Multitag sequencing ecogenomics analysis

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 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 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;
• elongating primer templates formed thereby to from a first population of tagged polynucleotides, each of said 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 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 a fourth tag sequence and said second probe sequence specific for said second target sequence;
• elongating primer templates formed thereby to from a second population of tagged polynucleotides, each of said 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;
• determining sequences of polynucleotides in said mixture, 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 a tag sequence and a probe sequence specific to a target sequence, wherein:

• (A) in each of said primers the probe sequence is 3′ to the tag sequence, but not necessarily adjacent thereto;
• (B) in each of said primers:
• the tag sequence is different from the tag sequence of the other 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 sequences 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 a tag sequence and a probe sequence specific to a target sequence, wherein:

• (A) in each of said primer the probe sequence is 3′ to the tag sequence, but not necessarily adjacent thereto;
• (B) in each of said primers:
• the tag sequence is different from the tag sequence of the other 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 sequences 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 primers is disposed separately from the others in said kit.