Detection of nucleic acid hybrids by prolonged chemiluminescence

Claim 1

A method for determining a particular single stranded polynucleotide sequence in a test medium, comprising the steps of: (a) immobilizing on a solid support single stranded nucleic acids in the test medium, (b) contacting the immobilized nucleic acids with a polynucleotide probe having a base sequence substantially complementary to the sequence to be determined and said contacting being under conditions favorable to hybridization between the probe and the sequence to be determined, wherein the probe is labeled with a chemiluminescence enhancer, (c) separating the immobilized hybrids from the unhybridized probe, (d) initiaiing a chemiluminescent reaction by contacting the separated, labeled, immobilized hybrids with an oxidant, a 2,3-dihydro-1,4-phthalazinedione chemiluminescence precursor, and a peroxidase enzyme, (e) detecting the resulting light emission, and (f) relating the amount of emitted light to the amount of the single stranded polynucleotide sequence, the chemiluminescence enhancer being selected from the group consisting of luciferin and dehydroluciferin and wherein the chemiluminescent precursor is a 2,3-dihydro-1,4-phthalazinedione of the formula ##STR12## wherein R 1 is amino, and R 2 , R 3 and R 4 are all H and wherein R 1 is amino and each of R 2 , R 3 and R 4 is unsubstituted C 1 -C 6 -alkyl or alkenyl or C 1 C 6 -alkyl or alkenyl substituted by a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol; hydroxyl; C 1 -C 6 -alkoxy; carboxyl or amino or R 2 is an amino or substituted amino and each of R 1 , R 3 and R 4 is H, unsubstituted C 1 -C 6 -alkyl or alkenyl or C 1 -C 6 -alkyl or alkenyl substituted by a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol; hydroxyl; C 1 -C 6 -alkoxy or carboxyl, amino, or R 1 and R 2 together with the ring carbon atoms they are bonded to form a fused benz ring which is substituted with an amino group or a substituted amino group, said substituted amino group being substituted by a substituent selected from the group consisting of carboxy, C 1 -C 10 alkyl and C 2 -C 10 alkenyl, and each of R 3 and R 4 is H, unsubstituted C 1 -C 6 -alkyl or alkenyl or C 1 -C 6 -alkyl or alkenyl substituted by a substituent slected from the group cossisting of chlorine, fluorine, bromine, iodine, hydroyy, carboxy, nitro, cyano and thiol; hydroxyl; C 1 -C 6 -alkoxy; carboxyl or amino, said hydroxyl being unsubstituted or substituted by a substituent selected from the group consisting of C 1 -C 10 alkyl and C 2 -C 10 alkenyl.

Claim 2

A method for determining a particular single stranded polynucleotide sequence in a test medium, comprising the steps of: (a) immobilizing on a solid support single stranded nucleic acids in the test medium, (b) contacting the immobilized nucleic acids with a polynucleotide probe having a base sequence substantially complementary to the sequence to be determined and said contacting being under conditions favorable to hybridization between the probe and the sequence to be determined, wherein the probe is labeled with a chemiluminescence enhancer, (c) separating the immobilized hybrids from the unhybridized probe, (d) initiaiing a chemiluminescent reaction by contacting the separated, labeled, immobilized hybrids with an oxidant, a 2,3-dihydro-1,4-phthalazinedione chemiluminescence precursor, and a peroxidase enzyme, (e) detecting the resulting light emission, and (f) relating the amount of emitted light to the amount of the single stranded polynucleotide sequence, the chemiluminescence enhancer being selected from the group consisting of luciferin and dehydroluciferin and wherein the chemiluminescent precursor is a 2,3-dihydro-1,4-phthalazinedione of the formula ##STR12## wherein R 1 is amino, and R 2 , R 3 and R 4 are all H and wherein R 1 is amino and each of R 2 , R 3 and R 4 is unsubstituted C 1 -C 6 -alkyl or alkenyl or C 1 C 6 -alkyl or alkenyl substituted by a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol; hydroxyl; C 1 -C 6 -alkoxy; carboxyl or amino or R 2 is an amino or substituted amino and each of R 1 , R 3 and R 4 is H, unsubstituted C 1 -C 6 -alkyl or alkenyl or C 1 -C 6 -alkyl or alkenyl substituted by a substituent selected from the group consisting of chlorine, fluorine, bromine, iodine, hydroxy, carboxy, nitro, cyano and thiol; hydroxyl; C 1 -C 6 -alkoxy or carboxyl, amino, or R 1 and R 2 together with the ring carbon atoms they are bonded to form a fused benz ring which is substituted with an amino group or a substituted amino group, said substituted amino group being substituted by a substituent selected from the group consisting of carboxy, C 1 -C 10 alkyl and C 2 -C 10 alkenyl, and each of R 3 and R 4 is H, unsubstituted C 1 -C 6 -alkyl or alkenyl or C 1 -C 6 -alkyl or alkenyl substituted by a substituent slected from the group cossisting of chlorine, fluorine, bromine, iodine, hydroyy, carboxy, nitro, cyano and thiol; hydroxyl; C 1 -C 6 -alkoxy; carboxyl or amino, said hydroxyl being unsubstituted or substituted by a substituent selected from the group consisting of C 1 -C 10 alkyl and C 2 -C 10 alkenyl.