Method for the quantitative determination of terminal deoxynucleotidyl transferase in biological samples

Claim 1

A method for quantifying Terminal Deoxynucleotidyl Transferase (TdT) in a biological sample containing solubilized DNA polymerases, by incubating at from about 35 to about 37° C. a buffered mixture of said sample with oligodeoxynucleotide primers and deoxynucleotide substrates, wherein the primer or the substrate is labeled for detection, in the presence of a divalent metal to produce by DNA synthesis labeled acid insoluble polydeoxynucleotides which are insoluble from said incubation mixture and from the quantification of which the amount of TdT in the extract is determinable, wherein the improvement comprises: incubating said enzyme sample with labeled oligodeoxynucleotide primers or labeled deoxynucleotide substrates sufficiently chemically modified at one or more of the base moieties of said primers or substrates by one or more nonhydrogen bonding or bulky substituents groups to impair or destroy the ability of such primers or substrates to form nucleoside base pairs by hydrogen bonding, thereby permitting direct quantification of TdT in the sample without interference by other DNA polymerases present in the sample by rendering such primers or substrates selective to utilization by TdT in the synthesis of labeled polydeoxynucleotides.

Claim 2

A method for quantifying Terminal Deoxynucleotidyl Transferase (TdT) in a biological sample containing solubilized DNA polymerases, by incubating at from about 35 to about 37° C. a buffered mixture of said sample with oligodeoxynucleotide primers and deoxynucleotide substrates, wherein the primer or the substrate is labeled for detection, in the presence of a divalent metal to produce by DNA synthesis labeled acid insoluble polydeoxynucleotides which are insoluble from said incubation mixture and from the quantification of which the amount of TdT in the extract is determinable, wherein the improvement comprises: incubating said enzyme sample with labeled oligodeoxynucleotide primers or labeled deoxynucleotide substrates sufficiently chemically modified at one or more of the base moieties of said primers or substrates by one or more nonhydrogen bonding or bulky substituents groups to impair or destroy the ability of such primers or substrates to form nucleoside base pairs by hydrogen bonding, thereby permitting direct quantification of TdT in the sample without interference by other DNA polymerases present in the sample by rendering such primers or substrates selective to utilization by TdT in the synthesis of labeled polydeoxynucleotides.