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Specific amplification of fetal DNA sequences from a mixed, fetal-maternal source

A specific, DNA probe technology, applied in the field of selective amplification of fetal DNA sequences, can solve the problems of maternal DNA contamination, large-scale or systematic research that has not been reported

Inactive Publication Date: 2009-04-29
THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Second, the test is limited to trisomy 21 and trisomy 18
The third problem is only 95% sensitivity
However, the DNA obtained by these methods is likely to be highly contaminated with maternal DNA
Furthermore, no large-scale or systematic studies have been reported

Method used

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  • Specific amplification of fetal DNA sequences from a mixed, fetal-maternal source
  • Specific amplification of fetal DNA sequences from a mixed, fetal-maternal source
  • Specific amplification of fetal DNA sequences from a mixed, fetal-maternal source

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 demonstrates the successful use of adapter-mediated amplification on DNA isolated from maternal plasma. Purified DNA was digested with the CpG methylation-sensitive enzyme HpyCh4-IV prior to amplification. After digestion, the adapters were annealed and ligated to the digested DNA, and finally the top strand of the adapter pair was used for PCR according to published protocols. See Example 1 and Guillaud-Bataille, M., et al., Nucleic Acids Res. 32:e112 (2004). Notably, it has been determined that no formaldehyde should be involved in maternal blood collection methods.

Embodiment 2

[0042] Example 2 demonstrates successful adapter-mediated methylation-specific amplification of trophoblast / fetal DNA. Trophoblast / fetal DNA and DNA samples from whole blood were digested with the CpG methylation sensitive enzyme Acll. Similar to Example 1, after enzyme digestion, the adapters were annealed and ligated to the digested DNA, and finally the upper strand of the adapter pair was used for PCR according to the same PCR protocol as described in Example 1. Notably, trophoblast / fetal DNA consistently yielded more and apparently different PCR products than whole blood. However, it has been determined that despite the fact that CpG methylation sensitive enzymes are used, non-trophoblast / fetal DNA (ie DNA from whole blood) is still amplified. Therefore, the inventors determined that adapter-mediated PCR amplification alone is not sufficient for specific amplification of trophoblast / fetal DNA.

[0043] Thus, in the adapter-mediated PCR step of the present invention, a mi...

Embodiment 4

[0060] The invention also provides methods of identifying fetal-specific amplicons. See embodiment 5 for details. The method comprises separately preparing methylation-sensitive representatives from fetal DNA and whole blood DNA using the method for selectively amplifying fetal DNA described above. Fetal-specific amplicons refer to amplicons that will be amplified from trophoblast / fetal DNA but not other DNA using the methods described herein. Trophoblast / fetal DNA is hypomethylated DNA compared to adult DNA. Methylation-sensitive restriction enzymes will cleave hypomethylated fetal loci but not methylated maternal loci.

[0061] labeling a methylation-sensitive representative from fetal DNA with a first fluorochrome, and labeling whole blood DNA with a second fluorochrome different from the first fluorochrome to generate a labeled fetal DNA probe and a labeled whole blood DNA probe . Labeled probes are hybridized to an array of oligonucleotides corresponding to predicted ...

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Abstract

The present invention provides a method of selectively amplifying fetal DNA sequences from a mixed, fetal-maternal source. This method utilizes differential methylation to allow for the selective amplification of trophoblast / fetal specific sequences from DNA mixtures that contain a high proportion of non- trophoblast / fetal DNA. The invention also provides methods of using the amplified fetal DNA sequences for aneuploidy detection.

Description

technical field [0001] The present invention provides methods for selectively amplifying fetal DNA sequences from mixed fetal-maternal sources. The method exploits differential methylation to allow selective amplification of trophoblast / fetal-specific sequences from DNA mixtures containing a high proportion of non-trophoblast / fetal DNA. The present invention also provides methods for aneuploidy detection using amplified fetal DNA sequences. Background technique [0002] Numerous studies have shown that whole-chromosomal aneuploidy occurs in 1 to 2% of newborns. Hsu, A.M. (ed.) Genetic Disorders and the Fetus, pp. 179-248 (1998). This chromosomal abnormality is an important cause of prenatal morbidity and mortality and a major cause of severe developmental delay in long-term survivors. Given the maternal age dependence of common trisomies and the marked increase in mean age at childbearing, it is clear that the importance of screening for aneuploidy will continue to increa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34C12N15/87C12Q1/68
CPCC12Q2600/156C12Q2600/154C12Q1/6881C12Q1/6883C12N15/87C12P19/34
Inventor 史蒂芬·布朗
Owner THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK
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