Multiplex amplification detection assay II
Inventors
Allawi, Hatim T. • Weisburg, William G. • Lidgard, Graham P. • Kaiser, Michael W. • Vaccaro, Abram M. • Shea, Gracie
Assignees
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Publication Number
US-12173362-B2
Publication Date
2024-12-24
Expiration Date
Abstract
Provided herein is technology relating to the amplification-based detection of bisulfite-treated DNAs and particularly, but not exclusively, to methods and compositions for multiplex amplification of low-level sample DNA prior to further characterization of the sample DNA. The technology further provides methods for isolating DNA from blood or blood product samples, e.g., plasma samples.
Core Innovation
The invention provides a method of analyzing a sample for multiple target nucleic acids. The method amplifies bisulfite-treated DNA by PCR in a single reaction using a plurality of different primer pairs to produce a pre-amplified mixture, and the pre-amplified mixture is then partitioned into a plurality of different detection assay reaction mixtures, where each detection assay reaction mixture contains a portion of the pre-amplified mixture.
For each partition, the method conducts a detection assay for multiple target nucleic acids using PCR-flap assays. The PCR-flap assays employ flap oligonucleotides and an additional amount of a primer pair selected from the plurality of different primer pairs used in the pre-amplification step, wherein the selected primer pair is not a nested primer pair or a semi-nested primer pair.
In described embodiments, flap oligonucleotides include a target-specific region of at least 13 bases and nucleotides capable of making non-Watson-Crick base pairs. The described system further includes primer/probe sequence designs and FRET-labeled assay formats, including Z-QuARTS and inosine-containing probes.
Claims Coverage
The independent claim covers a multi-target workflow with an initial single-reaction PCR using multiple primer pairs on bisulfite-treated DNA, followed by partitioned PCR-flap detection assays. The inventive features are centered on the single-reaction multi-primer pre-amplification, partitioning into multiple assay mixtures, and PCR-flap detection using flap oligonucleotides plus an additional amount of a selected primer pair explicitly excluding nested or semi-nested primer pairs.
Single reaction PCR pre-amplification of bisulfite-treated DNA with multiple primer pairs
amplifying a sample comprising bisulfite-treated DNA by PCR in a single reaction using a plurality of different primer pairs to produce a pre-amplified mixture
Partitioning pre-amplified mixture into multiple detection assay reaction mixtures
partitioning the pre-amplified mixture into a plurality of different detection assay reaction mixtures, wherein each detection assay reaction mixture comprises a portion of the pre-amplified mixture
PCR-flap detection with flap oligonucleotides and additional selected primer pair excluding nested formats
conducting a plurality of detection assays on the detection assay reaction mixtures, wherein the detection assays are PCR-flap assays that employ flap oligonucleotides and an additional amount of a primer pair selected from said plurality of different primer pairs of step (a), wherein the primer pair selected is not a nested primer pair or a semi-nested primer pair
Across the independent claim, the coverage is directed to a multiplexing architecture that combines a single-reaction multi-primer PCR pre-amplification of bisulfite-treated DNA with partitioned PCR-flap detection assays. The PCR-flap detection is further characterized by use of flap oligonucleotides and an additional amount of a selected primer pair chosen from the pre-amplification primer set, explicitly excluding nested and semi-nested primer pairs.
Stated Advantages
Avoids nested primer designs and semi-nested primer designs by using the same primer pairs for pre-amplification and PCR-flap detection.
Improves assay performance in multiplexed pre-amplification plus PCR-flap assay configurations.
Provides faster reactions and improved detection described in comparisons of Z-QuARTS versus TELQAS and Z-QuARTS versus QuARTS.
Claims of lower Cp and linear standard curve behavior are stated for comparative formats.
No non-specific signal and no background in no-target controls are stated for the comparisons described.
Improved detection/strand counts are stated in the comparison of Z-QuARTS versus QuARTS.
Documented Applications
Analyzing samples for multiple target nucleic acids using multiplex amplification detection, including sample types referenced in the document such as cell lines, stool, and plasma (cfDNA).
Detecting methylation across sample types while using bisulfite-treated DNA and the partitioned PCR-flap detection format.
Multiplex analysis of a sample for multiple target nucleic acids using bisulfite-treated DNA and partitioned PCR-flap assays is explicitly described.
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