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Publication Number
US-11857940-B2
Publication Date
2024-01-02
Expiration Date
Abstract
The present disclosure provides a “looping amplification” method to increase the specificity of nucleic acid amplification. This increased specificity facilitates multiplexing to a much higher degree than was previously possible.
Core Innovation
The invention provides a method for amplifying one or more target nucleic acids by contacting sample nucleic acids with a forward primer and a reverse primer for each target nucleic acid. Each primer comprises a target-specific portion, a common sequence 5′ of the target-specific portion, a DNA sequencing primer binding site 5′ of the common sequence, and a first or second nucleotide tag 5′ of the DNA sequencing primer binding site, where the first and second nucleotide tags in the forward and reverse primers are different. The common sequence is at least 8 nucleotides in length, and the forward primer or the reverse primer additionally comprises a first flow cell attachment site 5′ of the first nucleotide tag.
Target amplicons are produced such that a target nucleotide sequence is flanked by the common sequence on one end and its reverse complement on the other end, whereby a single strand of the target amplicon can form a stem loop structure. The amplification is carried out using a third primer, and the third primer comprises a portion specific for the second nucleotide tag, a barcode nucleotide sequence 5′ of the tag-specific portion, and a second flow cell attachment site 5′ of the barcode nucleotide sequence. The resulting target amplicons comprise an architecture with first flow cell attachment site, first nucleotide tag, DNA sequencing primer binding site, common sequence, target nucleotide sequence, reverse complement of common sequence, second DNA sequencing primer binding site, second nucleotide tag, barcode nucleotide sequence, and second flow cell attachment site.
The described approach is used to increase nucleic-acid amplification specificity for highly multiplexed targeted re-sequencing by employing looping amplification in which stem-loop structure suppresses cross-hybridization during amplification. In sequencing-compatible embodiments, primers include target-specific regions plus a shared common sequence derived from a transposon sequence, and the common sequence and primer architecture are arranged to promote hairpin formation during annealing. The document further describes multiplexing scaling using tag and barcode schemes, including 1-step 3-primer and 2-step PCR barcoding schemes, and microfluidic matrix devices with multiplexed reaction chambers.
Claims Coverage
The independent claim recites a multi-primer amplification method that includes primer composition, stem-loop producing amplicon flanking arrangement, and a third-primer barcode/flow-cell attachment architecture.
Primer architecture with target-specific portions, common sequence, sequencing primer binding site, and distinct nucleotide tags
A method where contacting sample nucleic acids with a forward primer and a reverse primer for each target nucleic acid uses primers comprising a target-specific portion, a common sequence 5′ of the target-specific portion, a DNA sequencing primer binding site 5′ of the common sequence, and a first or second nucleotide tag 5′ of the DNA sequencing primer binding site, where the first and second nucleotide tags in the forward and reverse primers are different, and the common sequence is at least 8 nucleotides in length, with a flow cell attachment site 5′ of the first nucleotide tag in the forward primer or the reverse primer.
Stem-loop forming flanked amplicon using the common sequence and its reverse complement
A requirement that amplifying the target nucleic acid(s) produces at least one target amplicon in which a target nucleotide sequence is flanked by the common sequence on one end and its reverse complement on the other end, whereby a single strand of the target amplicon can form a stem loop structure.
Third primer adding tag-specific portion, barcode, and second flow cell attachment site
Amplification is carried out using a third primer comprising a portion specific for the second nucleotide tag, a barcode nucleotide sequence 5′ of the tag-specific portion, and a second flow cell attachment site 5′ of the barcode nucleotide sequence, producing target amplicons comprising a structure that includes first flow cell attachment site, first nucleotide tag, first DNA sequencing primer binding site, common sequence, target nucleotide sequence, reverse complement of common sequence, second DNA sequencing primer binding site, second nucleotide tag, barcode nucleotide sequence, and second flow cell attachment site.
Overall claim coverage centers on looping amplification that integrates a shared common sequence and distinct tags across forward and reverse primers, requires stem-loop formation by the resulting amplicon flanking arrangement, and uses a third primer to add a barcode linked to a tag-specific portion together with sequencing flow-cell attachment sites.
Stated Advantages
Improves amplification specificity for highly multiplexed targeted re-sequencing by suppressing amplification cross-hybridization via stem-loop (hairpin) formation.
Supports high mapping specificity in highly multiplexed embodiments.
Provides improved GC coverage in described embodiments.
Enables high-plex amplification with sequencing specificity in described single-tube amplification.
Documented Applications
Highly multiplexed targeted re-sequencing using looping amplification to increase nucleic-acid amplification specificity and suppress amplification cross-hybridization.
DNA sequencing library generation using sequencing-compatible library architectures that include tags, barcodes, and Illumina flow-cell attachment sites.
Sequencing of target amplicons using bridge sequencing-compatible architecture as described.
Multiplexing scaling using microfluidic matrix devices with multiplexed reaction chambers for large numbers of reactions or targets.
Large-plex targeted re-sequencing amplification and mapping.
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