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Highly multiplex PCR methods and compositions

a pcr method and composition technology, applied in the direction of instruments, library screening, directed macromolecular evolution, etc., can solve the problems of increasing the risk of generating such products, limiting the use of amplified products for further analysis and/or assays, and generating non-target amplification products. to achieve the effect of increasing the chances of a successful pregnancy

Inactive Publication Date: 2014-04-03
NATERA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a method for selectively amplifying DNA at specific locations in the genome, which can be useful for identifying specific genetic markers. The method involves using a special technique called ligation-mediated PCR to attach DNA probes to the targeted areas and then increasing the number of copies of those areas using amplification. This results in a selective enrichment of DNA at those specific locations. The method may have applications in genetic testing or the development of personalized medicine.

Problems solved by technology

However, when multiple pairs are added to the same PCR reaction, non-target amplification products may be generated, such as amplified primer dimers.
The risk of generating such products increases as the number of primers increases.
These non-target amplicons significantly limit the use of the amplified products for further analysis and / or assays.
Unfortunately, standard methods have either poor accuracy, or involve an invasive procedure that carries a risk of miscarriage.
Methods based on maternal blood hormone levels or ultrasound measurements are non-invasive, however, they also have low accuracies.
Methods such as amniocentesis, chorion villus biopsy and fetal blood sampling have high accuracy, but are invasive and carry significant risks.

Method used

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  • Highly multiplex PCR methods and compositions
  • Highly multiplex PCR methods and compositions
  • Highly multiplex PCR methods and compositions

Examples

Experimental program
Comparison scheme
Effect test

experiment 1

[0583]The objective was to show that a Bayesian maximum likelihood estimation (MLE) algorithm that uses parent genotypes to calculate fetal fraction improves accuracy of non-invasive prenatal trisomy diagnosis compared to published methods.

[0584]Simulated sequencing data for maternal cfDNA was created by sampling reads obtained on trisomy-21 and respective mother cell lines. The rate of correct disomy and trisomy calls were determined from 500 simulations at various fetal fractions for a published method (Chiu et al. BMJ 2011; 342:c7401) and our MLE-based algorithm. We validated the simulations by obtaining 5 million shotgun reads from four pregnant mothers and respective fathers collected under an IRB-approved protocol. Parental genotypes were obtained on a 290K SNP array. (See FIG. 14)

[0585]In simulations, the MLE-based approach achieved 99.0% accuracy for fetal fractions as low as 9% and reported confidences that corresponded well to overall accuracy. We validated these results u...

experiment 2

[0587]The objective was to improve non-invasive detection of fetal trisomy 18, 21, and X particularly in samples consisting of low fetal fraction by using a targeted sequencing approach combined with parent genotypes and Hapmap data in a Bayesian Maximum Likelihood Estimation (MLE) algorithm.

[0588]Maternal samples from four euploid and two trisomy-positive pregnancies and respective paternal samples were obtained under an IRB-approved protocol from patients where fetal karyotype was known. Maternal cfDNA was extracted from plasma and roughly 10 million sequence reads were obtained following preferential enrichment that targeted specific SNPs. Parent samples were similarly sequenced to obtain genotypes.

[0589]The described algorithm correctly called chromosome 18 and 21 disomy for all euploid samples and normal chromosomes of aneuploid samples. Trisomy 18 and 21 calls were correct, as were chromosome X copy numbers in male and female fetuses. The confidence produced by the algorithm w...

experiment 3

[0591]The objective was to determine if trisomy is detectable with high confidence on a triploid fetus, using novel informatics to analyze SNP loci of free floating fetal DNA in maternal plasma.

[0592]20 mL of blood was drawn from a pregnant patient following abnormal ultrasound. After centrifugation, maternal DNA was extracted from the buffy coat (DNEASY, QIAGEN); cell-free DNA was extracted from plasma (QIAAMP QIAGEN). Targeted sequencing was applied to SNP loci on chromosomes 2, 21, and X in both DNA samples. Maximum-Likelihood Bayesian estimation selected the most likely hypothesis from the set of all possible ploidy states. The method determines fetal DNA fraction, ploidy state and explicit confidences in the ploidy determination. No assumptions are made about the ploidy of a reference chromosome. The diagnostic uses a test statistic that is independent of sequence read counts, which is the recent state of the art.

[0593]The instant method accurately diagnosed trisomy of chromoso...

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Abstract

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. Utility Ser. No. 13 / 683,604, filed Nov. 21, 2012, which is a continuation-in-part of U.S. Utility application Ser. No. 13 / 300,235, filed Nov. 18, 2011, and is a continuation-in-part of U.S. Utility application Ser. No. 13 / 110,685, filed May 18, 2011. U.S. Utility application Ser. No. 13 / 110,685 claims the benefit of U.S. Provisional Application Ser. No. 61 / 395,850, filed May 18, 2010; U.S. Provisional Application Ser. No. 61 / 398,159, filed Jun. 21, 2010; U.S. Provisional Application Ser. No. 61 / 462,972, filed Feb. 9, 2011; U.S. Provisional Application Ser. No. 61 / 448,547, filed Mar. 2, 2011; and U.S. Provisional Application Ser. No. 61 / 516,996, filed Apr. 12, 2011. U.S. Utility application Ser. No. 13 / 300,235 claims the benefit of U.S. Provisional Application Ser. No. 61 / 571,248, filed Jun. 23, 2011 and U.S. Provisional Application Ser. No. 61 / 542,508, filed Oct. 3, 2011. U.S. Utility application...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G16B5/20G16B20/10G16B20/20G16B20/40G16B30/10G16B40/10
CPCC12Q1/68C12Q1/6844C12Q2600/16G16B20/00G16B5/00G16B30/00G16B40/00G16B30/10G16B40/10G16B20/20G16B5/20G16B20/40G16B20/10C12Q1/686C12N15/1089C12Q1/6883C12Q1/6876C12Q1/6886C12Q2600/156
Inventor ZIMMERMANN, BERHARDHILL, MATTHEWLACROUTE, PHILIPPEDODD, MICHAEL
Owner NATERA
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