Single molecule amplification and detection of DNA length

Abstract

Methods and systems for performing single molecule amplification for detection, quantification and statistical analysis of nucleic acids are provided. Methods and systems are provided for determining and quantifying lengths of nucleic acids of interest.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This Application is a Continuation in Part of Non-Provisional patent application U.S. Ser. No. 10 / 741,162, “SINGLE MOLECULE AMPLIFICATION AND DETECTION OF DNA IN A MICROFLUIDIC FORMAT”, filed Dec. 12, 2003 by Knapp, et al., which is a regular utility application corresponding to prior filed Provisional Patent Application U.S. Ser. No. 60 / 518,431 “SINGLE MOLECULE AMPLIFICATION AND DETECTION OF DNA IN A MICROFLUIDIC FORMAT” by Knapp et al., filed Nov. 6, 2003, Provisional Patent Application U.S. Ser. No. 60 / 462,384 “SINGLE MOLECULE AMPLIFICATION AND DETECTION OF DNA IN A MICROFLUIDIC FORMAT” by Knapp et al., filed Apr. 11, 2003, and Provisional Patent Application U.S. Ser. No. 60 / 436,098 “SINGLE MOLECULE AMPLIFICATION AND DETECTION OF DNA IN A MICROFLUIDIC FORMAT” by Knapp et al., filed Dec. 20, 2002. The subject application claims priority to and benefit to each of these prior applications, which are incorporated herein by reference in t...

AI Technical Summary

Benefits of technology

[0051] Samples containing unknown amounts of a nucleic acid of interest can be quantified by comparing to their signal peaks to sets of standard signal peaks. For example, a nucleic acid of interest in a sample can be quantified by amplifying a dilution series of standard materials containing known amounts of the nucleic acid of interest through a certain number of amplification cycles, detecting signals associated with standard amplicons produced from the standard materials, amplifying the sample nucleic acid of interest the number of amplification cycles, detecting a signal associated with sample amplicons produced from the sample nucleic acid of interest, and comparing one or more standard amplicon signals to the sample amplicon signal to determine a concentration value for the nucleic acid of interest in the sample. Sample and standard signal parameters for comparison can include, e.g., the shape of their signal peaks, points of inflection on the signal peaks, slopes of the signal peaks, signal peak amplitudes, signal peak areas, signal peak widths at half height, and/or the like. The reliability of results can be enhanced through various schemes of repeated testing. For example, the amplifying, detecting, and comparing steps can be repeated one or more times, with different numbers of amplification cycles, to determine additional concentration values for the sample nucleic acid of interest for statistical evaluation providing more precise or more accurate concentration value results for the nucleic acid of interest in the sample.

[0052] Improved assay results can be obtained by gathering signal data after amplifications through two or more different numbers of cycles. A major benefit of running the quantitative assay at different amplifications is to broaden the usable range of the assay. Typically, statistical evaluation of the additional data provided by analysis at multiple amplification levels can enhance other assay parameters, such as precision, accuracy, and sensitivity. Quantifying a nucleic acid of interest in a sample based on detection of multiple amplifications can include: amplifying the nucleic acid of interest through more than one number of amplification cycles, detecting signals associated with amplicons produced from two or more of the amplification cycle numbers, preparing a sample curve of a signal parameter versus number of amplification cycles, a

Problems solved by technology

Despite the wide-spread use of amplification technologies and the adaptation of these technologies to truly high throughput systems, certain technical difficulties persist in amplifying and detecting nucleic acids, particularly rare copy nucleic acids.

For example, if a set of primers hybridizes to a high copy nucleic acid, as well as to a low copy nucleic acid in a given sample, the geometric amplification of the high copy nucleic acid proportionately dominates the amplification reaction and it is difficult or impossible to identify the low copy nucleic acid in any resulting population of amplified nucleic acids.

Thus, low copy number alleles of a gene can be very difficult to detect, e.g., where a primer set cannot easily be identified that only amplifies the rare nucleic acid (and the practitioner will realize that perfect reagent specificity is rare or non-existent in practice).

It is worth noting that these problems simply have not been addressed by the prior art.

(2001) Anal. Chem. 73:565-570), none of these prior approaches are suitable for detection of rare copy nucleic acids in samples.

That is, none of these approaches are suitable to high throughput automation and the devices in the prior art cannot be adapted to practicably detect rare copy nucleic acids.

This cumbersome process results in few amplification reactions being made and analyzed in any useful time period and required almost continuous user intervention to make the system operate.

In addition, none of the prior methods unambiguously determine the length of a nucl

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