Multitag sequencing ecogenomics analysis-us

A multiplex method for the quantitative determination of polynucleotides in two or more samples, comprising:

• hybridizing a first primer pair to polynucleotides in a first sample, the first primer of said first primer pair comprising, in 5′ to 3′order, an emulsion PCR linker sequence, a first tag sequence and a first probe sequences specific for a first target sequence and the second primer of said first primer pair comprising , in 5′ to 3′ order, an emulsion PCR linker sequence, a second tag sequence and a second probe sequence specific for a second target sequence, wherein the first and the second probe sequences flank and hybridize to opposite strands of a variable genetic region such that one strand of said variable genetic region is the template strand for elongation of each primer, wherein each of said emulsion PCR linker sequences is effective for binding to a solid support and for emulsion PCR amplification, and wherein said emulsion PCR linker sequences of said first and second primers may be the same or different;
• amplifying primer templates formed thereby to form a first population of tagged polynucleotides, each of said tagged polynucleotides comprising:
• (a) the sequence of said first primer of said first primer pair, a sequence of said variable genetic region, and a sequence complementary to the sequence of said second primer of said first primer pair or (b) a sequence complementary to the sequence of said first primer of said first primer pair, a sequence of said variable genetic region and the sequence of said second primer of said first primer pair;
• hybridizing a second primer pair to polynucleotides in a second sample, the first primer of said second primer pair comprising, in 5′ to 3′ order, an emulsion PCR linker sequence, a third tag sequence and said first probe sequences specific for said first target sequence and the second primer of said second primer pair comprising, in 5′ to 3′ order, an emulsion PCR linker sequence, a fourth tag sequence and said second probe sequence specific for said second target sequence, wherein each of said emulsion PCR linker sequences is effective for binding to a solid support and for emulsion PCR amplification, and wherein said emulsion PCR linker sequences of said first and second primers may be the same or different;
• and may be the same or different from those of said first and second primers of said first primer pair;
• amplifying primer templates formed thereby to from a second population of tagged polynucleotides, each of said tagged polynucleotides comprising:
• (a) the sequence of said first primer of said second primer pair, a sequence of said variable genetic region, and a sequence, complementary to the sequence of said second primer of said second primer pair or (b) a sequence complementary to the sequence of said first primer of said second primer pair, a sequence of said variable genetic region and the sequence of said second primer of said second primer pair;
• mixing said first and second populations together;
• binding individual polynucleotides to a solid support via said linker sequence effective for said binding and amplifying said bound polynucleotides by emulsion PCR, determining sequences of thus amplified polynucleotides comprising the tag sequences and the variable genetic element;
• from the tag sequences comprised in the polynucleotide sequences thus determined identifying the sample in which polynucleotide sequences occurred;
• from the sequences of the variable genetic region comprised in the polynucleotide sequences thus determined identifying particular variants of said variable genetic element;
• from this information determining the number of times given variants occur in each sample, and
• from the number for each variant in the polynucleotides thus determined, quantifying said polynucleotides in said samples.
• wherein said sequences are determined without Southern blot transfer and/or without size-separating primer extension products and/or without electrophoresis.

A kit comprising a plurality of two or more primers, each primer in said plurality comprising, in 5′ to 3′ order, an emulsion PCR linker sequence, a tag sequence and a probe sequence specific to a target sequence, wherein:

• (A) each emulsion PCR linker sequence is effective for binding to a solid support and for emulsion PCR amplification, wherein the emulsion PCT linker sequences may be the same or different in said primers;
• (B) in each of said primers:
• the tag sequence is different from the tag sequence of the other primers in the plurality;
• the tag sequence is not the complementary sequence to any other tag sequence in the plurality;
• the tag sequence does not contain any homodinucleotide sequences;
• the junction sequence between the tag sequence and the adjacent parts of the primer, if any, is not a homodinucleotide sequence;
• (C) in each of said primers the probe sequence is complementary to the target sequence and the target sequence is located 3′ to a variable genetic region, and
• (D) each of said primers is disposed separately from the others in containers in said kit.

A kit comprising a plurality of two or more primers pairs, each primer in said plurality comprising, in 5′ to 3′ order, an emulsion PCR linker sequence, a tag sequence and a probe sequence specific to a target sequence, wherein:

• (A) wherein each emulsion PCR linker sequence is effective for binding to a solid support and for emulsion PCR amplification;
• (B) in each of said primers:
• the tag sequence is different from the tag sequence of the other primers in the plurality;
• the tag sequence is not the complementary sequence to any other tag sequence in the plurality;
• the tag sequence does not contain any homodinucleotide sequences;
• the junction sequence between the tag sequence and the adjacent parts of the primer, if any, is not a homodinucleotide sequence;
• (C) in each of said primers the probe sequence is complementary to the target sequence,
• (D) in each primer pair the probe sequences are specific to target sequences that flank a variable genetic region.;
• (E) each of said primer pairs or each of said primers is disposed separately from others in said kit.