Cloning of Immunoglobulin Chromosomal Translocations by Long-Distance Inverse Polymerase Chain Reaction

Abstract Junctional sequences created by chromosomal translocations in mature B- cell neoplasms, which involve immunoglobulin gene loci (IG) and putative proto-oncogenes on reciprocal partner chromosomes, are unique to neoplastic cells characterized by particular histological and immunological phenotypes. To establish a rapid and sensitive method to detect neoplastic cells carrying a specific chromosomal translocation, we have developed a novel strategy based on long-distance polymerase chain reaction (LD-PCR) amplification. Genomic DNA was extracted from tumor cells carrying t(14;19)(q32;q13), t(8;14)(q24;q32), t(3;22)(q27;q11), t(2;3)(p12;q27), or t(3;14)(q27;q32). Thirty-two to 35-mer oligonucleotide primer pairs were designed to be complementary to exons or flanking sequences of the BCL3, c-MYC and BCL6 oncogenes, and to IG constant region genes. LD-PCR with a newly available Taq polymerase for longer product synthesis successfully amplified fragments representing BCL3/C alpha junctional sequences for t(14;19); c-MYC/C mu, c-MYC/C gamma, and c-MYC/C alpha for t(8;14); BCL6/C lambda for t(3;22); BCL6/C kappa for t(2;3); 5′-BCL6/C mu, and 5′-BCL6/C gamma for t(3;14). In Burkitt's lymphoma/leukemia, all materials in which c- MYC rearrangements were detectable by conventional Southern blot hybridization showed positive LD-PCR amplification. The sizes of the amplified fragments varied from 1.8 kb to 12 kb, and these were specific to each material. Serial dilution of tumor cells or DNA in negative materials demonstrated a single band on agarose gel electrophoresis stained with ethidium bromide at a level of sensitivity of 10(-3), and hybridization with radioactive probe improved the level by one order of magnitude (1 cell in 10(4)), indicating that this LD- PCR approach is a sensitive technique capable of detecting minimal residual disease. Thus, the present study provided a useful tool for diagnosis and subsequent management of B-cell neoplasms characterized by specific chromosomal translocations.

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Application of long-distance polymerase chain reaction to detection of junctional sequences created by chromosomal translocation in mature B- cell neoplasms

Blood ◽

10.1182/blood.v88.3.985.bloodjournal883985 ◽

1996 ◽

Vol 88 (3) ◽

pp. 985-994 ◽

Cited By ~ 41

Author(s):

T Akasaka ◽

M Muramatsu ◽

H Ohno ◽

I Miura ◽

E Tatsumi ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

B Cell ◽

Tumor Cells ◽

Chromosomal Translocation ◽

Pcr Amplification ◽

Chromosomal Translocations ◽

Chain Reaction ◽

Long Distance ◽

Neoplastic Cells ◽

Polymerase Chain

Junctional sequences created by chromosomal translocations in mature B- cell neoplasms, which involve immunoglobulin gene loci (IG) and putative proto-oncogenes on reciprocal partner chromosomes, are unique to neoplastic cells characterized by particular histological and immunological phenotypes. To establish a rapid and sensitive method to detect neoplastic cells carrying a specific chromosomal translocation, we have developed a novel strategy based on long-distance polymerase chain reaction (LD-PCR) amplification. Genomic DNA was extracted from tumor cells carrying t(14;19)(q32;q13), t(8;14)(q24;q32), t(3;22)(q27;q11), t(2;3)(p12;q27), or t(3;14)(q27;q32). Thirty-two to 35-mer oligonucleotide primer pairs were designed to be complementary to exons or flanking sequences of the BCL3, c-MYC and BCL6 oncogenes, and to IG constant region genes. LD-PCR with a newly available Taq polymerase for longer product synthesis successfully amplified fragments representing BCL3/C alpha junctional sequences for t(14;19); c-MYC/C mu, c-MYC/C gamma, and c-MYC/C alpha for t(8;14); BCL6/C lambda for t(3;22); BCL6/C kappa for t(2;3); 5′-BCL6/C mu, and 5′-BCL6/C gamma for t(3;14). In Burkitt's lymphoma/leukemia, all materials in which c- MYC rearrangements were detectable by conventional Southern blot hybridization showed positive LD-PCR amplification. The sizes of the amplified fragments varied from 1.8 kb to 12 kb, and these were specific to each material. Serial dilution of tumor cells or DNA in negative materials demonstrated a single band on agarose gel electrophoresis stained with ethidium bromide at a level of sensitivity of 10(-3), and hybridization with radioactive probe improved the level by one order of magnitude (1 cell in 10(4)), indicating that this LD- PCR approach is a sensitive technique capable of detecting minimal residual disease. Thus, the present study provided a useful tool for diagnosis and subsequent management of B-cell neoplasms characterized by specific chromosomal translocations.

Download Full-text