Use of markers including nucleotide sequence based codes to monitor methods of detection and identification of genetic material

摘要

Disclosed is the use of artificially-generated nucleic acid coded markers to monitor nucleic acid amplification and sequencing reactions designed to detect or analyze biological samples. The markers generally include, along with a unique sequence preferably including coded section designed to represent one or more factors of interest, primer annealing sequences so that the marker may be amplified and sequenced in the same process and using the same amplification and sequencing primers as for the sample target. The invention also relates to the marker itself, and other uses, such as identifying the origin of various materials or products.

主权项

1. A method of testing for the presence of a target nucleic acid in a sample, the method comprising:
(i) performing amplification, comprising combining the sample, a marker nucleic acid, and a forward and a reverse amplification primer, wherein:
the forward and reverse primers are fully complementary to forward and reverse primer annealing sites on the target nucleic acid and on the marker nucleic acid,the target nucleic acid comprises a characteristic sequence, andthe marker nucleic acid comprises a known unique sequence, which is different from the characteristic sequence and contains a coding sequence and a homonucleotide sequence upstream of the coding sequence, the homonucleotlde sequence comprising at least as many bases in length as the characteristic sequence,thereby generating a marker amplicon containing the unique sequence, and, when the target nucleic acid sequence is present in the sample, a target amplicon containing the characteristic sequence; (ii) performing sequencing, thereby identifying the sequence of at least part of the coding sequence and, when the target nucleic acid is present in the sample, at least part of the characteristic sequence, wherein:
the sequencing comprises combining the amplicon or amplicons generated in step (i) and a sequencing primer that is complementary to a sequencing primer annealing site in the marker ampiicon and to a sequencing primer annealing site in the target amplicon, andthe sequencing further comprises a detection step using an automated sequencing machine, wherein a channel for detecting a nucleotide in the homonucleotide sequence is turned off for at least a portion of the detection step;thereby determining the presence or absence of the target nucleic acid sequence in the sample.