Multiplex method for nucleic acid detection

A method for determining the presence or absence of a specific base in a nucleic acid target sequence in a sample to be assayed that comprises the steps of:

• (A) admixing a sample to be assayed with a plurality of nucleic acid probes to form a hybridization composition, wherein the 3′-terminal region of at least one of said nucleic acid probes (i) is substantially complementary to said nucleic acid target sequence and comprises at least one predetermined nucleotide at an interrogation position, and (ii) includes an identifier nucleotide, and wherein said nucleic acid target sequence comprises at least one specific base whose presence or absence is to be determined;
• (B) maintaining said hybridization composition for a time period sufficient to form a treated sample, wherein said interrogation position of the probe is a nucleotide that is aligned with said specific base to be identified in said target sequence, when present, so that base pairing can occur;
• (C) admixing the treated sample with an enzyme whose activity is to release one or more nucleotides from the 3′-terminus of a hybridized nucleic acid probe to depolymerize the hybrid and form a treated reaction mixture;
• (D) maintaining the treated reaction mixture for a time period sufficient to release an identifier nucleotide therefrom;
• and
• (E) analyzing for the presence or absence of released identifier nucleotide to obtain an analytical output that indicates the presence or absence of said specific base to be identified.

A method for determining the presence or absence of a plurality of first nucleic acid targets in a nucleic acid sample containing those targets or a plurality of substantially identical second targets that comprises the steps of:

• (A) admixing said sample to be assayed with one or more nucleic acid probes to form a hybridization composition, wherein said first and second nucleic acid targets comprise a region of sequence identity except for at least a single nucleotide at a predetermined position that differs between the targets, and wherein said nucleic acid probe (i) is substantially complementary to said nucleic acid target region of sequence identity and comprises at least one nucleotide at an interrogation position, said interrogation position of the probe being aligned with said predetermined position of a target when a target and probe are hybridized and (ii) includes an identifier nucleotide in the 3′-terminal region;
• (B) maintaining said hybridization composition for a time period sufficient to form a treated sample wherein the nucleotide at said interrogation position of said probe is aligned with the nucleotide at said predetermined position of said target in said region of identity;
• (C) admixing the treated sample with a depolymerizing amount an enzyme whose activity is to release one or more nucleotides from the 3′-terminus of a hybridized nucleic acid probe to form a treated reaction mixture;
• (D) maintaining the treated reaction mixture for a time period sufficient to release identifier nucleotide and depolymerize said hybridized nucleic acid probe;
• and
• (E) analyzing for the presence of released identifier nucleotide to obtain an analytical output, said analytical output indicating the presence or absence of said nucleotide at said predetermined region and thereby the presence or absence of a first or second nucleic acid target.

A method for determining the presence or absence of a plurality of nucleic acid target sequences, each containing an interrogation position, in a nucleic acid sample that comprises the steps of:

• (A) providing a treated sample that contains a nucleic acid sample that may include said nucleic acid target sequences hybridized with their respective nucleic acid probes, each probe being comprised of three sections, (i) a first section that contains the probe 3′-terminal about 10 to about 30 nucleotides that are complementary to its nucleic acid target sequence at positions beginning about 1 to about 30 nucleic acids downstream of said interrogation position of the target sequence, (ii) a 5′-terminal region of about 10 to about 200 nucleic acids in length and having the identical sequence of said nucleic acid target sequence, and (iii) an optional third section that contains zero to about 50 nucleic acids that are not complementary to said nucleic acid sample;
• (B) extending said nucleic acid probes in a 3′ direction to form second probes hybridized to the nucleic acid sample as second hybrids;
• (D) denaturing said second hybrids to separate said second probes from said nucleic acid target sequences;
• (E) renaturing said aqueous composition to form hairpin structures from said second probes;
• (F) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′-terminus of a nucleic acid hybrid to form a treated reaction mixture;
• (G) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release one or more nucleotides from the 3′-terminus therefrom;
• and
• (H) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said nucleic acid target sequences.

A method for determining the presence or absence of a plurality of nucleic acid target sequences, or a specific base within the target sequences, in a nucleic acid sample, that comprises the steps of:

• (A) providing a treated sample that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences hybridized with their respective first nucleic acid probes as a first hybrid, said first probes each being comprised of at least two sections, a first section containing the probe 3′-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position, a second section of the first probe containing about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 to about 30 nucleotides downstream of the interrogation position that does not hybridize to said first section of the probe, and an optional third section of the probe located between the first and second sections of the probe that is zero to about 50 nucleotides in length and comprises a sequence that does not hybridize to either the first or second section of the probe;
• (B) extending the first hybrid in the treated sample at the 3′-end of the first probes, thereby extending the first probes past the interrogation position and forming an extended first hybrid that includes an interrogation position;
• (C) denaturing an aqueous composition of the extended first hybrid to separate the two nucleic acid strands and form an aqueous composition containing separated target nucleic acids and separated extended first probes;
• (D) annealing to each of the extended first probes second probes that are about 10 to about 30 nucleotides in length and are complementary to the extended first probes at a position beginning about 5 to about 2000 nucleotides downstream of the interrogation position in the extended first probes, thereby forming a second hybrid;
• (E) extending the second hybrid at the 3′-end of the second probes until that extension reaches the 5′-end of the extended first probes, thereby forming a second extended hybrid containing a second extended probe whose 3′-region includes an identifier nucleotide;
• (F) denaturing an aqueous composition of the extended second hybrid to separate the nucleic acid strands and form an aqueous composition containing separated extended first and second probes;
• (G) cooling the aqueous composition to form a hairpin structure from the separated extended second probes to form a hairpin structure-containing composition;
• (H) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′-terminus of a nucleic acid hybrid to form a treated reaction mixture;
• (I) maintaining the reaction mixture for a time period sufficient to release 3′-terminal region identifier nucleotides;
• and
• (J) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said predetermined nucleic acid target sequence or a specific base within the target sequence.

A method for determining the presence or absence of a plurality of nucleic acid target sequences containing an interrogation position in a nucleic acid sample that comprises the steps of:

• (A) providing a treated sample that contains a nucleic acid sample that may include said plurality of nucleic acid target sequences, each hybridized with its respective nucleic acid probe that is comprised of three sections, (i) a first section that contains the probe 3′-terminal about 10 to about 30 nucleotides that are complementary to the nucleic acid target sequence at positions beginning about 1 to about 30 nucleic acids downstream of said interrogation position of the target sequence, (ii) a 5′-terminal region of about 10 to about 200 nucleic acids in length and having the identical sequence of said nucleic acid target sequence, and (iii) an optional third section that contains zero to about 50 nucleic acids that are not complementary to said nucleic acid sample, and;
• (B) extending said nucleic acid probes in a 3′ direction to form second probes hybridized to the nucleic acid sample as a second hybrid;
• (D) denaturing said second hybrid to separate said second probes from said nucleic acid target sequences;
• (E) renaturing said aqueous composition to form hairpin structures from said second probes;
• (F) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′-terminus of a nucleic acid hybrid to form a treated reaction mixture;
• (G) maintaining the treated reaction mixture for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release one or more nucleotides from the 3′-terminus therefrom;
• and
• (H) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said nucleic acid target sequences.

A method for determining the presence or absence of a plurality of nucleic acid target sequences, or a specific base within a target sequence, in a nucleic acid sample, that comprises the steps of:

• (A) providing a treated sample that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences, each hybridized with its respective first nucleic acid probe as a first hybrid, said first probes being comprised of at least two sections, a first section containing the probe 3′-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position, a second section of the first probe containing about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 to about 30 nucleotides downstream of the interrogation position that does not hybridize to said first section of the probe, and an optional third section of the probe located between the first and second sections of the probe that is zero to about 50 nucleotides in length and comprises a sequence that does not hybridize to either the first or second section of the probe;
• (B) extending the first hybrid in the treated sample at the 3′-end of the first probes, thereby extending the first probes past the interrogation position and forming an extended first hybrid that includes an interrogation position;
• (C) denaturing an aqueous composition of the extended first hybrid to separate the two nucleic acid strands and form an aqueous composition containing separated target nucleic acids and a separated extended first probes;
• (D) annealing to the extended first probes a second probe that is about 10 to about 30 nucleotides in length and is complementary to the extended first probe at a position beginning about 5 to about 2000 nucleotides downstream of the interrogation position in the extended first probes, thereby forming a second hybrid;
• (E) extending the second hybrid at the 3′-end of the second probes until that extension reaches the 5′-end of the extended first probe, thereby forming a second extended hybrid containing a second extended probe whose 3′-region includes an identifier nucleotide;
• (F) denaturing an aqueous composition of the extended second hybrid to separate the two nucleic acid strands and form an aqueous composition containing separated extended first and second probes;
• (G) cooling the aqueous composition to form a hairpin structure from the separated extended second probe to form a hairpin structure-containing composition;
• (H) admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3′-terminus of a nucleic acid hybrid to form a treated reaction mixture;
• (I) maintaining the reaction mixture for a time period sufficient to release 3′-terminal region identifier nucleotides;
• and
• (J) analyzing for the presence of released identifier nucleotide to obtain an analytical output, the analytical output indicating the presence or absence of said predetermined nucleic acid target sequences or a specific base within a target sequence.

A composition of matter for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample comprising an aqueous solution that contains:

• (A) a purified and isolated enzyme whose activity in the presence of pyrophosphate is to release identifier nucleotide as a nucleoside triphosphate from hybridized nucleic acid probe;
• (B) adenosine 5′ diphosphate;
• (C) pyrophosphate;
• (D) a purified and isolated nucleoside diphosphate kinase;
• and
• (E) a plurality of nucleic acid probes, each of said nucleic acid probe being complementary to its respective predetermined nucleic acid target sequence.