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Non-invasive method for isolation and detection of fetal dna

Non-invasive method for isolation and detection of fetal dna description/claims

This application is a continuation-in-part application of U.S. Ser. No. 07/772,689, filed on Oct. 7, 1991, which is a continuation-in-part application of U.S. Ser. No. 07/706,393, filed on May 28, 1991, now abandoned, which is a continuation-in-part of U.S. Ser. No. 07/436,057, filed on Nov. 13, 1989, now abandoned, all being entitled “Non-Invasive Method for Isolation and Detection of Fetal DNA” by Diana W. Bianchi. The contents of all of the forementioned applications are hereby expressly incorporated by reference.

Work described herein was supported by the National Institute of Health and Children's Hospital Medical Center.

A variety of fetal cell types—platelets, trophoblasts, erythrocytes and leucocytes—cross the placenta and circulate transiently within maternal blood (Schroder, J., J. Med. Genet. 12:230-242 (1975); Douglas G. W. et al., Am. J. Obstet. Gynec. 78:960-973 (1959)). There have been numerous reports of efforts to separate fetal cells from maternal cells present in maternal blood, but none has been successful in isolating cells subsequently shown to contain fetal DNA. Distinguishing fetal cells from maternal cells has not been successful for several reasons, including the small number of fetal cells in a maternal blood sample and the fact that morphological differences are slight (e.g., trophoblasts are the only fetal cells which can be distinguished from maternal cells by morphology alone).

Others report screening the peripheral blood of pregnant women for cells of fetal origin. Fetal identification relied on the presence of a single cytogenetic marker, the Y chromosome. Lymphocytes with a putative “XY” karyotype were found in the maternal circulation as early as 14 weeks gestation (Walknowska, J., et al., The Lancet, 1119-1122 (1979)).

The availability of flow cytometry has led many to suggest that fetal cells could be obtained through the use of a flow cytometer and that such cells could be exploited for prenatal genetic diagnosis. However, although cells sorted in this manner have been said to be of fetal origin, based on analysis of cell surface antigens, morphology, or cytogenetic criteria, there has not been confirmation that the cells contain fetal DNA. A method by which fetal DNA could be obtained from maternal blood during pregnancy would be valuable, particularly if it made it possible to carry out prenatal diagnosis by a noninvasive technique.

The present invention is based, at least in part, on the discovery that fetal nucleated cells are present in the peripheral blood of a pregnant woman at a level which allows them to be useful in prenatal diagnostic methods. The method of the present invention is non-invasive because a peripheral blood sample from pregnant women, not fetal blood, is used as the source of the fetal DNA. The fetal DNA is derived from fetal nucleated cells present in the peripheral blood of a pregnant woman. The method of the present invention can be used to assess fetal characteristics (e.g. fetal sex and chromosomal abnormalities) or can be used to diagnose whether a fetus has a prenatal disease at an early stage of the gestational period. The non-invasive method of the present invention does not expose the fetus or mother to risks, e.g. infection, fetal injury, and miscarriage, associated with invasive methods such as amniocentesis.

The present invention pertains to a method of detecting the presence or absence of a fetal DNA sequence of interest in fetal DNA derived from a sample of peripheral blood obtained from a pregnant woman. The method involves obtaining a sample peripheral blood from a pregnant woman, treating the sample of peripheral blood such that the fetal DNA present in the fetal nucleated cells is made available for detection and detecting the presence or absence of the fetal DNA sequence of interest in the available fetal DNA. The proportion of fetal nucleated cells present in the sample of peripheral blood can be increased forming a sample enriched in fetal nucleated cells prior to the detection step. The fetal DNA sequence of interest can be detected by treating the peripheral blood sample such that fetal DNA present in the sample is made available for hybridization with a DNA probe and subsequently contacting the available fetal DNA with a DNA probe hybridizable to fetal DNA of interest under hybridization conditions. The presence or absence of hybridization between the DNA probe and the fetal DNA of interest is detected as an indication of the presence or absence of the fetal DNA of interest.

The method of the present invention can be used to determine the sex of a fetus by contacting the peripheral blood sample from a woman pregnant with a fetus with a DNA probe hybridizable to fetal Y chromosomal DNA. The presence of hybridization between the DNA probe and the fetal Y chromosomal DNA can be detected as an indication of a male fetus or the absence of hybridization can be detected as an indication of a female fetus.

The method of the present invention also may be used for diagnosing a disease in a fetus. A sample of peripheral blood obtained from a woman pregnant with a fetus is contacted with DNA probe hybridizable to fetal DNA of interest associated with a disease under hybridization conditions. The presence or absence of hybridization between the DNA probe and the fetal DNA of interest is detected as an indication of whether the fetus has the disease.

The method of the present invention further can be used to detect a chromosomal abnormality in a fetus such as a chromosomal aneuploidy, e.g., trisomy 13, trisomy 18, or trisomy 21. A sample of peripheral blood from the woman pregnant with a fetus is obtained. The fetal nucleated cells are separated from the peripheral blood sample onto a solid support forming immobilized fetal nucleated material, e.g. metaphase or interphase nuclei. The immobilized fetal nucleated material is contacted with a DNA probe hybridizable to chromosomal fetal DNA of interest under hybridization conditions. The presence or absence of hybridization between the DNA probe and the chromosomal fetal DNA of interest is detected as an indication of the presence or absence of a chromosomal abnormality.

The method of the present invention further can be used to determine whether a pregnancy is at risk. Fetal blood hemhorrages into the maternal blood system typically occur when a pregnancy is at risk increasing the number of fetal cells present in the maternal blood. A peripheral blood sample can be obtained from a pregnant woman at a selected gestational age and the number of fetal cells present in the sample can be detected. This detected number of fetal cells can be compared to a known standard representative of the number of cells present at the selected gestational age during a normal pregnancy. The standard can be established by taking peripheral blood samples from a group of women at the selected gestational age believed to be having normal pregnancies.

Other aspects of this invention relate to methods of enriching the peripheral maternal blood sample and kits containing reagents used to conduct the described methods. These aspects are described in more detail below.

FIG. 1 is a schematic representation of the method of the present invention by which fetal nucleated cells are isolated from maternal cells and DNA within the fetal cells is assessed for the occurrence of a particular fetal DNA sequence.

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