Multiplex amplification for the detection of nucleic acid variations

A pre-amplification, digital amplification technology, applied in the field of digital amplification, can solve problems such as phenotype and cognitive abnormalities

Inactive Publication Date: 2012-11-14
THE UNIV OF BRITISH COLUMBIA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Presence of the third chromosome causes overexpression of genes involved in development, causing phenotypic and cognitive abnormalities
However, one of the advantages of digital amplification lies in the rapid evaluation of amplification products, which is lost if downstream separation steps are added
Therefore, the problem of non-specific noise is highly relevant
[0016] Consistent with these challenges with multiplexing, recently published digital amplification strategies for the detection of aneuploidy do not involve multiplex probe-based detection of target molecules.

Method used

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  • Multiplex amplification for the detection of nucleic acid variations
  • Multiplex amplification for the detection of nucleic acid variations
  • Multiplex amplification for the detection of nucleic acid variations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0141] Example 1 - Calculation of Digital PCR Accuracy

[0142] The theoretical precision of a digital PCR assay depends on the total number of chambers (N) and the expected number of molecules (λ) within each chamber. When Nλ>>1, the number of molecules present in each cavity is a random variable k, which is fully described as an independent Poisson process. The probability of detecting at least one molecule is P(k>0)=1-e -λ . The random variable x describes the number of cavities that have at least one molecule in a column of N cavities. Therefore it can be obtained by having the average N(1-e -λ ) and variable σ 2 =N(e -λ -e -2λ ) gives P(x) from the binomial distribution. For large N, this closely approximates a Gaussian distribution:

[0143] P ( x ) = 1 2 πN ( e ...

Embodiment 2

[0150] Example 2 - Calculation of the separation of the measured means of two alleles varying by 1% as a function of the number of discrete subsamples (e.g. cavities) using digital PCR

[0151] figure 2 Numerical calculations of the separation of measured means for two alleles varying by 1% as a function of cavity number using digital PCR are shown. Differences were normalized by the expected standard deviation (σ) determined by the combined effect of 5 random Poisson variables (curves). Calculate the template concentration corresponding to positive amplification in 50% of the wells. A 5σ separation (horizontal line) is achieved at about 1000000 cavities. Standard deviations were achieved with the number of compartments that could discern a 1% difference in DNA concentration at a fill factor of 0.5 in a digital PCR experiment.

Embodiment 3

[0152] Example 3 - Theoretical Calculation of Sample Molecular Content and Blood Sample Volume

[0153] The presence of fetal DNA in maternal blood has been reported to represent approximately 2-6% of the total DNA present in cell-free serum during the first trimester (Lo et al. 1997; Lo et al. 1998; Wachtel et al. 2001; Lee et al. 2002). However, later publications showed that the fetal contribution was 9.7%, 9.0%, and 20.4% in the first, second, and third trimesters, respectively (Lun et al. 2008). For example, each genome copy of the T21 segment contributes an additional copy of chromosome 21, which allows direct non-invasive maternal blood testing by measuring the ratio of chromosome copy numbers present in maternal serum. If one assumes 2% fetal DNA fraction in a maternal blood sample, the expected abundance of chromosome 21 relative to the other chromosomes in the pool is (0.02x 1) / (1x 2)=1%. Such a small difference in relative concentration is difficult to detect by t...

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Abstract

Kits, primers, and methods are provided herein for detecting relative target source to reference source ratios in a biological sample, by distributing the biological sample into discrete subsamples, wherein the biological sample includes, a plurality of target molecules on a target source; and a plurality of reference molecules on a reference source; providing target primers directed to one or more of the plurality of target molecules and reference primers directed to one or more of the plurality of reference molecules; performing digital amplification with the target primers and the reference primers; and detecting the presence or absence of amplified target products with target probes and detecting the presence or absence of amplified reference products with reference probes, wherein the ratio of amplified target products to amplified reference products is indicative of a relative amount of target source to reference source in a biological sample.

Description

field of invention [0001] The present invention relates to digital amplification methods. In particular, the present invention relates to methods for detecting the relative ratio of a target source to a control source in a biological sample. [0002] Related Applications Cited [0003] This application requires that the application serial number submitted on January 15, 2010 be No.61 / 282,298, and the title of the invention be "MULTIPLEX AMPLIFICATION USING A COMMON PRIMER-DERIVED INTERNAL PROBE SEQUENCE (multiple amplification using a common primer-derived internal probe sequence) method)” and two U.S. patent applications with application serial number No. 61 / 282,299 and the title of the invention “MULTIPLEX AMPLIFICATION USING A COMMON TEMPLATE-DERIVED INTERNAL PROBE SEQUENCE (Multiple Amplification Method Using Internal Probe Sequences Derived from a Common Template)” Priority of provisional application. Background of the invention [0004] Most genetic diseases in huma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34C40B30/04G01N33/50
CPCC12Q2600/156C12Q2600/16C12Q1/6883C12Q1/6851C12Q2537/143C12Q2537/165C12Q2563/159
Inventor 卡尔·L·G·汉森欧列·派特丽芙凯文·海里斯肯尼斯·J·里瓦克
Owner THE UNIV OF BRITISH COLUMBIA
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