DETECTION OF MINIMAL NUMBERS OF NEOPLASTIC CELLS CARRYING DNA TRANSLOCATIONS BY DNA SEQUENCE AMPLIFICATION

摘要

The method of the present invention typically includes the following steps. (1) Incubating a mixture comprising: (a) deoxyribonucleotide triphosphates including deoxyadenosine triphosphate, deoxythymidine triphosphate, deoxycytosine triphosphate and deoxyguanosine triphosphate; (b) DNA from cells, most particularly lymphoid cells from blood, bone marrow or lymph node, of the individual; (c) DNA polymerase, preferably the Klenow fragment of E. coli DNA polymerase or Thermus aquaticus DNA polymerase; (d) a first oligonucleotide primer, said first primer being identical to an upstream nucleotide sequence flanking coding DNA translocation derived from one chromosome; and (e) a second oligonucleotide primer, said second primer being complementary to a downstream nucleotide sequence flanking the coding DNA translocation derived from the other chromosome. The incubating is under conditions allowing annealing of the primers to the crossover site of translocation and synthesis of coding or non-coding translocation DNA sequences and a substantial portion of flanking DNA sequences. The incubation is then terminated by DNA denaturation. (2) The cycles of annealing, synthesis and denaturation are then repeated numerous times (preferably 20-50) to facilitate duplications of the original and previously synthesized translocated and substantial flanking DNA sequences. The repetition is carried out in a manner allowing exponential amplification of DNA sequences. (3) The exponentially amplified DNA is probed for the presence of oligonucleotide sequences characterizing the translocation region. Positive probings indicate the presence of neoplastic cells in the sample of the individual's cells, since DNA characterizing said neoplastic cells was specifically amplified and probed.