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Methods and compositions for assaying mutations and/or large scale alterations in nucleic acids

A large-scale, point mutation technology, applied in biochemical equipment and methods, microbial determination/testing, etc., can solve problems such as increased costs, single base substitution detection, unreliable small insertions and deletions, etc.

Inactive Publication Date: 2007-06-20
INSTITUT CURIE +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This means duplication of payments and increased costs
[0024] Other methods for detecting large-scale changes, such as real-time PCR or multiplex liquid chromatography (MP / LC) (Dehainault C, Lauge A, Caux-Moncoutier V, Pages-Berhouet S, Doz F, Desjardins L, Couturier J, Gauthier-Villars M, Stoppa-Lyonnet D, Houdayer C, 2004, Multiplex PCR / liquid chromatography assay for detection of gene rearrangements: application to RB1 gene, Nucleic Acids Res32 (18): e139) also cannot detect single base substitutions, Also not reliable for small insertions and deletions

Method used

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  • Methods and compositions for assaying mutations and/or large scale alterations in nucleic acids
  • Methods and compositions for assaying mutations and/or large scale alterations in nucleic acids
  • Methods and compositions for assaying mutations and/or large scale alterations in nucleic acids

Examples

Experimental program
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Effect test

Embodiment 1

[0218] Semiquantitative determination of DNA concentration using capillary electrophoresis with SYBRgreen I - Capillary-to-capillary variability

[0219] This experiment was carried out using multiplex M25BC2. The multiplex M25BC2 consists of 4 fragments of different sizes (290bp, 365bp, 412bp and 472bp), which correspond to exons 5, 21, 20 and 16 of the BRCA2 gene, respectively.

[0220] Injections were performed at 2kV for 20 seconds. Perform separations in the same capillary array, in the same run.

[0221] Figure 1 shows the capillary-to-capillary repeatability of the area measurements of the different peaks obtained in Example 1. All peak areas were normalized by the area of ​​the second fragment (exon 21). The largest differences observed were on the order of 10%, which is perfectly compatible with large rearrangement deletions. Indeed, markers representing duplications will cause at least a 50% increase in amplitude, while deletions will cause a decrease in ampli...

Embodiment 2

[0223] Semiquantitative determination of DNA concentration using capillary electrophoresis with SYBRgreen I -Variability of consecutive runs

[0224] The experiments in this group were carried out using multiplex M25BC2. The multiplex M25BC2 consists of 4 fragments of different sizes (290bp, 365bp, 412bp and 472bp), which correspond to exons 5, 21, 20 and 16 of the BRCA2 gene, respectively.

[0225] Injections were performed at 2kV for 20 seconds. Separation is performed continuously. 5 injections were performed.

[0226] Figure 2 shows the run-to-run repeatability of the mean normalized area determination of the different peaks in Example 2 over 5 run runs. Peak areas were normalized by the area of ​​the second fragment (exon 21). The average normalized area from consecutive runs is very reproducible. Error bars correspond to standard deviation. This also shows that false positives for large-scale mutations are very unlikely.

Embodiment 3

[0228] Simultaneous detection of large deletions in one patient and substitutions in different patients

[0229] This experiment was performed using polysome M1BC1. The multiplex M1BC1 consists of 4 fragments of different sizes (268bp, 334bp, 433bp and 519bp), which correspond to different exons of the BRCA1 gene (exon 19, exon 6, exon exon 22 and exon 11.5).

[0230] Multiplex M1BC1-A corresponds to patients without major chromosomal rearrangements but with a single base substitution in exon 11.5.

[0231] The multiplex M1BC1-B corresponds to patients with exon 3 to exon 16 large deletions.

[0232]It can be seen that the peaks corresponding to exon 6 and exon 11.5 of M1BC1-B are about 2-fold smaller than those of M1BC1-A. This indicated that exons 6 and 11.5 in M1BC1-B were deleted. It can also be seen that exon 11.5 of M1BC1-A has two peaks, while exon 11.5 of M1BC1-B has a single peak. This suggests a small variant in exon 11.5 of M1BC1-A. Sequencing of this fragme...

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Abstract

The present invention relates to a method for detecting point mutation(s) and / or large scale alteration(s) relative to at least one nucleic acid fragment said method comprising at least the steps of providing a sample liable to contain said nucleic acid fragment and at least a second nucleic acid fragment acting as a quantitative reference, subjecting said fragments to suitable conditions for obtaining a product containing homoduplexes and possible heteroduplexes, conducting on said product an analytical method suitable for obtaining at least signal(s) discriminating the existing duplex form(s) of the first nucleic acid fragment and relative quantitative data concerning said first nucleic acid fragment The invention further relates to the use of this method in the diagnosis of predisposition to genetic diseases and cancers and in the diagnosis and prognosis of said diseases and cancers, like human breast cancer.

Description

Background technique [0001] Recent advances in genomics have held great promise for advancing human health and improving biotechnology. [0002] In medicine, for example, the understanding and diagnosis of hereditary diseases and cancers or the study of infectious organisms increasingly relies on the analysis of DNA and nucleic acids. [0003] Biotechnology is also increasingly dependent on molecular genetic analysis and high-throughput analysis of nucleic acids. Mapping genetic differences between individuals is becoming increasingly important especially for forensic investigations, medical applications, biotechnology and the food industry. [0004] For example, detecting mutations that cause abnormal proteins may be necessary to determine the genetic origin of a disease. Using methods for structural analysis of DNA, a large number of genetic pathological conditions can be diagnosed before symptoms begin. In cancer research, for example, mutations in the BRCA1 and BRCA2 ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q2600/16C12Q1/6886C12Q2565/125C12Q2545/101C12Q2537/113C12Q2537/143
Inventor 让-路易·维奥伊克洛德·乌达耶多米尼克·斯托帕-利奥内热雷米·韦伯
Owner INSTITUT CURIE
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