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

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 985-994 ◽  
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

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.

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

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 985-994 ◽  
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.

scholarly journals Rapid Molecular Cloning of Rearrangements of the IGHJ Locus Using Long-Distance Inverse Polymerase Chain Reaction

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2456-2464 ◽  
Author(s):  
T.G. Willis ◽  
D.M. Jadayel ◽  
L.J.A. Coignet ◽  
M. Abdul-Rauf ◽  
J.G. Treleaven ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
B Cell ◽  
Pcr Amplification ◽  
Response To Therapy ◽  
Chain Reaction ◽  
Long Distance ◽  
Cluster Region ◽  
Translocation Breakpoints ◽  
Alu Repeat ◽  
Polymerase Chain

Abstract Clonal rearrangements of the Ig heavy chain (IGH ) locus consisting of either intrachromosomal (VDJ ) rearrangements or interchromosomal translocations are a consistent feature of all B-cell malignancies and may be used both diagnostically and to monitor response to therapy. Many of these rearrangements are targeted to the IGHJ segments, but only some can be amplified with regular polymerase chain reaction (PCR) techniques. To permit PCR amplification of potentially all IGHJ rearrangements, we have devised a method incorporating self-ligation of restriction endonuclease-digested DNA fragments with long-distance PCR (long-distance, inverse PCR [LDI-PCR]). We show here, using only 4 nested oligonucleotide primers, the successful amplification and DNA sequencing of all IGHJ rearrangements up to 5.4 kb in length from a panel of 13 cases and cell lines of various types of B-cell malignancy. In all cases, both VDJ and DJ IGH rearrangements and translocation breakpoints were amplified. Six cases exhibited t(14; 18)(q32; q21). All translocation breakpoints were cloned and sequenced. Three cases exhibited a rearrangement to the BCL2 major breakpoint region (MBR). However, 2 other cases exhibited rearrangements between the MBR and the minor cluster region (mcr). These 2 cases broke within 44 bp of each other, confirming the presence of an additional 3′ BCL2 breakpoint cluster region. The final case fell immediately 3′ of the 3′ UTR of the BCL2 gene adjacent to an Alu repeat. No other BCL2 breakpoints within this region have been reported. Four cases exhibited t(11; 14)(q13; q32). All 3 cases with translocations targeted to the IGHJ segments were successfully amplified and sequenced, including 1 case in which the BCL1 translocation could not be detected by DNA blot using the currently available probes. All three translocation breakpoints fell outside the BCL1 major translocation cluster between 20 and 40 kb telomeric and showed no clustering. Two of the three fell within or adjacent to Alu repeat regions. LDI-PCR is a simple and robust technique that allows PCR amplification of nearly all IGHJ rearrangements.

scholarly journals Rapid Molecular Cloning of Rearrangements of the IGHJ Locus Using Long-Distance Inverse Polymerase Chain Reaction

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2456-2464 ◽  
Author(s):  
T.G. Willis ◽  
D.M. Jadayel ◽  
L.J.A. Coignet ◽  
M. Abdul-Rauf ◽  
J.G. Treleaven ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
B Cell ◽  
Pcr Amplification ◽  
Response To Therapy ◽  
Chain Reaction ◽  
Long Distance ◽  
Cluster Region ◽  
Translocation Breakpoints ◽  
Alu Repeat ◽  
Polymerase Chain

Clonal rearrangements of the Ig heavy chain (IGH ) locus consisting of either intrachromosomal (VDJ ) rearrangements or interchromosomal translocations are a consistent feature of all B-cell malignancies and may be used both diagnostically and to monitor response to therapy. Many of these rearrangements are targeted to the IGHJ segments, but only some can be amplified with regular polymerase chain reaction (PCR) techniques. To permit PCR amplification of potentially all IGHJ rearrangements, we have devised a method incorporating self-ligation of restriction endonuclease-digested DNA fragments with long-distance PCR (long-distance, inverse PCR [LDI-PCR]). We show here, using only 4 nested oligonucleotide primers, the successful amplification and DNA sequencing of all IGHJ rearrangements up to 5.4 kb in length from a panel of 13 cases and cell lines of various types of B-cell malignancy. In all cases, both VDJ and DJ IGH rearrangements and translocation breakpoints were amplified. Six cases exhibited t(14; 18)(q32; q21). All translocation breakpoints were cloned and sequenced. Three cases exhibited a rearrangement to the BCL2 major breakpoint region (MBR). However, 2 other cases exhibited rearrangements between the MBR and the minor cluster region (mcr). These 2 cases broke within 44 bp of each other, confirming the presence of an additional 3′ BCL2 breakpoint cluster region. The final case fell immediately 3′ of the 3′ UTR of the BCL2 gene adjacent to an Alu repeat. No other BCL2 breakpoints within this region have been reported. Four cases exhibited t(11; 14)(q13; q32). All 3 cases with translocations targeted to the IGHJ segments were successfully amplified and sequenced, including 1 case in which the BCL1 translocation could not be detected by DNA blot using the currently available probes. All three translocation breakpoints fell outside the BCL1 major translocation cluster between 20 and 40 kb telomeric and showed no clustering. Two of the three fell within or adjacent to Alu repeat regions. LDI-PCR is a simple and robust technique that allows PCR amplification of nearly all IGHJ rearrangements.

scholarly journals Use of the polymerase chain reaction to monitor the effectiveness of ex vivo tumor cell purging

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 654-660
Author(s):  
RS Negrin ◽  
HP Kiem ◽  
IG Schmidt-Wolf ◽  
KG Blume ◽  
ML Cleary
Keyword(s):  
Polymerase Chain Reaction ◽  
Tumor Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Magnetic Beads ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Chain Reaction ◽  
Polymerase Chain

The polymerase chain reaction (PCR) was used to detect residual malignant disease before and after ex vivo purging with monoclonal antibodies and complement or immunomagnetic treatment of BM samples contaminated with known numbers of t(14;18)-carrying tumor cells. Sensitivity of the PCR was demonstrated by detecting a specific t(14;18) amplification product in DNA extracted from a preparation consisting of one tumor cell among 10(5) normal cells. When BM contaminated with 1% to 5% t(14;18)-carrying cells from the B-cell lymphoma line SU-DHL-4 was subjected to two rounds of anti-B-cell pool of antibodies and complement (Ab-C) treatment a 3- to 4-log reduction of the pretreatment PCR signal was observed. A similar log-cell kill was detected using an independent clonogenic assay confirming the utility of the PCR approach. BM contaminated with a second B-cell lymphoma cell line, OCI-Ly8, was more resistant because a third cycle of Ab-C treatment was required to obtain a similar reduction in the PCR signal. A similar 4 logs of tumor cell removal was obtained using anti- B-cell antibodies conjugated to magnetic beads. These studies demonstrate that the t(14;18) PCR can be used to detect levels of tumor cells as low as 0.001%. This approach can be used to determine the effectiveness of BM purging in patients undergoing autologous BM transplantation as well as to assess the biologic role of minimal marrow disease.

scholarly journals Use of the polymerase chain reaction to monitor the effectiveness of ex vivo tumor cell purging

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 654-660 ◽  
Author(s):  
RS Negrin ◽  
HP Kiem ◽  
IG Schmidt-Wolf ◽  
KG Blume ◽  
ML Cleary
Keyword(s):  
Polymerase Chain Reaction ◽  
Tumor Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Magnetic Beads ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
B Cell Lymphoma ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract The polymerase chain reaction (PCR) was used to detect residual malignant disease before and after ex vivo purging with monoclonal antibodies and complement or immunomagnetic treatment of BM samples contaminated with known numbers of t(14;18)-carrying tumor cells. Sensitivity of the PCR was demonstrated by detecting a specific t(14;18) amplification product in DNA extracted from a preparation consisting of one tumor cell among 10(5) normal cells. When BM contaminated with 1% to 5% t(14;18)-carrying cells from the B-cell lymphoma line SU-DHL-4 was subjected to two rounds of anti-B-cell pool of antibodies and complement (Ab-C) treatment a 3- to 4-log reduction of the pretreatment PCR signal was observed. A similar log-cell kill was detected using an independent clonogenic assay confirming the utility of the PCR approach. BM contaminated with a second B-cell lymphoma cell line, OCI-Ly8, was more resistant because a third cycle of Ab-C treatment was required to obtain a similar reduction in the PCR signal. A similar 4 logs of tumor cell removal was obtained using anti- B-cell antibodies conjugated to magnetic beads. These studies demonstrate that the t(14;18) PCR can be used to detect levels of tumor cells as low as 0.001%. This approach can be used to determine the effectiveness of BM purging in patients undergoing autologous BM transplantation as well as to assess the biologic role of minimal marrow disease.

scholarly journals VARIASI GENETIK MADIDIHANG (Thunnus albacares; Bonnaterre, 1788) DENGAN ANALISIS MIKROSATELIT DI PERAIRAN INDONESIA

2018 ◽  
Vol 24 (3) ◽  
pp. 157
Author(s):  
Irwan Jatmiko ◽  
Fathur Rochman ◽  
Maya Agustina
Keyword(s):  
Polymerase Chain Reaction ◽  
Sustainable Use ◽  
Pcr Amplification ◽  
Yellowfin Tuna ◽  
Thunnus Albacares ◽  
Migratory Species ◽  
Chain Reaction ◽  
Long Distance ◽  
North Sulawesi ◽  
Polymerase Chain

Madidihang (Thunnus albacares) merupakan spesies yang bermigrasi jauh yang distribusinya di perairan tropis hingga perairan subtropis. Spesies ini ditemukan di Samudra Atlantik, Hindia dan Pasifik. Informasi genetik ikan dengan migrasi jauh seperti tuna penting diketahui untuk kepentingan pemanfaatan secara lestari. Penelitian ini bertujuan untuk memperoleh informasi keragaman genetik dan struktur populasi yang dieksploitasi dan kekerabatan populasi madidihang di perairan Indonesia. Pengumpulan sampel genetik dilakukan di tiga lokasi yaitu di Barat Sumatra, Selatan Bali dan perairan Sulawesi Utara. Metode yang digunakan adalah analisis mikrosatelit yang terdiri dari ekstraksi, purifikasi, amplifikasi polymerase chain reaction (PCR) dan elektroforesis. Hasil analisis terhadap 3 loci DNA mikrosatelit menunjukkan bahwa tingkat kekerabatan ketiga kelompok sampel relatif dekat yaitu berkisar antara 0,132-0,206. Hal ini menunjukkan bahwa Populasi madidihang di perairan Indonesia merupakan stok tunggal dan terjadi perkawinan acak. Meskipun demikian, sebagai spesies yang bermigrasi jauh lintas negara, pengelolaan madidihang juga memerlukan kerjasama yang baik antar negara yang tergabung dalam organisasi pengelolaan perikanan tuna regional.Yellowfin tuna (Thunnus albacares) is a highly migratory species that distribute from tropical to subtropical waters. This species can be found in the Atlantic, Indian and Pacific Oceans. Genetic information in fish with long distance migration such as tuna is very important for sustainable use. This study aims to obtain information on genetic diversity and population structure exploited and kinship of yellowfin tuna populations in Indonesian waters. Genetic sampling of yellowfin tuna was conducted in three locations in Indonesian waters in western Sumatra, southern Bali and North Sulawesi waters. The methods used was microsatellite analysis which consist of extraction, purification, polymerase chain reaction (PCR) amplification and electrophoresis. The result of 3 microsatellite DNA locus analysis showed that the level of kinship between the three sample groups in Indonesian waters was relatively close, ranging from 0.132 to 0.206. This shows that yellowfin tuna population in Indonesian waters is a single stock and random copulation. However, as a highly migratory species that migrate across the nations, yellowfin tuna management also requires good cooperation among countries incorporated in regional tuna fisheries management organizations.

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