Method and device for detecting the presence of target nucleic acids in a sample, and microfluidic device for use in such methods

Abstract

A method comprising loading sample into at least two sample chambers, subjecting the sample portions in each sample chamber to at least a first amplification step, and determining whether each sample portion contains at least one molecule of a target nucleic acid. If a sample portion contains at least a single molecule of the target nucleic acid, it would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a method for detecting whether at least one molecule of a first target nucleic acid is present in a first sample and detecting whether at least one molecule of a second target nucleic acid is present in a second sample. Also, a microfluidic device comprising sample chambers comprising respective ingredients which will react with respective target nucleic acids if contacted with the target nucleic acids and subjected to a round of amplification.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation application of U.S. patent application Ser. No. 10 / 798,857 (the entirety of which is incorporated herein by reference), filed on Mar. 11, 2004, which is a divisional application of U.S. patent application Ser. No. 10 / 131,854, filed on Apr. 25, 2002, which is a divisional application of U.S. patent application Ser. No. 09 / 563,714, filed on May 2, 2000 (now U.S. Pat. No. 6,391,559), which is a divisional application of U.S. patent application Ser. No. 08 / 838,262, filed Apr. 17, 1997 (now U.S. Pat. No. 6,143,496).[0002]This application is also a continuation application of U.S. patent application Ser. No. 10 / 131,854 (the entirety of which is incorporated herein by reference), filed on Apr. 25, 2002, which is a divisional application of U.S. patent application Ser. No. 09 / 563,714, filed on May 2, 2000 (now U.S. Pat. No. 6,391,559), which is a divisional application of U.S. patent application Ser. No. 08 / 838,...

AI Technical Summary

Benefits of technology

The present invention provides methods and apparatus for performing nucleic acid amplification on a miniaturized scale with the sensitivity to detect the presence of a single target nucleic acid molecule. The invention also provides sample retaining means for isolating and retaining fluid samples with volumes of from about one microliter to about one picoliter or less. The isolated samples can be subject to PCR thermal cycling and can be collectively analyzed to determine the presence and initial concentration of multiple different nucleic acid target molecules. The invention also provides methods for detecting and quantifying DNA segments, carrying out polymerase chain reaction in a plurality of discrete nanoliter-sized samples, and separating and analyzing multiple minute portions of a sample of fluid medium.

Problems solved by technology

Unfortunately, problems exist in the application of PCR to clinical diagnostics.

Development has been slow due in part to: labor intensive methods for detecting PCR product; susceptibility of PCR to carryover contamination—false positives due to contamination of a sample with molecules amplified in a previous PCR; and difficulty using PCR to quantitate the number of target nucleic acid molecules in a sample.

Furthermore, the assays are not labor intensive and are easily automated.

These methods have the disadvantage that slight differences in amplification efficiency between the control and experimental nucleic acids can lead to large differences in the amounts of their products after the million-fold amplification characteristic of PCR and related technologies, and it is difficult to determine relative amplification rates accurately.

However, these assays also require assumptions about relative amplification efficiency in different samples during the exponential phase of PCR.

However, to accumulate detectable amounts of product from a single starting template molecule usually requires that two or more sequential PCRs have to be performed, often using nested sets of primers, and this accentuates problems with carryover contamination.

However, this strategy usually fails before getting to the limit of detecting single starting molecules due to the appearance of artifactual amplicons derived from the primers (so called “primer-dimers”) which interfere with amplification of the desired product.

Attempts have been made to miniaturize PCR assemblies but no one has developed a cost-effective PCR assembly which can carry out PCR in a nanoliter-sized sample.

Part of the problem with miniaturization is that evaporation occurs very rapidly with small sample volumes, and this problem is made worse by the need to heat samples to −90° C. during thermocycling.

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