Methods for normalizing and for identifying small nucleic acids

摘要

The present teachings are generally directed to methods for normalizing at least one species of small nucleic acid that is present in a population of small nucleic acid species, wherein the relative concentration of at least one small nucleic acid species is substantially greater than the relative concentration of at least one other small nucleic acid species in the population. At least one small nucleic acid species is normalized using a multiplicity of primers comprising degenerate sequences. In some embodiments, a small nucleic acid species is identified by inserting at least part of an extension product from a normalized population into a vector and subsequently sequencing the insert. In some embodiments, a small nucleic acid species is identified by determining the sequence of at least part of an extension product.

主权项

1. A method for normalizing a population of different small nucleic acid species of varying abundance, wherein each small nucleic acid species is less than thirty nucleotides long, the method comprising,
ligating a 3′ adapter and a 5′ adapter, respectively, to the 3′ and 5′ ends of at least some of the small nucleic acids in the population to form a multiplicity of different double adapter-modified molecules, wherein the adapters comprise a 3′ adapter comprising a first primer-binding site, and a 5′ adapter comprising a second primer-binding site; amplifying at least some of the multiplicity of different double adapter-modified molecules with a formulated relative concentration of primers to generate a normalized population, wherein the formulated relative concentration of primers comprises a multiplicity of primer species each comprising different degenerate sequences at their respective 3′-ends and a corresponding universal primer species, wherein at least one of the degenerate sequences comprises one, two, three, four, five, or six nucleotides, wherein the concentration of the universal primer species is at least ten times greater than the concentration of any one of the primer species comprising a degenerate sequence, and wherein the concentration of the universal primer is greater than the total concentration of the multiplicity of primers comprising different degenerate sequences, and wherein the amplifying comprises a polymerase chain reaction.