Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels

摘要

Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.

主权项

1. A method for processing a genomic DNA sample, comprising:
(a) hybridizing a genomic sample comprising a population of target DNA molecules with a plurality of primers that comprise:
(i) a 3′ target specific region that hybridizes to a sequence of a target DNA of the plurality of target DNA molecules;(ii) different label regions, each of which is 5′ to the target-specific region, wherein the label regions comprise nucleotides selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases and derivatized nucleotide bases; and(iii) a universal primer sequence that is 5′ to each of the label regions,thereby producing duplexes comprising a target DNA molecule of the population of target DNA molecules and a primer of the plurality of primers; (b) subjecting the duplexes of step (a) to primer extension to extend the primers in the duplexes, thereby producing a mixture comprising extension products comprising tagged copies of the target DNA molecules, wherein each of the extension products comprises a label region of the different label regions and the universal primer sequence of the plurality of primers; (c) removing primers of the plurality of primers that have not been extended in step (b) from the mixture of step (b) or degrading any of the plurality of primers that have not been extended in step (b) from the mixture of step (b); and (d) amplifying the extension products after step c), thereby producing a plurality of different amplicons, wherein each of the different amplicons comprises multiple copies of the same polynucleotide, and wherein:
(i) the amplifying step is done by polymerase chain reaction using a primer that hybridizes to the universal primer sequence of plurality of primers of step (a); and(ii) each of at least some of the plurality of different amplicons has a different label region of the label regions.