Patent ID: 6093543
Filing Date: 2000-07-25
Classification: G01N

Abstract:
A method for detecting the presence of malignant tissue or malignant cells comprising the steps of:(A) obtaining a multi-protein DNA replication complex capable of supporting DNA replication by the method comprising:(1) homogenizing a sample comprising tissue or cells to obtain a homogenate;(2) separating the homogenate obtained in step (1) into a nuclear pellet and a cytosolic extract;(3) extracting nuclei from said nuclear pellet obtained in step (2) to obtain a nuclear extract;(4) subjecting the cytosolic extract obtained in step (2) to repeated differential centrifugation to obtain a post-microsomal supernatant;(5) combining said nuclear extract obtained in step (3) with said post-microsomal supernatant obtained in step (4) in the presence of polyethylene glycol (PEG) to obtain a suspension;(6) subjecting the suspension obtained in step (5) to centrifugation to obtain a PEG-supernatant;(7) subjecting the PEG-supernatant obtained in step (6) to dialysis to obtain a dialyzate;(8) subjecting the dialyzate obtained in step (7) to centrifugation to clarify said dialyzate and obtain a supernatant solution;(9) subjecting the supernatant solution obtained in step (8) to sucrose centrifugation and collecting sucrose interface fractions resulting from said centrifugation;(10) subjecting the sucrose interface fractions obtained in step (9) to dialysis to obtain dialyzed fractions; and(11) collecting fractions containing DNA replication activity,wherein said sample in said method comprises tissue or cells suspected of being malignant, so as to obtain a test complex;(B) obtaining a multi-protein DNA replication complex capable of supporting DNA replication by the method comprising steps (1)-(11) above, exceptwherein said sample in said method comprises normal tissue or cells, so as to obtain a control complex;(C) contacting, in vitro, said test complex obtained in step (A) with a DNA template comprising an origin of replication recognized by said test complex under conditions to achieve DNA synthesis;(D) contacting, in vitro, said control complex obtained in step (B) with a DNA template comprising an origin of replication recognized by said control complex under conditions to achieve DNA synthesis; and(E) comparing the frequency of mutation occurrence in the presence of said test complex with the frequency of DNA mutation occurrence in the presence of said control complex so as to detect the presence of malignant tissue or malignant cells.