Digital amplification with primers of limited nucleotide composition
Inventors
Wang, Youxiang • Yang, Zhijie • Chen, Xin • Zhao, Yu • Wang, Rong
Assignees
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Publication Number
US-11739375-B2
Publication Date
2023-08-29
Expiration Date
Abstract
The invention provides methods of digital amplification using primers of limited nucleotide composition. Limited nucleotide composition means that the primers are underrepresented in at least one nucleotide type. Such primers have much reduced capacity to prime from each other or to extend initiated by mispriming from other than at their intended primer binding sites in a target nucleic acid.
Core Innovation
The invention relates to performing digital amplification of a target nucleic acid in a sample by partitioning the sample into aliquots, conducting amplification reactions in the aliquots, and detecting whether an amplified segment is present in each aliquot. The amplified segment is formed by extension of a pair of forward and reverse primers on the target nucleic acid if the target nucleic acid is present in the aliquot.
The primers are designed so that they are underrepresented in one or more of the four standard nucleotide types, with the underrepresented nucleotide type or types being the same in the primers. The underrepresented nucleotide type or types are present at up to 2 internal positions and optionally at a 5′ terminal position of each forward primer and each reverse primer, and the 3′ position of the forward primer and the 3′ position of the reverse primer are occupied by the complement of an underrepresented nucleotide type.
The invention further includes using an artificial sequence linked at the 5′ end of a forward primer and/or reverse primer, where the artificial sequence is underrepresented in a nucleotide type that is also underrepresented in the forward and reverse primers. The disclosed approach supports digital amplification formats and reduces primer dimer and mispriming extension through the limited nucleotide composition.
Claims Coverage
The document provides three independent claims centered on digital amplification by partitioning into aliquots, forward and reverse primer extension, and detection of amplified segments per aliquot. The inventive features are the underrepresented nucleotide composition of the primers, the shared underrepresented nucleotide type across primer pairs, the positional constraints on those nucleotide types, and the 5′-linked artificial sequence in one claim.
Underrepresented nucleotide primer constraint for digital amplification
In a method of performing a digital amplification on a target nucleic acid by partitioning the sample into aliquots, conducting amplification reactions such that an amplified segment is formed by extension of a pair of forward and reverse primers if the target nucleic acid is present, and detecting the amplified segment in each aliquot, the primers are underrepresented in one or more of the four standard nucleotide types; the underrepresented nucleotide type or types are the same in the primers; the underrepresented nucleotide type or types are present at up to 2 internal positions and optionally at a 5′ terminal position of each of the forward primer and the reverse primer; and the 3′ position of the forward primer and the 3′ position of the reverse primer are occupied by the complement of an underrepresented nucleotide type.
Multiple primer pairs with shared underrepresented nucleotide types across multiple targets
In a method of performing a digital amplification by partitioning a sample comprising a plurality of target nucleic acids into aliquots, conducting amplification reactions such that amplified segments are formed by extension of multiple pairs of forward and reverse primers corresponding to the respective target nucleic acids, and detecting an amplified segment in each aliquot, the primers are underrepresented in one or more of the four standard nucleotide types, the underrepresented nucleotide type or types being the same in the primers; the underrepresented nucleotide type or types being present at two or fewer internal positions and/or the 5′ end position; and each of the plurality of primer pairs is underrepresented in the same standard nucleotide type or types for the corresponding target nucleic acid.
Artificial 5′ sequence linked to underrepresented nucleotide type primer ends
In a method of performing a digital amplification by partitioning the sample into aliquots, conducting amplification reactions such that an amplified segment is formed by extension of a pair of forward and reverse primers if the target nucleic acid is present, and detecting the amplified segment in each aliquot, the primers are underrepresented in one or more of the four standard nucleotide types with the underrepresented nucleotide type or types being the same in the primers and being present at two or fewer internal positions and/or the 5′ end position; and the forward primer and/or reverse primer is linked at its 5′ end to an artificial sequence underrepresented in a nucleotide type that is underrepresented in the forward and reverse primers.
Across the independent claims, the claimed digital amplification method relies on partitioning into aliquots, primer extension by forward and reverse primers, and detecting amplified segments per aliquot, with the main inventive emphasis on underrepresented nucleotide primer design under defined positional constraints and, in one claim, a 5′-linked artificial sequence.
Stated Advantages
Reducing background fluorescence by lowering primer dimer and primer-mispriming extension.
Improving specificity and accuracy of digital amplification, including improved discrimination of trisomic versus euploid samples.
Enabling multiplex digital assays with reduced background.
Documented Applications
Non-invasive prenatal testing (NIPT), including fetal fraction determination from cell-free fetal DNA and detection of trisomy 21, trisomy 18, and trisomy 13 using multiplex ddPCR.
Copy-number variation detection using digital amplification.
SNP/indel detection using digital amplification.
Methylation state determination using bisulfite-driven conversion and quantification targeted to differentially methylated regions (DMRs).
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