped in 16 VH (350 members), 19 Vκ (164 members), 3 Vλ (3 members), 1 JH(4 members), 1 Jκ (5 members), and 1 Jλ (5 members) families according to the IMGT nomenclature. MacVector software was used to analyze each Ig gene family. Based on the nucleotide sequence alignment of individual families, we designed a set of external and internal primers targeting conserved motifs in the leader, framework region 1, joining and constant regions of H and L chain genes (FIG. 1).
The 112 oligonucleotide primers (SEQ ID NO: 1-112) designed for amplifying the mouse rearranged/expressed Ig genes are summarized in Tables 1-3. A panel of 17 VH external, 17 VH internal, 1 JH external and 2 JH internal primers were designed for the H chain. In addition, 5 CH external, 5 CH internal and 5 CH primers for reverse transcribing the Ig transcript (RTCH) were designed for the H chain isotypes IgM, IgG, IgA, IgE and IgD (Table 1). For κ L chain, a panel of 19 Vκ external, 20 Vκ internal, 1 Jκ external, 1 Jκ internal, 1 Cκ external, 1 Cκ internal and 1 RTCκ, primers were designed (Table 2). Likewise, for λ L chain, a panel of 3 Vλ external, 3 Vλ internal, 2 Jλ external, 2 Jλ internal, 1 Cλ external, 2 Cλ internal and 1 RTCλ primers were designed (Table 3). The designed primer pairs were tested for their specificity and compatibility using PCR simulating software, Amplify 3. The total number of genes in any given Ig family, sequence of the oligonucleotide primer, position of the primer binding sites of the external and internal primers, fold degeneracy and the number of genes in a given Ig family with 0, 1, 2, 3 or 4 mismatches relative to the primer sequence are detailed in Tables 1-3. The 5′ external and internal primers bind in the leader and framework region 1, respectively, in the case of 8 out of 16 (50%) VH families, 18 out of 19 (94.7%) Vκ families and all Vλ genes (100%). In the remaining V gene families, both 5′ external and internal primers hybridize to framework region 1. Primers designed for 7 out of 16 (43.7%) VH families, 14 out of 19 (73.7%) Vκ families and all Vλ genes (100%) allow amplification of full-length V genes.
The primers were designed to cover functional and non-functional (pseudogene) germline Ig genes. In some instances, certain pseudogenes could not be included in designing the primers either because the sequences were too divergent or the available sequence was truncated and did not cover the primer binding site(s) (Tables 1-3). In most cases, single external and internal primers were sufficient to cover all the members of an Ig gene family. However, there were some exceptions. Two external and internal primers were required to cover the 193 genes belonging to the VH1 family. Two internal primers were required to cover the JH gene segments. Two CH external and internal primers were required to cover all the IgG isotypes. The instances where 2 primers were required to cover all the members of a L chain family are: 2 Vκ1 family internal primers, 2 Jλ external primers, 2 Jλ internal primers and 2 Cλ internal primers. On the other hand, single external and internal primers were sufficient to cover Vλ 1 and Vλ2 genes.
The primers were 17 to 22 nucleotide long with a Tm of 56° C. to 64° C., with the exception of the primers used for the first strand cDNA synthesis, which were 16 to 17 nucleotide long (Tm=48° C. to 50° C.). Majority (71.4%) of the designed primers had no degeneracy and 93.7% percentage had 4-fold or less degeneracy. The proportions of germline Ig genes to which the primers bind with no mismatch, up to 1 mismatch and up to 2 mismatches are 59.7%, 84.1% and 94.9%, respectively. More than half (59%) of the primers ended with a ‘G’ or ‘C’ at their 3′ end, favouring the extension by the thermostable polymerase and consequently, the efficiency of amplification.
The 5′ primers, designed to work for both DNA and RNA templates, are located in the leader or the framework one region (FIG. 1). For genomic PCR, the 3′ primers are designed to hybridize to the J region. For amplifying the expressed V gene using RNA as a template, the 3′ primers anneal to the CH1 and CL domain of the H and L chain genes, respectively. The choice between using the J or C region primer depends on the intended application i.e. genomic PCR versus RT-PCR.
Designed Nested PCR Primers Allow Amplification of the Rearranged V Gene from all VH and VL Gene Families
A nested PCR strategy was employed for amplifying the rearranged VH and VL genes using genomic DNA from splenocytes (FIG. 1A). Primer pairs corresponding to each V gene family were tested individually. In total 16 VH, 19 Vκ and 2 VλPCRs were set up using V gene family targeting primers in conjunction with the corresponding J region primer(s). Amplified products of the expected size ( ̃350 bp) were obtained for 15 VH, 19 Vκ and 2 Vλ gene families (FIG. 2). The PCR products showed minor variation in size across families, depending upon the position of the 5′ internal primer binding site. The size of the PCR product obtained was generally consistent with the position of the binding site of the 5′ internal primer of the corresponding V gene family (Tables 1-3). It is highly unlikely that these bands were the result of PCR contamination. This was confirmed by cloning and sequencing the PCR product of 6 randomly selected VH and Vκ families, and all Vλ genes.
We tested the compatibility of the primers (SEQ ID NO: 1-112) by setting up 3 separate PCRs with pooled mixes comprising of primers directed at all H-, all κ- and all λ-gene families (FIG. 3, lanes 1, 3 and 5, respectively). The PCRs once again yielded single bands (with no primer dimers) indicating that the primers did not interfere with each other during the amplification process even when present as a complex primer mix. The negative control (water only) did not yield any band (FIG. 3, lanes 2, 4 and 6).
Our amplification strategy requires a second (nested) round of PCR but the additional effort is compensated by the enhanced specificity and sensitivity. Although the primers were designed for amplifying the rearranged/expressed Ig genes from C57BL/6 and BALB/c, they are highly likely to work efficiently for other mouse strains as well, owing to sufficiently high degree of sequence conservation across mice strains. This is illustrated by the amplification of all the VH and VL gene families from CBA/J mouse, the recently reported VH16 family being an exception (FIG. 2).
Designed Nested PCR Primers are V Gene Family-Specific
To test whether our primers were V gene family-specific we randomly selected 6 V gene families (VH1, VH5, VH8, Vκ3, Vκ4 and Vκ13) that had 12 to 193 members, 6 V gene families (VH4, VH11, VH15, Vκ11, Vκ14 and Vκ17) that had 2 to 8 members and the 3 Vλ genes (Tables 1-3). We constructed independent libraries using V gene family-specific primers and splenocyte genomic DNA as template for 6 VH and 6 Vκ gene families, and the 3 Vλ genes. Two independent libraries were generated for the Vλ genes, one for Vλ 1 and Vλ2 genes, and a second one for Vλ3 gene. The nucleotide sequence was determined for 4-6 recombinants recovered from each library. Sequence analysis revealed that in all instances the rearranged V genes recovered were functional and belonged to the targeted V gene family (Table 4). Analysis of rearranged V genes recovered from libraries generated for V gene families that had 12 or more members showed a broad V gene usage pattern, Vκ13 family being an exception. Vκ13 has 18 members of which 15 are pseudogenes. Eleven pseudogenes were not included in designing the primers as their sequences were too divergent (Table 2). gm33, one of the 3 functional members of the Vκ13 gene family, was observed in the 4 independent VκJκ rearrangements recovered from the library.
V gene usage was found to be limited in V gene families with 1 to 4 members (VH4, VH11, VH15, Vκ11, Vκ17, Vλ1⁄2 and Vλ3) (Table 4). We observed that all the independent rearrangements recovered from VH4, VH11, VH15, and Vκ11 gene family-specific libraries utilized a single V gene belonging to the corresponding V gene family.
All the 4 independent rearrangements recovered from the VH4 family-specific library utilized X24.2.50 gene. X24.2.50 gene reported (from C57BL/6 mice) in the IMGT and IgBLAST databases has an in-frame stop codon in framework region 2. All the 4 independent rearrangements lack this in-frame stop codon. It is highly unlikely that the nonsense codon somatically mutated to a sense codon in all 4 rearrangements. Our data thus suggests that the homolog of X24.2.50 gene in CBA/J mice, from which our V gene libraries were constructed, is a functional VH gene.
These data confirm that our primers are V gene family-specific. We have demonstrated this for V gene families with 12 or more members as well as V gene families that have only a couple of members.
Efficient Amplification and Rapid Identification of VH and VL Gene Family Expressed in Hybridomas
The designed primers were used to amplify the expressed VH and VL genes from total RNA isolated from a collection of 38 unique IgM and IgG expressing hybridomas available in our laboratory. We successfully amplified the VH and VL genes expressed in from all 38 hybridomas using the primer set (SEQ ID NO: 1-112) and PCR strategy. The RT-PCR amplified product was sequenced using the appropriate CH, Cκ or Cλ internal primer. Sequence comparison and V gene family assignment for the expressed H and L chain genes was done using IMGT and IgBLAST databases.
Once the identity of the family to which the expressed V gene belongs has been established, one can, if need be, use the corresponding 5′ internal primer to sequence the second strand of the purified PCR product. Nucleotide sequence analysis of the VH genes expressed in the 38 hybridomas indicated that they belonged to 8 out of the 16 VH families. In addition to the major VH gene families, VH1, VH5 and VH9, the primers amplified VH2, VH3, VH7, VH10 and VH14 minor VH gene families. A family with less than 5% of the total number of genes present in our VH database was classified as a minor family.
Similarly, the VL genes expressed in the 38 hybridomas were sequenced and analyzed. Thirty seven of the 38 hybridomas expressed κ L chain. The expressed Vκ genes belong to 14 out of the 19 Vκ families. They are Vκ1, Vκ2, Vκ3, Vκ 4, Vκ5, Vκ6, Vκ8, Vκ9, Vκ10, Vκ12, Vκ13, Vκ15, Vκ18 and Vκ19 families. Of these Vκ5, Vκ9, Vκ10, Vκ15, Vκ18 and Vκ19 families have 6 (3.7%) or fewer members. The lone λ L chain expressing hybridoma rearranged Vλ2 and Jλ2 gene segments. These data demonstrate that our Ig primers are capable of specifically amplifying the expressed VH, Vκ and Vλ gene families that are very diverse in sequence and size. V gene families that were not represented in the panel of hybridomas may not be involved in the immune response to the immunizing Ag or we may not have sampled enough clones to observe them.
Primers Allow Broad V Gene Family Coverage Under Conditions where Primer Mixes and Splenocytes were Used
PCR amplification of the rearranged/expressed VH and Vλ genes using genomic DNA and total RNA from splenocytes as starting template was carried out, the rearranged VH and Vλ genes from splenocyte genomic DNA were amplified using pooled primers that target all VH families and all Vλ genes, respectively (SEQ ID NO:1-112, Tables 1-3). The VH and Vλ PCR products were cloned and the recombinants recovered from the two libraries were sequenced. The nucleotide sequence was analyzed to identify the germline V gene and family utilized. VH and Vλ cDNA libraries were constructed using pooled constant region, and VH and Vλ primer mixes. The summary of the nucleotide sequence analysis is presented in Table 6. The 119 rearranged VH genomic clones analyzed represented 38 unique VH genes belonging to 12 of the 15 VH families present in CBA/J mice. Most of the remaining 81 rearranged VH genomic clones were independent VDJ rearrangements of the 38 unique VH genes. In some instances, we observed 2 or more identical copies of the same genomic clone. Of the 53 VH cDNA clones analyzed 22 were unique and 31 were either independent rearrangements involving the 22 unique VH genes or identical copies of cDNA clones (Table 6). The 22 unique cDNAs belonged to 10 VH families. Four VH genes were found as genomic and cDNA clones. VH genomic and cDNA clones, when put together, covered 13 VH families and 56 unique VH genes. For the λ light chain, all the 3 Vλ genes i.e. VL1, VL2 and VLx, were recovered as genomic and cDNA clones. In conclusion, VH and Vλ primer set (SEQ ID NO: 1-112) disclosed in the present invention provides broad V gene family coverage under conditions where primer mixes and splenocytes were used.
Cross-Family Priming and Mutations Introduced by PCR Primers
Use of degenerate primers and primer cocktails (e.g. pooled VH or VL primer mixes) can allow broad V family/member coverage. The potential disadvantage of using degenerate primers or primer mixes is that it can lead to introduction of mutation(s) in the annealing region as result of (a) degenerate bases present in the primer, (b) cross-family priming and (c) authentic somatic mutation(s) present in the primer binding site may get replaced by the corresponding nucleotide in the annealing primer during PCR. We studied cross-family priming in the Vκ and VH families. We RT-PCR amplified and cloned the expressed Vκ gene from 37 hybridomas using pooled Vκ primers. These hybridomas represent 14 of the 19 Vκ families and the number of hybridomas that belong to a given Vκ gene family ranged from 1 to 7. The nucleotide sequences from the hybridomas were analyzed to identify the Vκ internal primer that amplified the expressed Vκ gene. In 31 of the 37 (83.8%) hybridomas analyzed, the Vκ internal primer used and the amplified Vκ gene belonged to the same family indicating low cross-Vκ gene family priming and high degree of specificity of the primers. Of the 5 Vκ1 gene family expressing hybridomas, 2 were primed by Vκ4 primer. None of the 7 Vκ4 expressing hybridomas were primed by a Vκ1 primer. We also observed cross-Vκ gene family priming in 3 other cases.
The issue of cross-family priming was also examined for the VH gene families. This was addressed in two ways. One, by analyzing the recombinants recovered from a library generated using pooled VH primers and splenocyte genomic DNA. A VH cDNA library was generated in parallel using pooled VH primers and total RNA from splenocytes. The clones recovered from the VH genomic and cDNA libraries (listed in Table 6) were analyzed to identify the VH internal primer that amplified the expressed VH gene and family to which the expressed VH gene belongs. In the 38 VH genomic and 22 cDNA clones analyzed, the VH internal primer used and the amplified VH gene belonged to the same family with no exceptions. Thus, the VH primers exhibited high VH family specificity and no cross-family priming. We avoided the difficulties that can arise as a result of using degenerate primers and primer mixes by limiting the sequence analysis to the region internal to the 5′ and 3′ internal primer.
Pooled Constant Region Specific Primers Allow Efficient RT-PCR Amplification of H and L Chain Isotypes
The use of IgH, Igκ and Igλ specific constant region primers for reverse transcription allows subsequent amplification of only IgH, Igκ and Igλ chain, respectively. In situations where the isotype of the expressed H and L chain is not known e.g. when small numbers of B cells or single B cells are used, the CH and CL constant region primers have to be pooled. To test this, total RNA isolated from splenocytes was reverse transcribed using pooled IgM, IgG, IgA, Igκ and Igλ specific constant region primers (listed in Tables 1-3). Equal amount of cDNA was used as template for setting up 6 independent nested PCRs for amplifying IgM, IgG, IgA, Igκ and Igλ V gene transcripts. The IgG transcripts were covered in two separate reactions, one for IgG3 and the second one for the remaining IgG isotypes. The expressed H, κ and λ chain were amplified using the corresponding pooled VH, Vκ and Vλ primer cocktail, respectively. The second round PCR products were cloned and recombinants recovered from the resultant 6 cDNA isotype specific libraries were sequenced. Analysis of the recombinants revealed that each of the 6 libraries were indeed specific for the intended isotype. No non-specific amplification was observed in any of the isotype specific libraries. Thus, our pooled constant region primers allow efficient and specific RT-PCR amplification of IgM, IgG, IgA, Igκ and Igλ isotypes.
Expressed VH and VL Genes Belonging to Different V Gene Families can be Amplified from Pooled Hybridomas in a Representative Manner
Since we use pooled VH and VL primer sets to amplify the rearranged/expressed VH and VL genes, it was important to test for any possible preferential binding of individual primers to selected rearranged/expressed V genes as it can complicate B cell repertoire analysis. We analyzed this by pooling equal amounts of total RNA from 5 previously characterized IgM expressing hybridomas. The VH genes expressed in these hybridomas belonged to VH1, VH2, VH3, VH7 and VH14 gene families. The expressed VL genes present belonged to Vκ5, Vκ9, Vκ12, Vκ18 and Vλ2 gene families. We constructed VH and VL gene specific cDNA libraries starting with total RNA pooled from hybridomas as template, and cocktails of all VH and VL primers. The plasmid DNA recovered from the recombinants obtained from the VH and VL cDNA libraries were digested with restriction enzyme(s) that can distinguish the VH and VL genes expressed in the hybridomas pooled in the experiment. Using restriction profiling, we recovered the expected 5 expressed VH and 4 Vκ genes from the VH and VL cDNA libraries, respectively. The 5 expressed VH and 4 Vκ genes identified by restriction digestion were confirmed by sequencing. In addition to these 4 expressed Vκ genes, we found a rearranged Vκ1 family member that was rendered non-functional because of a frameshift mutation in framework region 1 and the presence of a nonsense codon in framework region 2. This VκJκ gene most likely represents a non-productively rearranged allele present in one of the 5 hybridomas. These data suggests that the primer set (SEQ ID NO: 1-112) and RT-PCR strategy amplifies the expressed VH and VL genes from a mixture of hybridoma cells in a representative manner.
Nested RT-PCR Amplification of the Expressed V Genes is Highly Sensitive
After establishing the broad coverage and specificity of the designed primer set, we set out to determine the sensitivity of our nested (RT-) PCR strategy and primers. We first tested the detection limit of our amplification strategy by performing RT-PCR for the expressed Vκ genes using a dilution series ranging from 100 to 1 hybridoma cell (FIG. 4A). Using pooled Vκ primer mix and Cκ primer, we were able to readily amplify the expressed κ L chain gene from a single hybridoma cell by RT-PCR. Similarly, we amplified the expressed VH gene from a single hybridoma cell using pooled VH and Cμ primers. Although we were able to detect a faint VH band from a single cell template input (FIG. 4B) but the yield was not sufficient for sequence analysis. We went on to test the effect of increasing the number of PCR amplification cycles for 25 per round of PCR to 50 on the detection limit of the expressed VH gene. Increasing the PCR cycles from 25 to 50 permitted easy detection of the amplified VH product from a single hybridoma cell (FIG. 4B, lower panel). Increasing the numbers of PCR cycles increased the yield of the Vκ RT-PCR product further. We did not detect any product when hybridoma cell or reverse transcriptase was omitted from the reaction mix (FIG. 4).
In addition to the hybridomas, we tested the ability of the designed primers to amplify the rearranged VH and VL genes from immunohistochemically stained B220+ B cells microdissected from splenic sections from naïve BALB/c mice. Using genomic DNA based nested PCR strategy and separately pooled VH and Vκ primer mixes we were able to amplify the rearranged VH and Vκ genes from 10 to 15 microdissected B220+ B cells (FIG. 5). On rare occasions we were able to amplify the rearranged V genes from 4 to 5 microdissected B cells but the amplification was not very consistent and reproducible (data not shown). B220+ B cells were microdissected from splenic sections using a hydraulic micromanipulator. The rearranged VH (A) and Vκ (B) genes were amplified using VH and Vκ primer mixes, respectively. The images of the stained section taken before and after the B cells were microdissected are shown in the left most and middle columns. The estimated number of B cells microdissected (encircled) from the splenic section is indicated. The amplified rearranged VH (H) and Vκ (κ) gene product obtained using the genomic DNA based nested PCR strategy along with the negative control (buffer only, C) is shown in the right most column (FIG. 5).
In accordance with the present invention in one embodiment there is provided a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 112.
In one embodiment there is provided the set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 112, wherein said immunoglobin genes are VH, Vκ and Vλ, JH, Jκ, Jλ, CH, Cκ and Cλ.
In one embodiment there is provided the set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 112, wherein said antibody is selected from the group consisting of immunoglobulin heavy chain isotypes IgG, IgM, IgA, IgE, and IgD; and light chain isotypes κ and λ.
In another embodiment of the present invention there is provided a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 1: SEQ ID NO: 1 to 37, SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108; and set 44: SEQ ID NO: 1 to 34, SEQ ID NO: 38 to 93, SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112.
In another embodiment of the present invention there is provided a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 1: SEQ ID NO: 1 to 37, SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108; and set 44: SEQ ID NO: 1 to 34, SEQ ID NO: 38 to 93, SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112, wherein said immunoglobin genes are VH, Vκ and Vλ genes.
One embodiment of the present invention provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 2: SEQ ID NO: 1 to 37, set 3: SEQ ID NO: 1 to 4 and SEQ ID NO: 35-37, set 4: SEQ ID NO: 5 to 6 and SEQ ID NO: 35 to 37, set 5: SEQ ID NO: 7 to 8 and SEQ ID NO: 35-37, set 6: SEQ ID NO: 9 to 10 and SEQ ID NO: 35-37, set 7: SEQ ID NO: 11 to 12 and SEQ ID NO: 35-37; set 8: SEQ ID NO: 13 to 14 and SEQ ID NO: 35-37; set 9: SEQ ID NO: 15 to 16 and SEQ ID NO: 35-37; set 10: SEQ ID NO: 17 to 18 and SEQ ID NO: 35-37; set 11: SEQ ID NO: 19 to 20 and SEQ ID NO: 35-37; set 12: SEQ ID NO: 21-22 and SEQ ID NO: 35-37; set 13: SEQ ID NO: 23 to 24 and SEQ ID NO: 35-37; set 14: SEQ ID NO: 25 to 26 and SEQ ID NO: 35-37; set 15: SEQ ID NO: 27 to 28 and SEQ ID NO: 29 to 30; set 16: SEQ ID NO: 29 to 30 and SEQ ID NO: 35-37; set 17: SEQ ID NO: 31 to 32 and SEQ ID NO: 35-37; and set 18: SEQ ID NO: 33-37.
Another embodiment of the present invention provides the set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 2: SEQ ID NO: 1 to 37, set 3: SEQ ID NO: 1 to 4 and SEQ ID NO: 35-37, set 4: SEQ ID NO: 5 to 6 and SEQ ID NO: 35 to 37, set 5: SEQ ID NO: 7 to 8 and SEQ ID NO: 35-37, set 6: SEQ ID NO: 9 to 10 and SEQ ID NO: 35-37, set 7: SEQ ID NO: 11 to 12 and SEQ ID NO: 35-37; set 8: SEQ ID NO: 13 to 14 and SEQ ID NO: 35-37; set 9: SEQ ID NO: 15 to 16 and SEQ ID NO: 35-37; set 10: SEQ ID NO: 17 to 18 and SEQ ID NO: 35-37; set 1: SEQ ID NO:19 to 20 and SEQ ID NO: 35-37; set 12: SEQ ID NO: 21-22 and SEQ ID NO: 35-37; set 13: SEQ ID NO: 23 to 24 and SEQ ID NO: 35-37; set 14: SEQ ID NO: 25 to 26 and SEQ ID NO: 35-37; set 15: SEQ ID NO: 27 to 28 and SEQ ID NO: 29 to 30; set 16: SEQ ID NO: 29 to 30 and SEQ ID NO: 35-37; set 17: SEQ ID NO: 31 to 32 and SEQ ID NO: 35-37; and set 18: SEQ ID NO: 33-37, wherein said immunoglobin genes are VH genes.
Still another embodiment of the present invention provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides are as set forth in set 19: SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108.
Still another embodiment of the present invention provides the set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides are as set forth in set 19: SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108, wherein said immunoglobin genes are VL genes.
Yet another embodiment of the present invention provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 20: SEQ ID NO: 55 to 95; set 21: SEQ ID NO: 55 to 57 and SEQ ID NO: 94 to 95; set 22: SEQ ID NO: 58 to 59 and SEQ ID NO: 94 to 95; set 23: SEQ ID NO: 60 to 61 and SEQ ID NO: 94 to 95; set 24: SEQ ID NO: 62 to 63 and SEQ ID NO: 94 to 95; set 25: SEQ ID NO: 64 to 65 and SEQ ID NO: 94 to 95; set 26: SEQ ID NO: 66 to 67 and SEQ ID NO: 94 to 95; set 27: SEQ ID NO: 68 to 69 and SEQ ID NO: 94 to 95; set 28: SEQ ID NO: 70 to 71 and SEQ ID NO: 94 to 95; set 29: SEQ ID NO: 72 to 73 and SEQ ID NO: 94 to 95; set 30: SEQ ID NO: 74 to 75 and SEQ ID NO: 94 to 95; set 31: SEQ ID NO: 76 to 77 and SEQ ID NO: 94 to 95; set 32: SEQ ID NO: 78 to 79 and SEQ ID NO: 94 to 95; set 33: SEQ ID NO: 80 to 81 and SEQ ID NO: 94 to 95; set 34: SEQ ID NO: 82 to 83 and SEQ ID NO: 94 to 95; set 35: SEQ ID NO: 84 to 85 and SEQ ID NO: 94 to 95; set 36: SEQ ID NO: 86 to 87 and SEQ ID NO: 94 to 95; set 37: SEQ ID NO: 88 to 89 and SEQ ID NO: 94 to 95; set 38: SEQ ID NO: 90 to 91 and SEQ ID NO: 94 to 95; and set 39: SEQ ID NO: 92 to 95.
Yet another embodiment of the present invention provides the set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 20: SEQ ID NO: 55 to 95; set 21: SEQ ID NO: 55 to 57 and SEQ ID NO: 94 to 95; set 22: SEQ ID NO: 58 to 59 and SEQ ID NO: 94 to 95; set 23: SEQ ID NO: 60 to 61 and SEQ ID NO: 94 to 95; set 24: SEQ ID NO: 62 to 63 and SEQ ID NO: 94 to 95; set 25: SEQ ID NO: 64 to 65 and SEQ ID NO: 94 to 95; set 26: SEQ ID NO: 66 to 67 and SEQ ID NO: 94 to 95; set 27: SEQ ID NO: 68 to 69 and SEQ ID NO: 94 to 95; set 28: SEQ ID NO: 70 to 71 and SEQ ID NO: 94 to 95; set 29: SEQ ID NO: 72 to 73 and SEQ ID NO: 94 to 95; set 30: SEQ ID NO: 74 to 75 and SEQ ID NO: 94 to 95; set 31: SEQ ID NO: 76 to 77 and SEQ ID NO: 94 to 95; set 32: SEQ ID NO: 78 to 79 and SEQ ID NO: 94 to 95; set 33: SEQ ID NO: 80 to 81 and SEQ ID NO: 94 to 95; set 34: SEQ ID NO: 82 to 83 and SEQ ID NO: 94 to 95; set 35: SEQ ID NO: 84 to 85 and SEQ ID NO: 94 to 95; set 36: SEQ ID NO: 86 to 87 and SEQ ID NO: 94 to 95; set 37: SEQ ID NO: 88 to 89 and SEQ ID NO: 94 to 95; set 38: SEQ ID NO: 90 to 91 and SEQ ID NO: 94 to 95; and set 39: SEQ ID NO: 92 to 95, wherein said immunoglobin genes are Vκ genes.
Further embodiment of the present invention provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 40: SEQ ID NO: 99 to 108; set 41: SEQ ID NO: 99 to 100 and SEQ ID NO: 105 to 108; set 42: SEQ ID NO: 101 to 102 and SEQ ID NO: 105 to 108; and set 43: SEQ ID NO: 103 to 108.
Further embodiment of the present invention provides the set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 40: SEQ ID NO: 99 to 108; set 41: SEQ ID NO: 99 to 100 and SEQ ID NO: 105 to 108; set 42: SEQ ID NO: 101 to 102 and SEQ ID NO: 105 to 108; and set 43: SEQ ID NO: 103 to 108, wherein said immunoglobin genes are Vλ genes.
The present invention further provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 45: SEQ ID NO: 1 to 34, SEQ ID NO: 38-54; set 46: SEQ ID NO: 1 to 34, SEQ ID NO: 38, SEQ ID NO: 43 to 44; set 47: SEQ ID NO: 1 to 34, SEQ ID NO: 39, SEQ ID NO: 45 to 48; set 48: SEQ ID NO: 1 to 34, SEQ ID NO: 40, SEQ ID NO: 49 to 50; set 49: SEQ ID NO: 1 to 34, SEQ ID NO: 41, SEQ ID NO: 51 to 52; set 50: SEQ ID NO: 1 to 34, SEQ ID NO: 42, SEQ ID NO: 53 to 54; set 51: SEQ ID NO: 1 to 4 and SEQ ID NO: 38 to 54; set 52: SEQ ID NO: 5 to 6 and SEQ ID NO: 38 to 54; set 53: SEQ ID NO: 7 to 8 and SEQ ID NO: 38 to 54; set 54: SEQ ID NO: 9 to 10 and SEQ ID NO: 38 to 54; set 55: SEQ ID NO: 11 to 12 and SEQ ID NO: 38 to 54; set 56: SEQ ID NO: 13 to 14 and SEQ ID NO: 38 to 54; set 57: SEQ ID NO: 15 to 16 and SEQ ID NO: 38 to 54; set 58: SEQ ID NO: 17 to 18 and SEQ ID NO: 38 to 54; set 59: SEQ ID NO: 19 to 20 and SEQ ID NO: 38 to 54; set 60: SEQ ID NO: 21 to 22 and SEQ ID NO: 38 to 54; set 61: SEQ ID NO: 23 to 24 and SEQ ID NO: 38 to 54; set 62: SEQ ID NO: 25 to 26 and SEQ ID NO: 38 to 54; set 63: SEQ ID NO: 27 to 28 and SEQ ID NO: 38 to 54; set 64: SEQ ID NO: 29 to 30 and SEQ ID NO: 38 to 54; set 65: SEQ ID NO: 31 to 32 and SEQ ID NO: 38 to 54; and set 66: SEQ ID NO: 33 to 34 and SEQ ID NO: 38 to 54.
The present invention further provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 45: SEQ ID NO: 1 to 34, SEQ ID NO: 38-54; set 46: SEQ ID NO: 1 to 34, SEQ ID NO: 38, SEQ ID NO: 43 to 44; set 47: SEQ ID NO: 1 to 34, SEQ ID NO: 39, SEQ ID NO: 45 to 48; set 48: SEQ ID NO: 1 to 34, SEQ ID NO: 40, SEQ ID NO: 49 to 50; set 49: SEQ ID NO: 1 to 34, SEQ ID NO: 41, SEQ ID NO: 51 to 52; set 50: SEQ ID NO: 1 to 34, SEQ ID NO: 42, SEQ ID NO: 53 to 54; set 51: SEQ ID NO: 1 to 4 and SEQ ID NO: 38 to 54; set 52: SEQ ID NO: 5 to 6 and SEQ ID NO: 38 to 54; set 53: SEQ ID NO: 7 to 8 and SEQ ID NO: 38 to 54; set 54: SEQ ID NO: 9 to 10 and SEQ ID NO: 38 to 54; set 55: SEQ ID NO: 11 to 12 and SEQ ID NO: 38 to 54; set 56: SEQ ID NO: 13 to 14 and SEQ ID NO: 38 to 54; set 57: SEQ ID NO: 15 to 16 and SEQ ID NO: 38 to 54; set 58: SEQ ID NO: 17 to 18 and SEQ ID NO: 38 to 54; set 59: SEQ ID NO: 19 to 20 and SEQ ID NO: 38 to 54; set 60: SEQ ID NO: 21 to 22 and SEQ ID NO: 38 to 54; set 61: SEQ ID NO: 23 to 24 and SEQ ID NO: 38 to 54; set 62: SEQ ID NO: 25 to 26 and SEQ ID NO: 38 to 54; set 63: SEQ ID NO: 27 to 28 and SEQ ID NO: 38 to 54; set 64: SEQ ID NO: 29 to 30 and SEQ ID NO: 38 to 54; set 65: SEQ ID NO: 31 to 32 and SEQ ID NO: 38 to 54; and set 66: SEQ ID NO: 33 to 34 and SEQ ID NO: 38 to 54, wherein said immunoglobin genes are expressed heavy chain genes.
Another embodiment of the present invention provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 67: SEQ ID NO: 55 to 93; SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112; set 68: SEQ ID NO: 55 to 93 and SEQ ID NO: 96 to 98, set 69: SEQ ID NO: 55 to 57 and SEQ ID NO: 96 to 98, set 70: SEQ ID NO: 58 to 59 and SEQ ID NO: 96 to 98, set 71: SEQ ID NO: 60 to 61 and SEQ ID NO: 96 to 98, set 72: SEQ ID NO: 62 to 63 and SEQ ID NO: 96 to 98, set 73: SEQ ID NO: 64 to 65 and SEQ ID NO: 96 to 98, set 74: SEQ ID NO: 66 to 67 and SEQ ID NO: 96 to 98, set 75: SEQ ID NO: 68 to 69 and SEQ ID NO: 96 to 98, set 76: SEQ ID NO: 70 to 71 and SEQ ID NO: 96 to 98, set 77: SEQ ID NO: 72 to 73 and SEQ ID NO: 96 to 98, set 78: SEQ ID NO: 74 to 75 and SEQ ID NO: 96 to 98, set 79: SEQ ID NO: 76 to 77 and SEQ ID NO: 96 to 98, set 80: SEQ ID NO: 78 to 79 and SEQ ID NO: 96 to 98; set 81: SEQ ID NO: 80 to 81 and SEQ ID NO: 96 to 98; set 82: SEQ ID NO: 82 to 83 and SEQ ID NO: 96 to 98; set 83: SEQ ID NO: 84 to 85 and SEQ ID NO: 96 to 98; set 84: SEQ ID NO: 86 to 87 and SEQ ID NO: 96 to 98; set 85: SEQ ID NO: 88 to 89 and SEQ ID NO: 96 to 98; set 86: SEQ ID NO: 90 to 91 and SEQ ID NO: 96 to 98; set 87: SEQ ID NO: 92 to 93 and SEQ ID NO: 96 to 98; set 88: SEQ ID NO: 99 to 104 and SEQ ID NO: 109 to 112; set 89: SEQ ID NO: 99 to 100 and SEQ ID NO: 109 to 112; set 90: SEQ ID NO: 101 to 102 and SEQ ID NO: 109 to 112; and set 91: SEQ ID NO: 103 to 104 and SEQ ID NO: 109 to 112.
Another embodiment of the present invention provides a set of oligonucleotides 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 nucleotide sequences of said set of oligonucleotides is selected from the group consisting of set 67: SEQ ID NO: 55 to 93; SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112; set 68: SEQ ID NO: 55 to 93 and SEQ ID NO: 96 to 98, set 69: SEQ ID NO: 55 to 57 and SEQ ID NO: 96 to 98, set 70: SEQ ID NO: 58 to 59 and SEQ ID NO: 96 to 98, set 71: SEQ ID NO: 60 to 61 and SEQ ID NO: 96 to 98, set 72: SEQ ID NO: 62 to 63 and SEQ ID NO: 96 to 98, set 73: SEQ ID NO: 64 to 65 and SEQ ID NO: 96 to 98, set 74: SEQ ID NO: 66 to 67 and SEQ ID NO: 96 to 98, set 75: SEQ ID NO: 68 to 69 and SEQ ID NO: 96 to 98, set 76: SEQ ID NO: 70 to 71 and SEQ ID NO: 96 to 98, set 77: SEQ ID NO: 72 to 73 and SEQ ID NO: 96 to 98, set 78: SEQ ID NO: 74 to 75 and SEQ ID NO: 96 to 98, set 79: SEQ ID NO: 76 to 77 and SEQ ID NO: 96 to 98, set 80: SEQ ID NO: 78 to 79 and SEQ ID NO: 96 to 98; set 81: SEQ ID NO: 80 to 81 and SEQ ID NO: 96 to 98; set 82: SEQ ID NO: 82 to 83 and SEQ ID NO: 96 to 98; set 83: SEQ ID NO: 84 to 85 and SEQ ID NO: 96 to 98; set 84: SEQ ID NO: 86 to 87 and SEQ ID NO: 96 to 98; set 85: SEQ ID NO: 88 to 89 and SEQ ID NO: 96 to 98; set 86: SEQ ID NO: 90 to 91 and SEQ ID NO: 96 to 98; set 87: SEQ ID NO: 92 to 93 and SEQ ID NO: 96 to 98; set 88: SEQ ID NO: 99 to 104 and SEQ ID NO: 109 to 112; set 89: SEQ ID NO: 99 to 100 and SEQ ID NO: 109 to 112; set 90: SEQ ID NO: 101 to 102 and SEQ ID NO: 109 to 112; and set 91: SEQ ID NO: 103 to 104 and SEQ ID NO: 109 to 112, wherein said immunoglobin genes are expressed light chain genes.
In yet another embodiment there is provided a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 54.
In yet another embodiment there is provided a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 54, wherein said immunoglobin genes are VH, JH, and CH 
In yet another embodiment there is provided a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 55 to 98.
In yet another embodiment there is provided a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 55 to 98, wherein said immunoglobin genes are Vκ, Jκ, and Cκ gene.
In yet another embodiment there is provided a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 98 to 112.
In yet another embodiment there is provided a set of oligonucleotides 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 nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 98 to 112, wherein said immunoglobin genes are Vλ, Jλ, and Cλ gene.
The set of oligonucleotides as disclosed in the present invention are useful for detection of cancer, wherein the cancer is B cell lymphoma. One embodiment provides a hybridoma cell for identifying clonality of the cells.
Further embodiment of the present invention provides 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 using the set of oligonucleotides as set forth in SEQ ID NO: 1-112.
The present invention also provides 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; the process comprises 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 oligonucleotide 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.
In another embodiment of the present invention there is provided 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; the process comprises 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 oligonucleotide 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: SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 and SEQ ID NO: 54; and detecting presence of an amplified product.
Yet another embodiment of the present invention relates to cancer, wherein said cancer is B cell lymphoma.
Yet another embodiment of the present invention relates to cell, wherein said cell is a hybridoma cell.
Yet another embodiment of the present invention relates to wherein said sample is a B cell.
Further there is provided a process for constructing library of polynucleotides encoding immunoglobulin genes, the process comprises amplifying immunoglobulin genes using the oligonucleotide sequences as set forth in SEQ ID NO: 1-112 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.
One embodiment of the present invention provides a prokaryotic expression vector.
Another embodiment of the present invention provides a host cell wherein the host cell is E. coli. 
In addition the present invention 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 said kit comprises nucleotide sequences of said oligonucleotides are as set forth in SEQ ID NO: 1 to 112.
In another embodiment of the present invention there is provided 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 1: SEQ ID NO: 1 to 37, SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108; and set 44: SEQ ID NO: 1 to 34, SEQ ID NO: 38 to 93, SEQ ID NO: 96 to 104 and SEQ ID NO: 109 to 112.
In still another embodiment of the present invention there is provided 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 2: SEQ ID NO: 1 to 37, set 3: SEQ ID NO: 1 to 4 and SEQ ID NO: 35-37, set 4: SEQ ID NO: 5 to 6 and SEQ ID NO: 35 to 37, set 5: SEQ ID NO: 7 to 8 and SEQ ID NO: 35-37, set 6: SEQ ID NO: 9 to 10 and SEQ ID NO: 35-37, set 7: SEQ ID NO: 11 to 12 and SEQ ID NO: 35-37; set 8: SEQ ID NO: 13 to 14 and SEQ ID NO: 35-37; set 9: SEQ ID NO: 15 to 16 and SEQ ID NO: 35-37; set 10: SEQ ID NO: 17 to 18 and SEQ ID NO: 35-37; set 11: SEQ ID NO: 19 to 20 and SEQ ID NO: 35-37; set 12: SEQ ID NO: 21-22 and SEQ ID NO: 35-37; set 13: SEQ ID NO: 23 to 24 and SEQ ID NO: 35-37; set 14: SEQ ID NO: 25 to 26 and SEQ ID NO: 35-37; set 15: SEQ ID NO: 27 to 28 and SEQ ID NO: 29 to 30; set 16: SEQ ID NO: 29 to 30 and SEQ ID NO: 35-37; set 17: SEQ ID NO: 31 to 32 and SEQ ID NO: 35-37; and set 18: SEQ ID NO: 33-37.
In still yet another embodiment of the present invention there is provided 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 said kit comprises set 19: SEQ ID NO: 55 to 95 and SEQ ID NO: 99 to 108.
In further embodiment of the present invention there is provided a 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 20: SEQ ID NO: 55 to 95; set 21: SEQ ID NO: 55 to 57 and SEQ ID NO: 94 to 95; set 22: SEQ ID NO: 58 to 59 and SEQ ID NO: 94 to 95; set 23: SEQ ID NO: 60 to 61 and SEQ ID NO: 94 to 95; set 24: SEQ ID NO: 62 to 63 and SEQ ID NO: 94 to 95; set 25: SEQ ID NO: 64 to 65 and SEQ ID NO: 94 to 95; set 26: SEQ ID NO: 66 to 67 and SEQ ID NO: 94 to 95; set 27: SEQ ID NO: 68 to 69 and SEQ ID NO: 94 to 95; set 28: SEQ ID NO: 70 to 71 and SEQ ID NO: 94 to 95; set 29: SEQ ID NO: 72 to 73 and SEQ ID NO: 94 to 95; set 30: SEQ ID NO: 74 to 75 and SEQ ID NO: 94 to 95; set 31: SEQ ID NO: 76 to 77 and SEQ ID NO: 94 to 95; set 32: SEQ ID NO: 78 to 79 and SEQ ID NO: 94 to 95; set 33: SEQ ID NO: 80 to 81 and SEQ ID NO: 94 to 95; set 34: SEQ ID NO: 82 to 83 and SEQ ID NO: 94 to 95; set 35: SEQ ID NO: 84 to 85 and SEQ ID NO: 94 to 95; set 36: SEQ ID NO: 86 to 87 and SEQ ID NO: 94 to 95; set 37: SEQ ID NO: 88 to 89 and SEQ ID NO: 94 to 95; set 38: SEQ ID NO: 90 to 91 and SEQ ID NO: 94 to 95; and set 39: SEQ ID NO: 92 to 95.
The present invention also provides 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 40: SEQ ID NO: 99 to 108; set 41: SEQ ID NO: 99 to 100 and SEQ ID NO: 105 to 108; set 42: SEQ ID NO: 101 to 102 and SEQ ID NO: 105 to 108; and set 43: SEQ ID NO: 103 to 108.
The present invention further provides 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 45: SEQ ID NO: 1 to 34, SEQ ID NO: 38-54; set 46: SEQ ID NO: 1 to 34, SEQ ID NO: 38, SEQ ID NO: 43 to 44; set 47: SEQ ID NO: 1 to 34, SEQ ID NO: 39, SEQ ID NO: 45 to 48; set 48: SEQ ID NO: 1 to 34, SEQ ID NO: 40, SEQ ID NO: 49 to 50; set 49: SEQ ID NO: 1 to 34, SEQ ID NO: 41, SEQ ID NO: 51 to 52; set 50: SEQ ID NO: 1 to 34, SEQ ID NO: 42, SEQ ID NO: 53 to 54; set 51: SEQ ID NO: 1 to 4 and SEQ ID NO: 38 to 54; set 52: SEQ ID NO: 5 to 6 and SEQ ID NO: 38 to 54; set 53: SEQ ID NO: 7 to 8 and SEQ ID NO: 38 to 54; set 54: SEQ ID NO: 9 to 10 and SEQ ID NO: 38 to 54; set 55: SEQ ID NO: 11 to 12 and SEQ ID NO: 38 to 54; set 56: SEQ ID NO: 13 to 14 and SEQ ID NO: 38 to 54; set 57: SEQ ID NO: 15 to 16 and SEQ ID NO: 38 to 54; set 58: SEQ ID NO: 17 to 18 and SEQ ID NO: 38 to 54; set 59: SEQ ID NO: 19 to 20 and SEQ ID NO: 38 to 54; set 60: SEQ ID NO: 21 to 22 and SEQ ID NO: 38 to 54; set 61: SEQ ID NO: 23 to 24 and SEQ ID NO: 38 to 54; set 62: SEQ ID NO: 25 to 26 and SEQ ID NO: 38 to 54; set 63: SEQ ID NO: 27 to 28 and SEQ ID NO: 38 to 54; set 64: SEQ ID NO: 29 to 30 and SEQ ID NO: 38 to 54; set 65: SEQ ID NO: 31 to 32 and SEQ ID NO: 38 to 54; and set 66: SEQ ID NO: 33 to 34 and SEQ ID NO:38 to 54.
The present invention further provides 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 said kit comprises at least one set of oligonucleotides selected from the group consisting of set 67: SEQ ID NO: 55 to 93; SEQ ID NO: 96 to 104 and SEQ ID NO:109 to 112; set 68: SEQ ID NO: 55 to 93 and SEQ ID NO: 96 to 98, set 69: SEQ ID NO: 55 to 57 and SEQ ID NO: 96 to 98, set 70: SEQ ID NO: 58 to 59 and SEQ ID NO: 96 to 98, set 71: SEQ ID NO: 60 to 61 and SEQ ID NO: 96 to 98, set 72: SEQ ID NO: 62 to 63 and SEQ ID NO: 96 to 98, set 73: SEQ ID NO: 64 to 65 and SEQ ID NO: 96 to 98, set 74: SEQ ID NO: 66 to 67 and SEQ ID NO: 96 to 98, set 75: SEQ ID NO: 68 to 69 and SEQ ID NO: 96 to 98, set 76: SEQ ID NO: 70 to 71 and SEQ ID NO: 96 to 98, set 77: SEQ ID NO: 72 to 73 and SEQ ID NO: 96 to 98, set 78: SEQ ID NO: 74 to 75 and SEQ ID NO: 96 to 98, set 79: SEQ ID NO: 76 to 77 and SEQ ID NO: 96 to 98, set 80: SEQ ID NO: 78 to 79 and SEQ ID NO: 96 to 98; set 81: SEQ ID NO: 80 to 81 and SEQ ID NO: 96 to 98; set 82: SEQ ID NO: 82 to 83 and SEQ ID NO: 96 to 98; set 83: SEQ ID NO: 84 to 85 and SEQ ID NO: 96 to 98; set 84: SEQ ID NO: 86 to 87 and SEQ ID NO: 96 to 98; set 85: SEQ ID NO: 88 to 89 and SEQ ID NO: 96 to 98; set 86: SEQ ID NO: 90 to 91 and SEQ ID NO: 96 to 98; set 87: SEQ ID NO: 92 to 93 and SEQ ID NO: 96 to 98; set 88: SEQ ID NO: 99 to 104 and SEQ ID NO: 109 to 112; set 89: SEQ ID NO: 99 to 100 and SEQ ID NO: 109 to 112; set 90: SEQ ID NO: 101 to 102 and SEQ ID NO: 109 to 112; and set 91: SEQ ID NO: 103 to 104 and SEQ ID NO: 109 to 112.
EXAMPLES
It should be understood that the following examples described herein are for illustrative purposes only and that various modifications or changes in light will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.
Example 1
BALB/c and CBA/J inbred strains of mice were from The Jackson Laboratory, and were bred and maintained in the Small Animal Facility of the National Institute of Immunology. Mice used for experiments were 6 to 8 wk old. Approval from the Institutional Animal Ethics Committee was obtained for all experimental procedures involving animals.
PCR Amplification of Rearranged VH and VL Genes from Splenocyte Genomic DNA
Genomic DNA was isolated from 2×107 splenocytes from CBA/J mice using a commercially available genomic DNA isolation kit following the manufacturer's instructions (Qiagen, Germany).
A nested PCR was performed for amplifying the rearranged VH and VL gene families. Family targeted PCRs were set up for each V gene family using H chain primer sets 3-18, κ L chain primer sets 21-39 and λ L chain primer sets 41-43 (Table 6) and genomic DNA from splenocytes. The 50 μl first round PCR mix consisted of 1× PCR buffer 1,200 μM dNTPs, 0.25 μM external 5′ (V gene) and 3′ (J region) primers each, and 2.5 U of AmpliTaq DNA polymerase (Applied Biosystems, USA).
Touchdown PCR was set up on a GeneAmp 2700 PCR System (Applied Biosystems, USA). The touchdown part of the PCR programme started with an initial denaturation step at 95° C. for 2 min. This was followed by denaturation at 94° C. for 1 min, annealing at 62° C. for 1 min and an extension at 72° C. for 1 min. The annealing temperature was decreased from 62° C. to 52° C. over 5 cycles at the rate of 2° C. per cycle. This was followed by 25 cycles consisting of denaturation at 94° C. for 1 min, annealing at 52° C. for 1 min and an extension at 72° C. for 1 min. A final extension step was carried out at 72° C. for 10 min. The composition of the reaction mixture and cycling parameters for the second round PCR were same as were used for the first round PCR except that 2 μl of the unpurified first round PCR product served as template for the second round PCR in place of splenocyte genomic DNA. The amplified products were analyzed on a 0.9% agarose gel.
The melting temperature (Tm in ° C.) of the primers was calculated using the formula [(4× number of G or C nucleotides in the primer sequence)+(2× number of A or T nucleotides in the primer sequence)].
Primers for First round of PCR: VH1 family SEQ ID NO. 1 and 2; VH2 family SEQ ID NO. 5; VH3 family SEQ ID NO. 7; VH4 family SEQ ID NO. 9; VH5 family SEQ ID NO. 11; VH6 family SEQ ID NO. 13; VH7 family SEQ ID NO. 15; VH8 family SEQ ID NO. 17; VH9 family SEQ ID NO. 19; VH10 family SEQ ID NO. 21; VH11 family SEQ ID NO. 23; VH12 family SEQ ID NO. 25; VH13 family SEQ ID NO. 27; VH14 family SEQ ID NO. 29; VH15 family SEQ ID NO. 31; VH16 family SEQ ID NO. 33 in conjunction with SEQ ID NO. 35 plus Vκ1 family SEQ ID NO. 55; Vκ2 family SEQ ID NO. 58; Vκ3 family SEQ ID NO. 60; Vκ4 family SEQ ID NO. 62; Vκ5 family SEQ ID NO. 64; Vκ6 family SEQ ID NO. 66; Vκ7 family SEQ ID NO. 68; Vκ8 family SEQ ID NO. 70; Vκ9 family SEQ ID NO. 72; Vκ10 family SEQ ID NO. 74; Vκ11 family SEQ ID NO. 76; Vκ12 family SEQ ID NO. 78; Vκ13 family SEQ ID NO. 80; Vκ14 family SEQ ID NO. 82; Vκ15 family SEQ ID NO. 84; Vκ16 family SEQ ID NO. 86; Vκ17 family SEQ ID NO. 88; Vκ18 family SEQ ID NO. 90; Vκ19 family SEQ ID NO. 92 in conjunction with SEQ ID NO 94 plus Vλ1 and Vλ2 SEQ ID NO. 99; Vλ3 SEQ ID NO. 101; Vλ4, Vλ5, Vλ6, Vλ7 and Vλ8 SEQ ID NO. 103 in conjunction with SEQ ID NO 105 and 106.
Primers for Second round of PCR: VH1 family SEQ ID NO. 3 and 4; VH2 family SEQ ID NO. 6; VH3 family SEQ ID NO. 8; VH4 family SEQ ID NO. 10; VH5 family SEQ ID NO. 12; VH6 family SEQ ID NO. 14; VH7 family SEQ ID NO. 16; VH8 family SEQ ID NO. 18; VH9 family SEQ ID NO. 20; VH10 family SEQ ID NO. 22; VH11 family SEQ ID NO. 24; VH12 family SEQ ID NO. 26; VH13 family SEQ ID NO. 28; VH14 family SEQ ID NO. 30; VH15 family SEQ ID NO. 32; VH16 family SEQ ID NO. 34 in conjunction with SEQ ID NO. 36 and 37 plus Vκ1 family SEQ ID NO. 56 and 57; Vκ2 family SEQ ID NO. 59; Vκ3 family SEQ ID NO. 61; Vκ4 family SEQ ID NO. 63; Vκ5 family SEQ ID NO. 65; Vκ6 family SEQ ID NO. 67; Vκ7 family SEQ ID NO. 69; Vκ8 family SEQ ID NO. 71; Vκ9 family SEQ ID NO. 73; Vκ10 family SEQ ID NO. 75; Vκ11 family SEQ ID NO. 77; Vκ12 family SEQ ID NO. 79; Vκ13 family SEQ ID NO. 81; Vκ14 family SEQ ID NO. 83; Vκ15 family SEQ ID NO. 85; Vκ16 family SEQ ID NO. 87; Vκ17 family SEQ ID NO. 89; Vκ18 family SEQ ID NO. 91; Vκ19 family SEQ ID NO. 93 in conjunction with SEQ ID NO. 95 plus Vλ1 and Vλ2 SEQ ID NO. 100; Vλ3 SEQ ID NO. 102; Vλ4, Vλ5, Vλ6, Vλ7 and Vλ8 SEQ ID NO. 104 in conjunction with SEQ ID NO. 107 and 108.
Example 2
Construction of Rearranged V Gene Family-Specific Libraries
V gene family-specific primers and AmpliTaq DNA polymerase were used for amplifying 6 randomly selected VH (VH1, VH4, VH5, VH8, VH11 and VH15) and 6 Vκ (Vκ3, Vκ4, Vκ11, Vκ13, Vκ14 and Vκ17) families and the 3 Vλ (i.e. Vλ1, Vλ2 and Vλ3) genes using splenocyte genomic DNA from CBA/J mice. The PCR products obtained were purified using columns (RBC, Taiwan), cloned in TOPO-TA cloning vector (Invitrogen, USA) and transformed into E. coli strain XL1 Blue. Four to six recombinants from each V gene family-specific library were sequenced using an automated DNA sequencer. IMGT and IgBLAST databases were used to assign the germline gene segments utilized in the H and L chain. L chains with the same VL and JL gene segments were considered independent rearrangements if the VL to JL junctions were different. Rearranged V genes recovered from the libraries that had identical sequences were scored once.
The VH1, VH4, VH5, VH8, VH11 and VH15 families were amplified by nested PCR using primer sets 3, 6, 7, 10, 13 and 17, respectively. The Vκ3, Vκ4, Vκ11, Vκ13, Vκ14 and Vκ17 families were amplified by nested PCR using primer sets 23, 24, 31, 33, 34 and 37, respectively. The Vλ1 and Vλ2, and Vλ3 gene were amplified by nested PCR using primer sets 41 and 42, respectively (Table 6).
A library targeting the complete complement of immunoglobulin H and L chain rearrangements can be constructed using H chain primer sets 3-18, κ L chain primer sets 21-39 and λ L chain primer sets 41-43 (Table 6).
Example 3
Preparation of Pooled 5′ External and Internal Primer Mixes
For applications where the identity of the rearranged/expressed VH and VL gene is not known, we used pooled primer mixes for (RT-) PCR. Separate external and internal primer mixes were made for VH, Vκ and Vλ gene families. The VH external primer mix was prepared by pooling 17 external primers targeting all the VH gene families at a concentration of 2 μM each (Table 1). Similarly, the VH internal primer mix was prepared by pooling the 17 internal primers targeting all the VH gene families. Likewise, external and internal primer mixes were made for the Vκ and Vλ gene families (Table 2 and Table 3).
Pooled 5′ external VH primer mix consisted of primers 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 and SEQ ID NO; 33.
Pooled 5′ external Vκ primer mix consisted of primers 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 and SEQ ID NO: 92.
Pooled 5′ external Vλ primer mix consisted of primers SEQ ID NO. 99, SEQ ID NO. 101 and SEQ ID NO: 103.
Pooled 5′ internal VH primer mix consisted of primers 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 and SEQ ID NO: 34.
Pooled 5′ internal Vκ primer mix consisted of primers 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 and SEQ ID NO: 93.
Pooled 5′ internal Vλ primer mix consisted of primers SEQ ID NO. 100, SEQ ID NO. 102 and SEQ ID NO: 104.
Each 5′ external and internal primer was used at a final concentration of 0.1 μM in the first and second round PCR. The same 5′ primer mixes were used for amplifying the rearranged/expressed VH and VL genes from splenocyte genomic DNA or total RNA from splenocytes/hybridomas. The 3′ were used at a final concentration of 0.25 μM as described above. JH or JL (for genomic DNA template) and CH or CL (for RNA template) primers were used as antisense primers.
For genomic DNA template:
JH external primer SEQ ID NO. 35; JH internal primers SEQ ID NO. 36 and SEQ ID NO. 37; Jκ external primer SEQ ID NO. 94; Jκ internal primer SEQ ID NO. 95; Jλ external primer SEQ ID NO. 105 and SEQ ID NO. 106; Jλ internal primer SEQ ID NO. 107 and SEQ ID NO. 108.
For RNA template:
Pooled 3′ RT primer mix: SEQ ID NOs. 38-42, SEQ ID NO. 96 and SEQ ID NO. 109.
Pooled 3′CH external primer mix comprised of SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 51 and SEQ ID NO. 53.
The 3′ Cκ external primer was SEQ ID NO. 97.
The 3′ Cλ external primer was SEQ ID NO. 110.
Pooled 3′CH internal primer mix comprised of SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, and SEQ ID NO; 54.
The 3′Cκ internal primer was SEQ ID NO. 98.
Pooled 3′ Cλ internal primer mix comprised of SEQ ID NO. 111 and SEQ ID NO: 112.
Example 4
cDNA Synthesis and PCR Amplification of Rearranged VH and VL Genes from Hybridomas
Total RNA was extracted from 107 hybridoma cells using a commercially available RNA isolation kit following the manufacturer's instructions (Qiagen, Germany). The purified RNA was quantitated spectrophotometrically. The first strand cDNA for the expressed H and L chain was synthesized using a commercially available RT-PCR kit (Applied Biosystems, USA) and CH or CL RT antisense primers (FIG. 1 and Tables 1-3, SEQ ID NO: 1-112). For H chain isotype specific RT primer was used for the reverse transcription reaction. The corresponding primers used for the κ and λ chains were RTCκ and RTCλ, respectively. The 20 μl reverse transcription reaction mixture consisted of 1×PCR buffer II, 0.75 μM antisense primer, 10 mM dNTP mix, 5 mM MgCl2, 1 U of RNase inhibitor, 50 U of MuLV reverse transcriptase (Applied Biosystems, USA) and 10 μg of total RNA. The reaction was performed at 42° C. for 1 h. Reverse transcriptase was inactivated by incubating at 99° C. for 5 min. Five μl of unpurified cDNA was used as a template for the first round PCR. The concentration of the components and cycling parameters were the same as described above for the genomic PCR except that PCR buffer 1, AmpliTaq DNA polymerase, and J region primers were replaced by Pfu Ultra Hotstart buffer, Pfu Ultra Hotstart DNA polymerase (Stratagene, USA) and C region primers, respectively. For experiments involving low copy templates e.g. small numbers of B cells or single B cells, Taq DNA polymerase was preferred over Pfu DNA polymerase because of its higher amplification efficiency.
Example 5
Determination of Detection Limit by RT-PCR
For determining the detection limit by RT-PCR, hybridoma cells (IgM, κ) suspended in diethyl pyrocarbonate treated PBS were first counted using a haemocytometer and used in the range of 1 to 100 cells. The expressed Vκ and VH gene was amplified using Vκ and VH pooled primers respectively, in conjunction with corresponding constant region primers, as described above. In addition, a second set of RT-PCRs were set up where the amplification cycles were increased from 25 to 50 for the first and second round PCR. The amplified product was analyzed on a 0.9% agarose gel.
Pooled 3′ RT primer mix: SEQ ID NOs. 38-42 and SEQ ID NO; 96.
Pooled 5′ external VH primer mix consisted of primers 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 and SEQ ID NO. 33.
Pooled 5′ external Vκ primer mix consisted of primers 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 and SEQ ID NO. 92.
Pooled 5′ internal VH primer mix consisted of primers 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 and SEQ ID NO. 34.
Pooled 5′ internal Vκ primer mix consisted of primers 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 and SEQ ID NO. 93.
Pooled 3′CH external primer mix comprised of SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 49, SEQ ID NO. 51 and SEQ ID NO. 53.
The 3′Cκ external primer mix was SEQ ID NO. 97.
Pooled 3′CH internal primer mix comprised of SEQ ID NO. 44, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, and SEQ ID NO. 54.
The 3′Cκ internal primer was SEQ ID NO. 98.
Example 6
Sequencing and Sequence Analysis of the (RT)-PCR Amplified Rearranged/Expressed VH and VL Genes
RT-PCR amplified VH and VL genes expressed in the hybridomas were column- or gel-purified (Qiagen, Germany) and sequenced using the appropriate CH, Cκ or Cλ internal antisense primer (FIG. 1 and Tables 1-3). The nucleotide sequence was analyzed with MacVector software. The family to which the expressed VH and VL genes belong was assigned using IMGT and IgBLAST databases. Once the V gene family was identified, we used the corresponding 5′ internal primer to sequence the second strand of the amplified product. In some experiments the (RT-) PCR product was cloned and recombinants from the resultant genomic/cDNA library were sequenced. Irrespective of whether the amplified product was sequenced directly or sequence was from a recombinant clone, the nucleotide sequence analysis was restricted to the sequence internal to the 5′ and 3′ internal primer. This was done to prevent mutations introduced by the primer during (RT-) PCR from affecting the sequence analysis.
Example 7
RT-PCR Amplification of H and L Chain Transcripts from Splenocytes Using Pooled Constant Region Specific Primers
Total RNA was isolated from splenocytes from BALB/c mice as described above for hybridomas. The RT-PCR protocol used for hybridomas was followed for splenocytes with the following modifications. First strand cDNA was synthesized for H and L chain transcripts from splenocytes using pooled constant region specific primers. RTCμ, RTCγ, RTCα, RTCκ and RTCλ were used at a final concentration of 0.15 μM each (Tables 1-3). Three μL of unpurified cDNA was used as template for amplifying IgM, IgG, IgA, Igκ and Igλ V gene transcripts in separate nested PCRs as described above. Independent PCRs were setup for IgG3 and the remaining IgG isotypes; IgD and IgE isotypes were not analyzed. The constant region specific primer cocktail was used in the first and second round PCR at a final concentration of 0.25 μM and each primer was present in equimolar concentration. The appropriate (external or internal) VH, Vκ and Vλ primer pool was used for PCR amplifying the expressed IgH, Igκ and Igλ V genes, respectively. The second round PCR product was cloned in pCR2.1-TOPO (Invitrogen, USA) and transformed into TOP10 E. coli strain. The nucleotide sequence of the recombinants obtained from the resultant 6 cDNA libraries was analyzed as described above.
Pooled 5′ external VH primer mix consisted of primers 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 and SEQ ID NO. 33.
Pooled 5′ external Vκ primer mix consisted of primers 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 and SEQ ID NO. 92.
Pooled 5′ external Vλ primer mix consisted of primers SEQ ID NO. 99, SEQ ID NO. 101 and SEQ ID NO. 103.
Pooled 5′ internal VH primer mix consisted of primers 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 and SEQ ID NO. 34.
Pooled 5′ internal Vκ primer mix consisted of primers 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 and SEQ ID NO: 93.
Pooled 5′ internal Vλ primer mix consisted of primers SEQ ID NO. 100, SEQ ID NO. 102 and SEQ ID NO. 104.
Example 8
Identification of the Expressed VH and VL Genes from Pooled Hybridomas
Equal amounts (200 ng/hybridoma) of total RNA from 5 previously characterized IgM expressing hybridomas were mixed, and cDNA synthesized using H and L chain constant region specific antisense primers in 2 separate reverse transcription reactions. The expressed VH and VL genes in the hybridomas belonged to VH1, VH2, VH3, VH7 and VH14, and Vκ5, Vκ9, Vκ12, Vκ18 and Vλ2 gene families. Separate cocktails comprising of all VH and VL (containing of both K and λ) primers and AmpliTaq DNA polymerase were used to amplify the expressed VH and VL genes from the cDNA pool. The H and L chain RT-PCR products were purified and separate libraries were constructed in TOPO-TA cloning vector. The ligated DNA was transformed in E. Coli strain XL1 Blue. Plasmid DNA isolated from the recombinants was digested with restriction enzyme(s) that can distinguish the VH and VL genes expressed in the 5 hybridomas. The recombinants selected after restriction profiling were confirmed by nucleotide sequencing.
Example 9
Staining, Microdissection and PCR Amplification of the Rearranged VH and VL Genes from Splenic B Cells
Spleen from naive BALB/c mice were embedded in OCT, frozen over liquid nitrogen vapours, and stored at −70° C. till further use. Seven micron sections were cut using a cryostat microtome (Thermo Shandon, UK), collected on glass slides and stored at −70° C. until required. For staining, the splenic sections were washed with PBS, blocked using 2% bovine serum albumin (Vector Laboratories, USA) and incubated with rat anti-mouse B220 primary Ab (GE Biosciences, USA). The splenic sections were further incubated with horseradish peroxidase labeled goat anti-rat Ig. The B220+ cells were visualized using 3,3′-diaminobenzidine (Vector Laboratories, USA) and H2O2. The sections were washed with water and dehydrated in ethanol (once in 75% ethanol for 30 s, once in 95% ethanol for 30 s and thrice in absolute ethanol for 2 min each). The splenic section was subjected to three changes of xylene. The microdissection was performed using a hydraulic micromanipulator (Narashige, Japan) assembled on an inverted microscope (Nikon, Japan). Ten to fifteen B220+ cells were microdissected from the immunohistochemically stained splenic section, transferred to a 0.2 ml PCR tube containing 10 μl of 1×PCR buffer (Applied Biosystems, USA) and stored at −20° C. until required. The rearranged VH and Vκ genes were directly PCR amplified from the microdissected B cells using pooled primer mixes comprising of primers targeting all the VH and Vκ gene families, and the corresponding J region primer(s) as described above. The number of B cells microdissected was estimated by comparing the image of the stained section taken before and after microdissection.
Example 10
The rearranged H chain were amplified by nested PCR using splenocyte genomic DNA as template. The 50 μl first round PCR mix consisted of 1×PCR buffer 1,200 μM dNTPs, 0.25 μM external 5′ (V gene) and 3′ (J region) primers each, and 2.5 units of AmpliTaq DNA polymerase (Applied Biosystems, USA). Touchdown PCR was set up on a GeneAmp 2700 PCR System (Applied Biosystems, USA). The touchdown part of the PCR programme started with an initial denaturation step at 95° C. for 2 min. This was followed by denaturation at 94° C. for 1 min, annealing at 62° C. for 1 min and an extension at 72° C. for 1 min. The annealing temperature was decreased from 62° C. to 52° C. over 5 cycles at the rate of 2° C. per cycle. This was followed by 25 cycles consisting of denaturation at 94° C. for 1 min, annealing at 52° C. for 1 min and an extension at 72° C. for 1 min. A final extension step was carried out at 72° C. for 10 min. The first round primer mix comprised of SEQ ID NOs: 1, 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35. The composition of the reaction mixture and cycling parameters for the second round PCR were same as were used for the first round PCR except that 2 μl of the unpurified first round PCR product served as template for the second round PCR in place of splenocyte genomic DNA. The second round primer set comprised of SEQ ID NOs. 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 37.
Example 10 describes the process of amplifying rearranged H chain by nested PCR using splenocyte genomic DNA as template. The same process can be repeated for amplifying the κ and λ L chains using the corresponding primers described in Table 1-3.
Example 11
The rearranged H chains were amplified by nested PCR using total or polyA+ RNA as template. The 20 μl reverse transcription reaction mixture consisted of 1×PCR buffer II, 0.75 μM antisense primer, 10 mM dNTP mix, 5 mM MgCl2, 1 U of RNase inhibitor, 50 U of MuLV reverse transcriptase (Applied Biosystems, USA) and 10 μg of total RNA. The reaction was performed at 42° C. for 1 h. Reverse transcriptase was inactivated by incubating at 99° C. for 5 min. The reverse transcription reaction was done using a primer mix comprising of SEQ ID NOs. 38-42.
The 50 μl first round PCR mix consisted of 1×PCR buffer 1,200 μM dNTPs, 0.25 μM external 5′ (V gene) and 3′ (C region) primers each, and 2.5 units of Pfu Ultra Hotstart buffer and Pfu Ultra Hotstart DNA polymerase (Stratagene, USA) (Applied Biosystems, USA). Touchdown PCR was set up on a GeneAmp 2700 PCR System (Applied Biosystems, USA). The touchdown part of the PCR programme started with an initial denaturation step at 95° C. for 2 min. This was followed by denaturation at 94° C. for 1 min, annealing at 62° C. for 1 min and an extension at 72° C. for 1 min. The annealing temperature was decreased from 62° C. to 52° C. over 5 cycles at the rate of 2° C. per cycle. This was followed by 25 cycles consisting of denaturation at 94° C. for 1 min, annealing at 52° C. for 1 min and an extension at 72° C. for 1 min. A final extension step was carried out at 72° C. for 10 min. The first round PCR was done using a primer set consisting of SEQ ID NO: 1, 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 43, 45, 46, 49, 51 and 53. Five μl of unpurified cDNA was used as a template for the first round PCR. The composition of the reaction mixture and cycling parameters for the second round PCR were same as were used for the first round PCR except that 2 μl of the unpurified first round PCR product served as template for the second round PCR in place of genomic DNA. The primer set for the second round PCR comprised of SEQ ID No. 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 44, 47, 48, 50, 52 and 54.
Example 11 describes the process of amplifying the rearranged H chain by nested PCR using total or polyA+ RNA as template. The same process can be used for amplifying the κ and λ L chains using the corresponding primers described in Table 1-3.
BIBLIOGRAPHY


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    Chardes, T., Villard, S., Ferrieres, G., Piechaczyk, M., Cerutti, M., Devauchelle, G. and Pau, B. (1999) Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family. FEBS Lett 452, 386-94.
    Chiang, Y. L., Sheng-Dong, R., Brow, M. A. and Larrick, J. W. (1989) Direct cDNA cloning of the rearranged immunoglobulin variable region. Biotechniques 7, 360-6.
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    Guo, L., Zhang, X., Zheng, B. and Han, S. (2008) IgM-mediated signaling is required for the development of a normal B cell memory response. Mol Immunol 45, 1071-7.
    Jacob, J., Kassir, R. and Kelsoe, G. (1991a) In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J Exp Med 173, 1165-75.
    Jacob, J., Kelsoe, G., Rajewsky, K. and Weiss, U. (1991b) Intraclonal generation of antibody mutants in germinal centres. Nature 354, 389-92.
    Jones, S. T. and Bendig, M. M. (1991) Rapid PCR-cloning of full-length mouse immunoglobulin variable regions. Biotechnology (N Y) 9, 88-9.
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    Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R. and Pluckthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods 201, 35-55.
    Lan, F., Umeda, M. and Inoue, K. (1996) Extended fitness of variable region primers by a novel PCR protocol. J Immunol Methods 195, 27-32.
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    LeBoeuf, R. D., Galin, F. S., Hollinger, S. K., Peiper, S. C. and Blalock, J. E. (1989) Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction. Gene 82, 371-7.
    Nicholls, P. J., Johnson, V. G., Blanford, M. D. and Andrew, S. M. (1993) An improved method for generating single-chain antibodies from hybridomas. J Immunol Methods 165, 81-91.
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    Ruberti, F., Cattaneo, A. and Bradbury, A. (1994) The use of the RACE method to clone hybridoma cDNA when V region primers fail. J Immunol Methods 173, 33-9.
    Sehgal, D., Schiaffella, E., Anderson, A. O. and Mage, R. G. (1998) Analyses of single B cells by polymerase chain reaction reveal rearranged VH with germline sequences in spleens of immunized adult rabbits: implications for B cell repertoire maintenance and renewal. J Immunol 161, 5347-56.
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TABLE 1







Oligonucleotide primers for PCR amplification of


rearranged VH genes from genomic DNA and total RNA















Total








Family
no. of








or
genes

Fold
Mismatchese

Fold
Mismatches






















genea
(%)b
External primersc
Degd
0
1
2
3
4
Internal primers
Deg
0
1
2
3
4

























VH1f
193 
agRtYcagctgcaRcagtct
8
103
42
10
6
3
gaRgatRtcctgYaaggcttc
8
126
37
18
2
1



(55)
aggtccaactgcagcagcc
1





aRgBtgtcctgcaagRcttc
12







 


VH2g
14
tctgcctggtgacWttccca
2
7

1
1

tgcagctgaagSagtcagga
2
9
1






(4)
















 


VH3
 9
gtgcagcttcaggagtcag
1
6
2


1
aaccttctcagWcactgtcc
2

4
5





(2.6)
















 


VH4
 3
gaggtgaagcttctcgagtc
1
2
1



ggaggtggcctggtgcag
1
3







(0.9)
















 


VH5h
52
gaagtgaagctggtggagtc
1
5
9
20
5

agcctggagggtccctgaa
1
2
34
6
1
4



(14.8
















 


VH6i
 6
atgKacttgggactgaRctgt
4
2
3



gaggagtctggaggaggctt
1
3
2
1





(1.7)
















 


VH7
 7
cagtgtgaggtgaagctggt
1
5
2



tctggaggaggcttggtaca
1
6
1






(2)
















 


VH8j
22
ccaggttactctgaaagagtc
1
11
2
3
3

ctgggatattgcagccctcc
1
4
8
6
1




(6.3)
















 


VH9k
21
tgtggaccttgctattcctga
1
13
4

1

acagatccagttggtgcagt
1
15
6






(6)
















 


VH10l
 8
tgttggggctgaagtgggttt
1
5
2



aggtgtgcattgtgaggtgc
1
7
1






(2.3)
















 


VH11
 3
atggagtgggaactgagctta
1
3




gaagtgcagctgttggagac
1
3







(0.9)
















 


VH12
 2
agcttcaggagtcaggacc
1
2




cctggtgaaaccctcacag
1
2







(0.6)
















 


VH13
 2
caggtgcagcttgtagagac
1
2




aggcttggtgaggcctgga
1
2







(0.6)
















 


VH14m
 5
atgcagctgggtcatcttctt
1
2
1
1


gaggttcagctgcagcagt
1
5







(1.4)
















 


VH15
 2
gactggatttggatcacKctc
2
1
1



caatcccaggttcacctacaa
1
2







(0.6)
















 


VH16
 1
tggagtttggacttagttggg
1
1




gtgaggtgcagctggtgga
1
1







(0.3)
















 


JH
 4
ctYacctgaggagacDgtga
6
2
1
1


gtggtccctgcgccccag
1
2
2














ggtBccttggccccagta
3







 


RTCμ
 1
gatgacttcagtgttgt
1
1













 


RTCγ
 5
cagggatccaKagttc
1
4
1












 


RTCα
 1
caggtcacattcatcg
1
1













 


RTCε
 1
cacagtgctcatgttca
1
1













 


RTCδn
 1
agtggctgacttccaa
1
1













 


Cμ
 1
catggccaccagattcttatc
1
1




agacatttgggaaggactgac
1
1






 


Cγ
 5
agggaaataRcccttgaccag
1
4

1


ggccagtggatagacHgatg
3
5








agggaagtagcctttgacaag
1





cagggaccaagggatagaca
1







 


Cα
 1
gaatcaggcagccgattatcac
1
1




tgggagtgtcagtgggtaga
1
1






 


Cε
 1
tcatggaagcagtgcctttac
1
1




tagagctgagggttcctgata
1
1






 


Cδn
 1
tctgagaggaggaacatgtc
1
1




tcacctgcaagacagatagtg
1
1





aThe 16 pseudogenes that are grouped as ‘unclassified’ in the mouse germline VH IgBLAST database were very diverse in sequence and were not analyzed for designing primers.


bThe number of germline sequences present in the family analyzed. The proportion of genes contributed by a given family to the total number of genes present in our VH database is given in parenthesis.


cAll oligonucleotide primers are listed in 5′ to 3′ direction. The degenerate bases are shown in upper case. IUPAC code for mix bases: M, a/c; R, a/g; W, a/t; Y, c/t; S, c/g; K, g/t; H, a/c/t; V, a/c/g; D, a/g/t; B, c/g/t; N, a/c/g/t.


dFold degeneracy.


eNumber of sequences with the indicated number of mismatches vis-à-vis the primer sequence.


fEight sequences (AF455996, AF455989, AF455290, AF458189, AF459867, AF455983, AF303885 and AY672647) were incomplete and were excluded from the analysis. In addition, 21 sequences (not listed here) were not considered for designing the external primer, as they were incomplete at the 5′ end. The pseudogene J558.57pg.152 was not considered for designing the internal primer, as it was too divergent.


gFour pseudogenes (Q52.12pg.39, Q52.6pg.17, Q52.4pg.12 and Q52.1pg.2) either lacked the leader sequence or were too divergent in sequence and were not considered for designing the primers. The database entry U53526 (a functional gene) was not complete at the 5′ end and was not considered while designing the external primer.


hFive pseudogenes (7183.3pg.5, 7183.5pg.7, 7183.11pg.19, 7183.17pg.31 and 7183.21pg.38) were too divergent in sequence and were not considered for designing the primers. The sequences of eight functional genes (M18314, AF120462, AF290963, AF428078, U04228, U04230, U04231 and X67409) were incomplete at the 5′ end and could not be included while designing the external primer.


iGenBank entry X03398 lacked the leader sequence and was not considered for designing the external primer.


jThree pseudogenes 3609.2pg.138, 3609.10pg.167 and 3609N.1pg.68 were too divergent in sequence and were excluded from the analysis.


kThe sequences of three functional genes (L14364, L14367 and L14368) were incomplete at the 5′ end and could not be included while designing the external primer.


lPseudogene VH10.2pg.89 was not included in designing the external primer, as the sequence was incomplete at the 5′ end.


mX55934 was not included for designing the external primer, as sequence was incomplete at the 5′ end.


nUnder special experimental settings (Guo et al., 2008) one may be interested in analyzing the IgD repertoire.













TABLE 2







Oligonucleotide primers for PCR amplification of


rearranged Vκ genes from genomic DNA and total RNA















Total








Family
no. of








or
genes

Fold
Mismatches

Fold
Mismatches






















gene
(%)
External primers
Deg
0
1
2
3
4
Internal primers
Deg
0
1
2
3
4

























Vκ1a
13
tgatgacccaRactccact
2
8
1
2


cctgtcagtcttggagatca
1
7
1
2
1




(7.9)







tttgtcggttaccattggacaa
1







 


Vκ2b
11
gcttgtgctctggatccc
1
3
3

1

SRgatattgtgatgacgcagg
4
3
1
1
2




(6.7)
















 


Vκ3
12
ctgctgctctgggttcc
1
10
1

1

attgtgctgacccaatctcc
1
8
1
1
2




(7.3)
















 


Vκ4c
33
cagcttcctgctaatcagtg
1
20
8
1

1
aWtgtKctcacccagtctcc
4
23
6


1



(20.1)
















 


Vκ5
 6
ctcagatccttggacttHtg
3

5

1

gtctccagccaccctgtc
1
5
1






(3.7)
















 


Vκ6
12
tggagtcacagacYcagg
2
1
6
4
1

tgatgacccagtctcMcaaat
2
4
6
2





(7.3)
















 


Vκ7
 1
tggagtttcagacccagg
1
1




gcctgtgcagacattgtgat
1
1







(0.6)
















 


Vκ8
13
ctgctMtgggtatctggt
2
7
6



cctgtggggacattgtgatg
1
7
1
4
1




(7.9)
















 


Vκ9
 6
cWtcttgttgctctggtttc
2
3
3



acatccRgatgacYcagtct
4
4
2






(3.7)
















 


Vκ10
 3
gatgtcctctgctcagttc
1
3




ccagatgtgatatccagatg
1
2
1






(1.8)
















 


Vκ11
 4
cctgctgagttccttggg
1
1

1
2

gccagatgtgatgtYcaaatg
2
1
3






(2.4)
















 


Vκ12d
13
ctgctgctgtggcttaca
1
8
1
1
1

atccagatgactcagtctcc
1
7
3
3





(7.9)
















 


Vκ13e
18
ccttctcaacttctgctct
1
2

1

4
cctgatatgtgacatccRVat
6
4
2
1




 


Vκ14f
 8
agggcccYtgctcagttt
2
1
3
1


Magatgacccagtctccatc
2
2
1
2





(4.9)
















 


Vκ15g
 5
atgagggtccttgctgag
1
1


1

tgagatgtgacatccagatga
1
2







(3.1)
















 


Vκ16
 1
gaggttccaggttcaggt
1
1




ccagtgtgatgtccagataac
1
1







(0.6)
















 


Vκ17
 3
ccatgaccatgYtctcact
2
2
1



acaactgtgacccagtctcc
1
2
1






(1.8)
















 


Vκ18
 1
atggaaactccagcttcattt
1
1




acacaggctccagcttctct
1
1







(0.6)
















 


Vκ19
 1
atgagaccgtctattcagtt
1
1




gtgctcagtgtgacatccag
1
1







(0.6)
















 


Jκ
 5
tStacttacgtttBatttcca
6
2
1
1
1

tttBatttccagcttggtScc
6
2

3




 


RTCκ
 1
tcaagaagcacacgac
1
1













 


Cκ
 1
gcacctccagatgttaactg
1
1




gatggtgggaagatggatac
1
1





Also refer to the Table 1 footnotes.


aTwo pseudogenes (AJ231202 and AJ231204) were not included in the analysis as the sequences were too divergent.


bFour pseudogenes (AJ132682, AJ231261, AJ231265 and AJ231266) were not included in the analysis as the sequences were too divergent.


cTwo pseudogenes (AJ231220, AJ231230) and a functional gene (AJ231226) were not included in the analysis as the sequences were too divergent.


dJ00546 could not be included in designing the external primer as the GenBank entry does not cover the leader sequence. The database entry AJ235950 could not be included in designing the external primer as the sequence was too divergent.


eEleven pseudogenes (AJ132671-76, AJ132678-81 and AJ231272) were not included in the analysis as the sequences were too divergent.


fThree pseudogenes (AJ231237, AJ231246 and AJ231249) were not included in the analysis as the sequences were too divergent.


gThree pseudogenes (AJ231251, AJ231268 and AJ231270) were not included in the analysis as the sequences were too divergent.













TABLE 3







Oligonucleotide primers for PCR amplification of


rearranged Vλ genes from genomic DNA and total RNA















Total








Family
no. of








or
genes

Fold
Mismatches

Fold
Mismatches
















Gene
(%)
External primers
Deg
0
1
Internal primers
Deg
0
2





Vλ1/2
2
gcctggaYttcacttatactc
2
2

tcccaggctgttgtgactc
1
2




(66.7)










 


Vλ3
1
tggcctggactcctctctt
1
1

caacttgtgctcactcagtc
1
1




(33.3)










 


Vλ4/5/6/
5
actcagccaagctctgtg
1
5

ctctaggaagcacagtcaaac
1
5



 


Jλb
5
gYcactYacctaggacag
4
3
1
taggacagtcagtttggttcc
1
3
1




aaactacttacctaggacag
1


aggacagtgaccttggttcc
1




 


RTCλ
4
acaccagtgtggcYtt
2
3
1






 


Cλ
4
agctcctcagRggaaggtg
2
3
1
gaaacacggtgagWgtggg
2
3
1








gaaacagggtgactgatgg
1





aThis set of genes are absent in C57BL/6, BALB/c and other common laboratory mice (IMGT; http://imgt.cines.fr). We did not detect any PCR product with CBA/J splenocyte genomic DNA.;


bThe pseudogene Jλ3P was not included in the analysis.













TABLE 4







Sequence analysis of rearranged V genes recovered from


VH, Vκ and Vλ gene family-specific libraries










Recomb-



Gene
inants
Independent rearrangements











family
analyzed
V
D
J










Heavy chain











VH1
6
J558.47.137
DSP2.x
JH4




J558.11
DFL16.1, DFL16.2a
JH4




V6
DSP2.9
JH3




V6
DST4.3, DST4.2,
JH4





DST4-BALB/ca




V6
DSP2.12
JH4




V23
DQ52-C57BL/6,
JH2





DQ52-BALB/ca


VH4
4
X24.2.50
DSP2.x
JH1




X24.2.50
DSP2.x
JH2




X24.2.50
DQ52-C57BL/6
JH1




X24.2.50
DSP2.9
JH2


VH5
4
VH7183.a24.40
DSP2.x
JH3




VH7183.a28.48
DFL16.1j
JH1




VH7183.1j
DSP2.2
JH4


VH8
4
3609.7.153
DSP2.x
JH4




3609.4.142
DQ52-BALB/c
JH1




3609.12.174
DSP2.x
JH2




3609.12.174
DSP2.x
JH3


VH11
5
VH11.1.48
DSP2.12
JH4




VH11.1.48
DSP2.13
JH1




VH11.1.48
DSP2.5, DSP2.7,
JH1





DSP2.8a




VH11.1.48
DSP2.x, DSP2.8a
JH4


VH15
5
VH15B
DQ52-C57BL/6
JH2




VH15B
DSP2.9
JH4




VH15B
DSP2.x
JH2




VH15B
DSP2.13
JH2







κ light chain










Vκ3
4
21-4
Jκ1




21-10
Jκ2




21-5
Jκ1


Vκ4
5
ko4
Jκ1




ko4
Jκ2




kk4
Jκ2




kn4
Jκ1




ah4
Jκ1


Vκ11
6
if11
Jκ1, Jκ5a




if11
Jκ1




if11
Jκ1




if11
Jκ1




if11
Jκ1




if11
Jκ2


Vκ13
4
gm33
Jκ1




gm33
Jκ1




gm33
Jκ1




gm33
Jκ2


Vκ14
2
ba9
Jκ2




cb9
Jκ1


Vκ17
4
bw20
Jκ2




bt20
Jκ2




bt20
Jκ2




bt20
Jκ4







λ light chain










Vλ1⁄2
4
VL1
Jλ2




VL2
Jλ1




VL2
Jλ2


Vλ3
4
VLx
Jλ2





aAny one of the 2 or 3 gene fragments listed may have been used in the Ig rearrangement.













TABLE 5







Comparative analysis of degree of degeneracy in


V gene primer sets designed












V genes
No. of
Degeneracy
Average


Reference
covered
primers
range
degeneracy














LeBoeuf et al., 1989
VH, Vκ
 2
432-576
504


Larrick et al., 1990
VH, Vκ
 2
 32-128
80


Coloma et al., 1991
VH
 4
 32-512
280


Lan et al., 1996
VH, Vκ
 4
 1-512
152.3


Wang et al., 2000
VH, Vκ
 8
 1-256
66.4


Kettleborough et al.,
VH, Vκ
17
 4-128
43


1993


Jones and Bendig,
VH, Vκ
23
 1-128
14.7


1991


Essono et al., 2003
VH, Vκ
31
 1-147456
6090.1


Chardes et al., 1999
VH, Vκ
63
 1-12
2.5


Seijen et al., 2001
VH
16
 1-3
1.6


The present
VH, Vκ, Vλ
 77a
 1-12
1.8


invention





aAn analysis of all our V gene primers (38 external and 39 internal) is presented here for comparison purposes.













TABLE 6







Summary










PCR


Primer Set


template
Gene family
Primer SEQ ID Number
No.





Genomic DNA
All rearranged VH, Vκ and Vλ
1-37 + 55-95 + 99-108
Set 1



All rearranged VH
 1-37
Set 2



VH1 family
 1-4 + 35-37
Set 3



VH2 family
 5-6 + 35-37
Set 4



VH3 family
 7-8 + 35-37
Set 5



VH4 family
 9-10 + 35-37
Set 6



VH5 family
11-12 + 35-37
Set 7



VH6 family
13-14 + 35-37
Set 8



VH7 family
15-16 + 35-37
Set 9



VH8 family
17-18 + 35-37
Set 10



VH9 family
19-20 + 35-37
Set 11



VH10 family
21-22 + 35-37
Set 12



VH11 family
23-24 + 35-37
Set 13



VH12 family
25-26 + 35-37
Set 14



VH13 family
27-28 + 35-37
Set 15



VH14 family
29-30 + 35-37
Set 16



VH15 family
31-32 + 35-37
Set 17



VH16 family
33-37
Set 18



All rearranged VL
 55-95 + 99-108
Set 19



All rearranged Vκ
55-95
Set 20



Vκ1 family
55-57 + 94-95
Set 21



Vκ2 family
58-59 + 94-95
Set 22



Vκ3 family
60-61 + 94-95
Set 23



Vκ4 family
62-63 + 94-95
Set 24



Vκ5 family
64-65 + 94-95
Set 25



Vκ6 family
66-67 + 94-95
Set 26



Vκ7 family
68-69 + 94-95
Set 27



Vκ8 family
70-71 + 94-95
Set 28



Vκ9 family
72-73 + 94-95
Set 29



Vκ10 family
74-75 + 94-95
Set 30



Vκ11 family
76-77 + 94-95
Set 31



Vκ12 family
78-79 + 94-95
Set 32



Vκ13 family
80-81 + 94-95
Set 33



Vκ14 family
82-83 + 94-95
Set 34



Vκ15 family
84-85 + 94-95
Set 35



Vκ16 family
86-87 + 94-95
Set 36



Vκ17 family
88-89 + 94-95
Set 37



Vκ18 family
90-91 + 94-95
Set 38



Vκ19 family
92-95
Set 39



All rearranged Vλ
 99-108
Set 40



Vλ1 and Vλ2 genes
 99-100 + 105-108
Set 41



Vλ3 gene
101-102 + 105-108
Set 42



Vλ4, Vλ5, Vλ6, Vλ7 and Vλ8
103-108
Set 43



genes


Total or
Total expressed VH, Vκ and Vλ
1-34 + 38-93 + 96-104 +
Set 44


poly A+

109-112


RNA



All expressed H chains
 1-34 + 38-54
Set 45



IgM isotype
1-34 + 38 + 43-44
Set 46



IgG isotype
1-34 + 39 + 45-48
Set 47



IgA isotype
1-34 + 40 + 49-50
Set 48



IgE isotype
1-34 + 41 + 51-52
Set 49



IgD isotype
1-34 + 42 + 53-54
Set 50



VH1 family (all VH isotypes)
 1-4 + 38-54
Set 51



VH2 family (all VH isotypes)
 5-6 + 38-54
Set 52



VH3 family (all VH isotypes)
 7-8 + 38-54
Set 53



VH4 family (all VH isotypes)
 9-10 + 38-54
Set 54



VH5 family (all VH isotypes)
11-12 + 38-54
Set 55



VH6 family (all VH isotypes)
13-14 + 38-54
Set 56



VH7 family (all VH isotypes)
15-16 + 38-54
Set 57



VH8 family (all VH isotypes)
17-18 + 38-54
Set 58



VH9 family (all VH isotypes)
19-20 + 38-54
Set 59



VH10 family (all VH isotypes)
21-22 + 38-54
Set 60



VH11 family (all VH isotypes)
23-24 + 38-54
Set 61



VH12 family (all VH isotypes)
25-26 + 38-54
Set 62



VH13 family (all VH isotypes)
27-28 + 38-54
Set 63



VH14 family (all VH isotypes)
29-30 + 38-54
Set 64



VH15 family (all VH isotypes)
31-32 + 38-54
Set 65



VH16 family (all VH isotypes)
33-34 + 38-54
Set 66



All expressed L chains
55-93 + 96-104 + 109-112
Set 67



All expressed κL chains
55-93 + 96-98
Set 68



Vκ1 family
55-57 + 96-98
Set 69



Vκ2 family
58-59 + 96-98
Set 70



Vκ3 family
60-61 + 96-98
Set 71



Vκ4 family
62-63 + 96-98
Set 72



Vκ5 family
64-65 + 96-98
Set 73



Vκ6 family
66-67 + 96-98
Set 74



Vκ7 family
68-69 + 96-98
Set 75



Vκ8 family
70-71 + 96-98
Set 76



Vκ9 family
72-73 + 96-98
Set 77



Vκ10 family
74-75 + 96-98
Set 78



Vκ11 family
76-77 + 96-98
Set 79



Vκ12 family
78-79 + 96-98
Set 80



Vκ13 family
80-81 + 96-98
Set 81



Vκ14 family
82-83 + 96-98
Set 82



Vκ15 family
84-85 + 96-98
Set 83



Vκ16 family
86-87 + 96-98
Set 84



Vκ17 family
88-89 + 96-98
Set 85



Vκ18 family
90-91 + 96-98
Set 86



Vκ19 family
92-93 + 96-98
Set 87



All expressed λL chains
 99-104 + 109-112
Set 88



Vλ1 and Vλ2 genes
 99-100 + 109-112
Set 89



Vλ3 gene
101-102 + 109-112
Set 90



Vλ4, Vλ5, Vλ6, Vλ7 and Vλ8
103-104 + 109-112
Set 91



genes



















agRtYcagctgcaRcagtct
SEQ ID NO: 1


 



aggtccaactgcagcagcc
SEQ ID NO: 2


 



gaRgatRtcctgYaaggcttc
SEQ ID NO: 3


 



aRgBtgtcctgcaagRcttc
SEQ ID NO: 4


 



tctgcctggtgacWttccca
SEQ ID NO: 5


 



tgcagctgaagSagtcagga
SEQ ID NO: 6


 



gtgcagcttcaggagtcag
SEQ ID NO: 7


 



aaccttctcagWcactgtcc
SEQ ID NO: 8


 



gaggtgaagcttctcgagtc
SEQ ID NO: 9


 



ggaggtggcctggtgcag
SEQ ID NO: 10


 



gaagtgaagctggtggagtc
SEQ ID NO: 11


 



agcctggagggtccctgaa
SEQ ID NO: 12


 



atgKacttgggactgaRctgt
SEQ ID NO: 13


 



gaggagtctggaggaggctt
SEQ ID NO: 14


 



cagtgtgaggtgaagctggt
SEQ ID NO: 15


 



tctggaggaggcttggtaca
SEQ ID NO: 16


 



ccaggttactctgaaagagtc
SEQ ID NO: 17


 



ctgggatattgcagccctcc
SEQ ID NO: 18


 



tgtggaccttgctattcctga
SEQ ID NO: 19


 



acagatccagttggtgcagt
SEQ ID NO: 20


 



tgttggggctgaagtgggttt
SEQ ID NO: 21


 



aggtgtgcattgtgaggtgc
SEQ ID NO: 22


 



atggagtgggaactgagctta
SEQ ID NO: 23


 



gaagtgcagctgttggagac
SEQ ID NO: 24


 



agcttcaggagtcaggacc
SEQ ID NO: 25


 



cctggtgaaaccctcacag
SEQ ID NO: 26


 



caggtgcagcttgtagagac
SEQ ID NO: 27


 



aggcttggtgaggcctgga
SEQ ID NO: 28


 



atgcagctgggtcatcttctt
SEQ ID NO: 29


 



gaggttcagctgcagcagt
SEQ ID NO: 30


 



gactggatttggatcacKctc
SEQ ID NO: 31


 



caatcccaggttcacctacaa
SEQ ID NO: 32


 



tggagtttggacttagttggg
SEQ ID NO: 33


 



gtgaggtgcagctggtgga
SEQ ID NO: 34


 



ctYacctgaggagacDgtga
SEQ ID NO: 35


 



gtggtccctgcgccccag
SEQ ID NO: 36


 



ggtBccttggccccagta
SEQ ID NO: 37


 



gatgacttcagtgttgt
SEQ ID NO: 38


 



cagggatccaKagttc
SEQ ID NO: 39


 



caggtcacattcatcg
SEQ ID NO: 40


 



cacagtgctcatgttca
SEQ ID NO: 41


 



agtggctgacttccaa
SEQ ID NO: 42


 



catggccaccagattcttatc
SEQ ID NO: 43


 



agacatttgggaaggactgac
SEQ ID NO: 44


 



agggaaataRcccttgaccag
SEQ ID NO: 45


 



agggaagtagcctttgacaag
SEQ ID NO: 46


 



ggccagtggatagacHgatg
SEQ ID NO: 47


 



cagggaccaagggatagaca
SEQ ID NO: 48


 



gaatcaggcagccgattatcac
SEQ ID NO: 49


 



tgggagtgtcagtgggtaga
SEQ ID NO: 50


 



tcatggaagcagtgcctttac
SEQ ID NO: 51


 



tagagctgagggttcctgata
SEQ ID NO: 52


 



tctgagaggaggaacatgtc
SEQ ID NO: 53


 



tcacctgcaagacagatagtg
SEQ ID NO: 54


 



tgatgacccaRactccact
SEQ ID NO: 55


 



cctgtcagtcttggagatca
SEQ ID NO: 56


 



tttgtcggttaccattggacaa
SEQ ID NO: 57


 



gcttgtgctctggatccc
SEQ ID NO: 58


 



SRgatattgtgatgacgcagg
SEQ ID NO: 59


 



ctgctgctctgggttcc
SEQ ID NO: 60


 



attgtgctgacccaatctcc
SEQ ID NO: 61


 



cagcttcctgctaatcagtg
SEQ ID NO: 62


 



aWtgtKctcacccagtctcc
SEQ ID NO: 63


 



ctcagatccttggacttHtg
SEQ ID NO: 64


 



gtctccagccaccctgtc
SEQ ID NO: 65


 



tggagtcacagacYcagg
SEQ ID NO: 66


 



tgatgacccagtctcMcaaat
SEQ ID NO: 67


 



tggagtttcagacccagg
SEQ ID NO: 68


 



gcctgtgcagacattgtgat
SEQ ID NO: 69


 



ctgctMtgggtatctggt
SEQ ID NO: 70


 



cctgtggggacattgtgatg
SEQ ID NO: 71


 



cWtcttgttgctctggtttc
SEQ ID NO: 72


 



acatccRgatgacYcagtct
SEQ ID NO: 73


 



gatgtcctctgctcagttc
SEQ ID NO: 74


 



ccagatgtgatatccagatg
SEQ ID NO: 75


 



cctgctgagttccttggg
SEQ ID NO: 76


 



gccagatgtgatgtYcaaatg
SEQ ID NO: 77


 



ctgctgctgtggcttaca
SEQ ID NO: 78


 



atccagatgactcagtctcc
SEQ ID NO: 79


 



ccttctcaacttctgctct
SEQ ID NO: 80


 



cctgatatgtgacatccRVat
SEQ ID NO: 81


 



agggcccYtgctcagttt
SEQ ID NO: 82


 



Magatgacccagtctccatc
SEQ ID NO: 83


 



atgagggtccttgctgag
SEQ ID NO: 84


 



tgagatgtgacatccagatga
SEQ ID NO: 85


 



gaggttccaggttcaggt
SEQ ID NO: 86


 



ccagtgtgatgtccagataac
SEQ ID NO: 87


 



ccatgaccatgYtctcact
SEQ ID NO: 88


 



acaactgtgacccagtctcc
SEQ ID NO: 89


 



atggaaactccagcttcattt
SEQ ID NO: 90


 



acacaggctccagcttctct
SEQ ID NO: 91


 



atgagaccgtctattcagtt
SEQ ID NO: 92


 



gtgctcagtgtgacatccag
SEQ ID NO: 93


 



tStacttacgtttBatttcca
SEQ ID NO: 94


 



tttBatttccagcttggtScc
SEQ ID NO: 95


 



tcaagaagcacacgac
SEQ ID NO: 96


 



gcacctccagatgttaactg
SEQ ID NO: 97


 



gatggtgggaagatggatac
SEQ ID NO: 98


 



gcctggaYttcacttatactc
SEQ ID NO: 99


 



tcccaggctgttgtgactc
SEQ ID NO: 100


 



tggcctggactcctctctt
SEQ ID NO: 101


 



caacttgtgctcactcagtc
SEQ ID NO: 102


 



actcagccaagctctgtg
SEQ ID NO: 103


 



ctctaggaagcacagtcaaac
SEQ ID NO: 104


 



gYcactYacctaggacag
SEQ ID NO: 105


 



aaactacttacctaggacag
SEQ ID NO: 106


 



taggacagtcagtttggttcc
SEQ ID NO: 107


 



aggacagtgaccttggttcc
SEQ ID NO: 108


 



acaccagtgtggcYtt
SEQ ID NO: 109


 



agctcctcagRggaaggtg
SEQ ID NO: 110


 



gaaacacggtgagWgtggg
SEQ ID NO: 111


 



gaaacagggtgactgatgg
SEQ ID NO: 112






All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

## Claims

1. A set of oligonucleotides 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 set comprises:
a. 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104; and
b. 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.

2. 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, wherein said process comprises:
a. providing a sample;
b. providing 5′ oligonucleotides as set forth in SEQ ID NO: 1-34, SEQ ID NO: 55-93 and SEQ ID NO: 99-104;
c. 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;
d. performing a polymerase chain reaction; and
e. detecting presence of an amplified product.

3. 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; said process comprising
a. providing a sample;
b. 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;
c. performing a first round of polymerase chain reaction to obtain first product;
d. 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
e. detecting presence of an amplified product;
or said process comprising:
a. providing a sample;
b. performing reverse transcription 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;
c. 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: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, 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: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, and SEQ ID NO: 110;
d. performing a first round of polymerase chain reaction using cDNA to obtain first product;
e. 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: 44, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, 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: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 111 and SEQ ID NO: 112; and
f. detecting presence of an amplified product.

4. The process of claim 2 or 3, wherein said cancer is B cell lymphoma.

5. The process of claim 2 or 3, wherein said cell is a hybridoma cell and/or a B cell.

6. A process for constructing a library of polynucleotides encoding immunoglobulin genes, said process comprising:
a. 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;
b. cloning said amplified product in an expression vector to obtain a recombinant expression vector; and
c. transforming said recombinant expression vector in a host cell.

7. The process of claim 6, wherein the vector is a prokaryotic expression vector and the host cell is E. coli.

8. 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 said 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.