Non-invasive detection of fetal genetic traits

a fetal gene and non-invasive technology, applied in the field of non-invasive detection of fetal gene traits, can solve the problems of determining other, more complex fetal gene loci, such as chromosomal aberrations, aneuploidies or chromosomal aberrations, etc., and achieve the effect of facilitating the non-invasive detection of fetal genes

Inactive Publication Date: 2005-07-28
SEQUENOM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004] This surprising finding forms the basis of the present invention according to which separation of circulatory extracellular DNA fragments which are smaller than approximately 500 base pairs provides a possibility to enrich for fetal DNA sequences from the vast bulk of circulatory extracellular maternal DNA.
[0005] This selective enrichment, which is based on size discrimination of circulatory DNA fragments of approximately 500 base pairs or less, leads to a fraction which is largely constituted by fetal extracellular DNA. This permits the analysis of fetal genetic traits including those in

Problems solved by technology

The determination of other, more complex fetal genetic loci (e.g. chromosomal aberrations such as aneuploidies or chromosomal aberrations associated with Down's syndrome, or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith, such as single gene disorders, e.g. cystic fibrosis or the hemoglobinopathies) is, however, more pr

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Detection of Male Fetal DNA in Maternal Plasma by Real-Time Quantitative Polymerase Chain Reaction (PCR) After Size Fractionation of DNA by Agarose Gel Electrophoresis

Materials and Methods

[0021] Subjects and Sample Processing

[0022] Seven women pregnant in the third trimester with a male fetus were recruited for this study. 16-18 ml blood samples were collected into EDTA tubes. 6-9 ml of plasma were obtained after centrifugation at 1600 g for 10 minutes and a second centrifugation of the supernatant at 16000 g for 10 minutes.

[0023] DNA Isolation

[0024] DNA from 5-7 ml plasma was extracted using the QIAgen Maxi kit, according to the manufacturers' protocol. DNA was eluted in a volume of 1.5 ml.

[0025] DNA Precipitation [0026] 1. To the plasma DNA were added: 1 / 10 volume NaAc (3M, pH 5.2), 2 volumes absolute ethanol, MgCl2 to a final concentration of 0.01 M and Glycogen to a final concentration of 50 μg / ml. The solution was thoroughly mixed by vortexing. [0027] 2. The solution was...

example 2

Detection of Fetal DNA After Agarose Gel Electrophoresis by Polymerase Chain Reaction (PCR) of Microsatellite Markers, also Called “Short Tandem Repeats” (STRs)

Materials and Methods

[0044] Subjects and Samples

[0045] 18 ml blood samples from pregnant women and 9 ml blood from their partners were collected into EDTA tubes and plasma separated by centrifugation as described in example 1. The maternal buffy coat (i.e. the white colored top layer of the cell pellet obtained after the first centrifugation of 1600 g for 10 min.) was washed twice with PBS.

[0046] DNA Isolation

[0047] DNA from the plasma was extracted using a modification of the High Pure DNA template kit from Roche, the whole sample was passed through the filter usually used for 200 μl using a vacuum. The DNA was eluted in a volume of 50 μl elution buffer.

[0048] Paternal DNA was extracted from 400 μl paternal whole blood, using the High Pure DNA template kit, and eluted into 100 μl. Maternal DNA was isolated from the bu...

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Abstract

Blood plasma of pregnant women contains fetal and (generally>90%) maternal circulatory extracellular DNA. Most of said fetal DNA contains ≦500 base pairs, said maternal DNA having a greater size. Separation of circulatory extracellular DNA of <500 base pairs results in separation of fetal from maternal DNA. A fraction of a blood plasma or serum sample of a pregnant woman containing, due to size separation (e.g. by chromatography, density gradient centrifugation or nanotechnological methods), extracellular DNA substantially comprising ≦500 base pairs is useful for non-invasive detection of fetal genetic traits (including the fetal RhD gene in pregnancies at risk for HDN; fetal Y chromosome-specific sequences in pregnancies at risk for X chromosome-linked disorders; chromosomal aberrations; hereditary Mendelian genetic disorders and corresponding genetic markers; and traits decisive for paternity determination) by e.g. PCR, ligand chain reaction or probe hybridization techniques, or nucleic acid arrays.

Description

BACKGROUND OF THE INVENTION [0001] The presence of circulatory extracellular DNA in the peripheral blood is a well established phenomenon. In this context, it has been shown that in the case of a pregnant woman extracellular fetal DNA is present in the maternal circulation and can be detected in maternal plasma or serum. Studies have shown that this circulatory fetal genetic material can be used for the very reliable determination, e.g. by PCR (polymerase chain reaction) technology, of fetal genetic loci which are completely absent from the maternal genome. Examples of such fetal genetic loci are the fetal RhD gene in pregnancies at risk for HDN (hemolytic disease of the fetus and newborn) or fetal Y chromosome-specific sequences in pregnancies at risk for an X chromosome-linked disorder e.g. hemophilia or fragile X syndrome. [0002] The determination of other, more complex fetal genetic loci (e.g. chromosomal aberrations such as aneuploidies or chromosomal aberrations associated wit...

Claims

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Application Information

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IPC IPC(8): G01N33/53B01J20/281C12N15/09C12Q1/68G01N30/88G01N37/00
CPCC12Q1/6806C12Q2600/156C12Q1/6883C12Q2565/125
Inventor HAHN, SINUHEHOLZGREVE, WOLFGANGZIMMERMANN, BERNHARDLI, YING
Owner SEQUENOM INC
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