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Non-invasive fetal genetic screening by sequencing analysis

A fetal and sequencing technology, applied in the field of non-invasive technology, can solve the problems of sample consumption, differentiation, and digital analysis that do not allow the detection of single nucleotide polymorphisms in fetuses, so as to achieve improved clinical performance and high detection rate Effect

Inactive Publication Date: 2016-04-06
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, such methods are optimized for the analysis of a single target in a biological sample and, moreover, consume the sample without interrogating the identity of other targets in the sample
Furthermore, digital PCR is limited to optical detection
Therefore, multiplex analysis using digital PCR is not feasible due to the difficulty of distinguishing the optical signals of multiple targets in a given well
Furthermore, routine digital analysis does not allow detection of fetal single nucleotide polymorphisms (SNPs) that are suggestive of a wide range of fetal diseases or pathological conditions

Method used

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  • Non-invasive fetal genetic screening by sequencing analysis
  • Non-invasive fetal genetic screening by sequencing analysis
  • Non-invasive fetal genetic screening by sequencing analysis

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0035] A. Biological sample preparation

[0036] The methods of the invention involve non-invasive detection. In a specific embodiment of the method, the starting material is maternal blood. In order to obtain sufficient DNA for testing, preferably 10-20 mL of blood is drawn to obtain approximately at least 10,000 genome equivalents of total DNA. This sample size was estimated based on fetal DNA present at approximately 25 genome equivalents / mL of maternal plasma in early pregnancy, a concentration of fetal DNA of approximately 3.4% of total plasma DNA. However, for genetic screens requiring less statistical significance, or DNA samples in which fetal DNA is enriched, less blood may be drawn.

[0037] It should be noted that although fetal DNA is referred to throughout this specification, fetal RNA found in maternal blood can also be analyzed. hPL (human placental lactogen) and hCG (human chorionic gonadotropin) mRNA transcripts, as analyzed using the respective real-time ...

Embodiment 1

[0093] Example 1: Detection of fetal genetic abnormalities in maternal blood

[0094] The amount of fetal DNA in maternal blood varies widely and increases as pregnancy progresses. In early pregnancy, the levels of fetal DNA present in maternal blood are insufficient to discern differences in the ratios of maternal and fetal DNA. The difference in the proportion of maternal and fetal DNA in maternal blood can be estimated.

[0095] The finite target allele counts according to the Poisson distribution are calculated as follows:

[0096] Interpretation:

[0097] N T =#total alleles=N M +N W ,

[0098] N M = # mutant allele, N W =# wild type allele

[0099] The above formula is shown in figure 2 , which shows the theoretical fetal count mean, assuming maternal carrier of the mutation, ε is the fraction of detectable alleles equal to the difference between wild-type and mutant alleles in homozygous fetuses.

[0100] Homozygous case: N M –N W =ε*N T

[0101] Cas...

Embodiment 2

[0114] Example 2: Avoiding amplification bias when screening a panel of genetic mutations

[0115]Amplification bias is a known problem in multiplex PCR. Amplification bias can be addressed by referring to one genotype within the amplicon relative to another. However, in order to obtain the fraction of fetal DNA in a sample, comparisons between different amplicons are necessary, each of which will experience amplification bias (e.g., the amount of two different targets that are quantitatively different before amplification equal, 2-fold or higher difference after amplification).

[0116] Preliminary evidence suggests that digital PCR produces less amplification bias than Poisson sampling error for single amplicons. If a multiplex PCR reaction is to be spiked with a known or equal number of reference targets with known sequences, the reference targets can then be used to detect relative or absolute amounts of targets in different samples.

[0117] For example, if a multipl...

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Abstract

The present invention provides a non-invasive technique for differentially detecting polygenotypes and / or mutations of multiple target genes in biological samples containing genetic material from different genomic sources. Performed using multiplex amplification of multiple target sequences from biological samples, sequencing is used to detect and count genetic mutations and chromosomal abnormalities at the single nucleotide level.

Description

[0001] related application [0002] This patent application claims the benefit of and priority to US Provisional Patent Application Serial No. 61 / 406,809, filed October 26, 2010, which is hereby incorporated by reference in its entirety. [0003] Statement Regarding Federally Sponsored Research or Development [0004] This invention was made with United States Government support under agreement OD000251 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0005] The present invention relates to the field of quantitative nucleic acid analysis from biological samples containing genetic material from different genomic sources. More specifically, the present invention provides a non-invasive technique for the differential detection of genotypes in biological samples containing genetic material from different genomic sources. Background technique [0006] Fetal DNA shed from the placenta can be detected in mate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/48C12Q1/68
CPCC12Q1/6809C12Q1/6881C12Q1/6883C12Q2600/156Y02A90/10C12Q2537/143C12Q2545/101
Inventor S·R·夸克W·顾H-M·C·范
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
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