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Restriction endonuclease enhanced polymorphic sequence detection

An endonuclease, nucleic acid technology, applied in the field of alleles, can solve problems such as lack of multiple tests

Active Publication Date: 2013-10-23
SEQUENOM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Competitive PCR with allele-specific fluorescent probes lacks the ability to multiplex more than 2-3 assays in a single tube format
[0004] In addition, similar methods that exploit methylation differences between DNA species (for example, U.S. Patent Published Application No. 20070059707 entitled "Methods for prenatal diagnosis of chromosome abnormalities" , which case is incorporated herein by reference) is invalid under low copy number genomic DNA

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  • Restriction endonuclease enhanced polymorphic sequence detection
  • Restriction endonuclease enhanced polymorphic sequence detection
  • Restriction endonuclease enhanced polymorphic sequence detection

Examples

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example

[0133] The following examples are provided to further illustrate various embodiments of the invention and not to limit it.

example 1

[0134] Example 1 : Restriction endonuclease-enhanced polymorphic sequence detection using Hpych4v and Nlaiii

[0135] The efficacy of restriction endonuclease-enhanced polymorphic sequence detection was demonstrated using several restriction endonucleases (REs), including HpyCH4V and NlaIII (from New England Biolabs, Inc. (New England BioLabs, Inc)). The ability of the two enzymes to specifically cut off one allele of a given polymorphism while performing PCR amplification of the remaining alleles of the polymorphism was tested separately in multiplex genotyping reactions. See Table 2 for polymorphisms tested with each enzyme.

[0136] Two CEPH DNA samples were mixed in different ratios to generate DNA samples consisting of 0%, 2%, 5%, 20%, 50% and 100% DNA heterozygous for both alleles of the SNP , the rest of the DNA is the DNA recognized by the RE as being homozygous for the allele. Table 3 shows the DNA samples and corresponding genotype information used in these stud...

example 2

[0162] Example 2 : Restriction Endonuclease Enhanced Polymorphic Sequence Detection Using Tfii

[0163]Similar experiments were carried out as described in Example 1 using a different restriction endonuclease TfiI. In this experiment, the TfiI restriction endonuclease selectively recognizes and cleaves the 'C' allele of the 'C / T' SNP, rs4487973. SNP rs4487973 occurs in the following genomic sequence on chromosome 1: CACACAGTTAGGATT[C / T]ACCTGAGCTTGTCCC (SEQ ID NOs: 28). For these studies, two CEPH DNA samples (one homozygous 'C' for rs4487973 SNP and 'C / T' heterozygous for rs4487973 SNP) were mixed at different ratios to generate samples containing 0%, 1 %, 2.5%, 10%, 50% DNA mixtures of the rs4487973T allele. TfiI restriction endonuclease was added or not added to each mixture to determine the effect of the endonuclease on polymorphic sequence detection. In the mixture not digested with TfiI enzyme, the rs4487973T allele was detected in the 10% and 50% T allele mixture bu...

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Abstract

The present invention provides an improved method for detecting specific polymorphic alleles in mixed DNA populations. The method comprises enriching the relative percentage of a given polymorphic allele that is exponentially amplified by PCR. The invention also provides methods for selectively enriching nucleic acid of interest (eg, fetal nucleic acid in a maternal sample). In the case of detection of fetal nucleic acid in maternal samples, restriction enzymes are introduced that can discriminate alleles of polymorphic sites. Preferably, the maternal allele is digested and the nucleic acid comprising the paternal allele is relatively enriched.

Description

technical field [0001] Provided herein are methods for detecting specific alleles in a mixed nucleic acid sample. The method can be used to detect the presence of fetal nucleic acid in a maternal sample. Background technique [0002] The use of circulating nucleic acid analysis for the non-invasive diagnosis, monitoring and prediction of many clinical conditions has been revealed. For example, for prenatal applications, circulating fetal-specific sequences have been detected and form part of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of target sequences, analytical sensitivity, and specificity. Robust and reliable discrimination of sequence differences (eg, single nucleotide polymorphisms or SNPs) between circulating DNA species is technically challenging and requires analytical methods with high sensitivity and specificity. [0003] Current techniques for detecting sequence differences ...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/683C12Q1/6879C12Q1/686C12Q2537/143C12Q2527/137C12Q2521/301C12Q2531/113
Inventor 马蒂亚斯·埃里希迪尔克·J·范登博姆
Owner SEQUENOM INC