Detection of minimal numbers of neoplastic cells carrying DNA translocations by DNA sequence amplification

Claim 1

A method for detecting neoplastic cells in an individual, said neoplastic cells being characterized by a t(14;18) chromosomal translocation related to a specific breakpoint clustering region, the method comprising the steps of: incubating a mixture comprising deoxyribonucleotide triphosphates, DNA from a cell sample of the individual, DNA polymerase, a first oligonucleotide primer and a second oligonucleotide primer, consisting essentially of base sequence 5'-TTTGACCTTTAG-3' or an oligonucleotide competing with said first primer for hybridizing to at least a portion of (-)-strand DNA of chromosome 18q21 immediately 5' to a molecular breakpoint hot spot region located in exon 3 and said second primer consisting essentially of base sequence 3'-CAGAGGAGTCCA-5' or an oligonucleotide competing with said second primer for hybridizing to a sequence 3' to an end of segment J H of chromosome 14 immunoglobulin heavy chain gene wherein is located a breakpoint clustering region, said incubating promoting annealing of the primers to the translocation crossover site and synthesis of coding or non-coding translocation DNA sequences and a substantial portion of flanking DNA sequences, the incubation then being terminated by DNA denaturation; repeating said incubation step without adding further cell sample in a cyclic manner to facilitate duplication of the original and newly synthesized translocated and substantial flanking DNA sequences, said repeating being carried out in a manner exponentially amplifying DNA sequences between and including DNA regions complementary to the first and second oligonucleotide primers; and analyzing the exponentially amplified DNA for oligonucleotide sequences characterizing the translocation region by separating the exponentially amplified DNA and hybridizing the amplified DNA with the first and second primers and identifying neoplastic cells by the presence of said sequences.

Claim 2

A method for detecting neoplastic cells in an individual, said neoplastic cells being characterized by a t(14;18) chromosomal translocation related to a specific breakpoint clustering region, the method comprising the steps of: incubating a mixture comprising deoxyribonucleotide triphosphates, DNA from a cell sample of the individual, DNA polymerase, a first oligonucleotide primer and a second oligonucleotide primer, consisting essentially of base sequence 5'-TTTGACCTTTAG-3' or an oligonucleotide competing with said first primer for hybridizing to at least a portion of (-)-strand DNA of chromosome 18q21 immediately 5' to a molecular breakpoint hot spot region located in exon 3 and said second primer consisting essentially of base sequence 3'-CAGAGGAGTCCA-5' or an oligonucleotide competing with said second primer for hybridizing to a sequence 3' to an end of segment J H of chromosome 14 immunoglobulin heavy chain gene wherein is located a breakpoint clustering region, said incubating promoting annealing of the primers to the translocation crossover site and synthesis of coding or non-coding translocation DNA sequences and a substantial portion of flanking DNA sequences, the incubation then being terminated by DNA denaturation; repeating said incubation step without adding further cell sample in a cyclic manner to facilitate duplication of the original and newly synthesized translocated and substantial flanking DNA sequences, said repeating being carried out in a manner exponentially amplifying DNA sequences between and including DNA regions complementary to the first and second oligonucleotide primers; and analyzing the exponentially amplified DNA for oligonucleotide sequences characterizing the translocation region by separating the exponentially amplified DNA and hybridizing the amplified DNA with the first and second primers and identifying neoplastic cells by the presence of said sequences.

Claim 3

A method for detecting a t(14;18) translocation in neoplastic cells in an individual, said translocation derived from region J of chromosome 14 immunoglobulin heavy chain gene, the method comprising the steps of: incubating a mixture comprising deoxyribonucleotide triphosphates, a DNA sample from tissue or cells of the individual, DNA polymerase and at least one set of oligonucleotide primers, each set of oligonucleotide primers consisting of a first oligonucleotide primer and a second oligonucleotide primer, said first oligonucleotide primer consisting essentially of base sequence 5'-TTTGACCTTTAG-3' or an oligonucleotide competing with said first primer for hybridizing to (-)-strand DNA of chromosome 18q21 within 1000 base pairs 5' to mbr and said second primer consisting essentially of base sequence 3'-CAGAGGAGTCCA-5' or an oligonucleotide competing with said second primer for hybridizing to a sequence 3' to the immunoglobulin heavy chain chromosome 14 J H region breakpoint said incubating being under conditions promoting annealing of the primers to the crossover sites of the translocation, the incubation then being terminated by DNA denaturation; repeating said incubation step without further adding of cell sample in a cyclic manner to facilitate further synthesis of the original DNA and of newly synthesized DNA, said repeating being carried out in a manner facilitating repeated primer extensions and exponential amplification of DNA sequences at the crossover sites of the translocations; separating amplified DNA by fractionation means; hybridizing the exponentially amplified DNA with a probe wherein the probe base sequence is identical to at least a portion of 18q21(+) at a point 3' to the first primer; and analyzing the hybridized exponentially amplified DNA for the presence of oligonucleotide sequences characterizing the t(14;18) translocation region and identifying therefrom the presence of neoplastic cells by the presence of said sequences.

Claim 4

A method for detecting a t(14;18) translocation in neoplastic cells in an individual, said translocation derived from region J of chromosome 14 immunoglobulin heavy chain gene, the method comprising the steps of: incubating a mixture comprising deoxyribonucleotide triphosphates, a DNA sample from tissue or cells of the individual, DNA polymerase and at least one set of oligonucleotide primers, each set of oligonucleotide primers consisting of a first oligonucleotide primer and a second oligonucleotide primer, said first oligonucleotide primer consisting essentially of base sequence 5'-TTTGACCTTTAG-3' or an oligonucleotide competing with said first primer for hybridizing to (-)-strand DNA of chromosome 18q21 within 1000 base pairs 5' to mbr and said second primer consisting essentially of base sequence 3'-CAGAGGAGTCCA-5' or an oligonucleotide competing with said second primer for hybridizing to a sequence 3' to the immunoglobulin heavy chain chromosome 14 J H region breakpoint said incubating being under conditions promoting annealing of the primers to the crossover sites of the translocation, the incubation then being terminated by DNA denaturation; repeating said incubation step without further adding of cell sample in a cyclic manner to facilitate further synthesis of the original DNA and of newly synthesized DNA, said repeating being carried out in a manner facilitating repeated primer extensions and exponential amplification of DNA sequences at the crossover sites of the translocations; separating amplified DNA by fractionation means; hybridizing the exponentially amplified DNA with a probe wherein the probe base sequence is identical to at least a portion of 18q21(+) at a point 3' to the first primer; and analyzing the hybridized exponentially amplified DNA for the presence of oligonucleotide sequences characterizing the t(14;18) translocation region and identifying therefrom the presence of neoplastic cells by the presence of said sequences.