“[0003] An examination of circulatory extracellular fetal DNA and circulatory extracellular maternal DNA in maternal plasma has now shown that, surprisingly, the majority of the circulatory extracellular fetal DNA has a relatively small size of approximately 500 base pairs or less, whereas the majority of circulatory extracellular maternal DNA in maternal plasma has a size greater than approximately 500 base pairs. Indeed, in certain instances the circulatory DNA material which is smaller than approximately 300 base pairs appears to be almost entirely fetal. Circulatory extracellular fetal DNA in the maternal circulation has thus been found to be smaller in size (approximately 500 base pairs or less) than circulatory extracellular maternal DNA (greater than approximately 500 base pairs). [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 or less 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 involved in chromosomal aberrations (e.g. aneuploidies or chromosomal aberrations associated with Down’s syndrome) or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies), the determination of which had, as mentioned above, so far proved difficult, if not impossible. Size separation of extracellular fetal DNA in the maternal circulation thus facilitates the non-invasive detection of fetal genetic traits, including paternally inherited polymorphisms which permit paternity testing.”
“[0008] The sample-fraction thus obtained not only permits the subsequent determination of fetal genetic traits which had already been easily detectable in a conventional manner such as the fetal RhD gene in pregnancies at risk for HDN (hemolytic disease of the fetus and the newborn), or fetal Y chromosomespecific sequences in pregnancies at risk for an X chromosomelinked disorder such as hemophilia, fragile X syndrome or the like, but also the determination of other, more complex fetal genetic loci, including but not limited to - chromosomal aberrations (e.g aneuploidies or Down’s syndrome) or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies); and - fetal genetic traits which may be decisive when paternity is to be determined. [0009] Such determination of fetal genetic traits can be effected by methods such as, for example, PCR (polymerase chain reaction) technology, ligase chain reaction, probe hybridisation techniques, nucleic acid arrays (so-called ‘DNA chips’) and the like.”
“[0019] Table 1 shows that in the five pregnancies examined, DNA fragments originating from the fetus were almost completely of sizes smaller than 500 base pairs with around 70 % being of fetal origin for sizes smaller than 300 base pairs. [0020] These results demonstrate that free DNA of fetal origin circulating in the maternal circulation can be specifically enriched by size separation of the total free DNA in the maternal blood. Depending on the downstream application the DNA size chosen for the enrichment of fetal DNA will be smaller than 300 or smaller than 500 bases.”
“A fraction of a sample of the blood plasma or serum of a pregnant woman in which, as the result of said sample having been submitted to a DNA extraction, followed by a size separation, of the extracellular DNA, the extracellular DNA present therein substantially consists of DNA consisting of 500 base pairs or less.”
“Frequency at which foetal DNA is present in maternal plasma: Difference by fragment length Objective: It has been reported that foetus-derived free DNA is present in maternal plasma, and this has been confirmed to be unmistakably true in our study as well. In this study, we hypothesised that there would be more short-length DNA fragments, because foetal DNA is degraded by DNAse in maternal plasma. Method: Using plasma samples from 9 women pregnant with boys, at 30 to 39 weeks' gestation, DNA was extracted with QIAamp DNA Blood Kit manufactured by QIAGEN. Quantitative PCR was performed using Roche's LightCycler. Primers targeting beta-globin (amplicon sizes of 110 bp and 196 bp: SYBR Green method) were prepared to estimate the amount of maternal DNA. In order to estimate the amount of foetusderived DNA, we created our own primers and probes targeting SRY (amplicon sizes of 114 bp and 186 bp: Probe hybridization method), which exists only as a single copy on the Y chromosome. By studying these 4 regions, we examined DNA copy number and the percentage of the foetus-derived DNA at around 110 bp and around 190 bp. Results: The frequency of SRY relative to beta-globin present in the maternal plasma was 19.6% for 114 bp and 9.8% for 186 bp. For overall beta-globin, 110 bp was detected 1.5 times more than 196 bp. Conclusion: This study suggests the possibility that short-length DNA fragments are more prevalent. In the future, prenatal genetic diagnosis is likely to be carried out using foetus-derived DNA in maternal plasma, but it is possible that it will be more advantageous in terms of sensitivity and specificity to prepare PCR primers with the amplicon size as short as possible.”
“In addition, for overall beta-globin, 110 bp was detected 1.5 times more than 196 bp.”
“231. First, it is not the law that a specification must necessarily enable the skilled person to make or perform all of the embodiments of a claimed invention. Were it otherwise, claims would be insufficient if they covered inventive improvements. But, as the decision in Polypeptide expression/Genentech I makes clear, in appropriate cases, a claim may embrace variants which may be provided or invented in the future and which achieve the same effect in a manner which could not have been envisaged without the invention. 232. Secondly, the assessment of insufficiency must be sensitive to the nature of the invention and the facts of the particular case. If the character of the invention is one of general methodology or is such that the invention is of general application then it may be permissible to claim it in general terms, even though the specification does not enable every way of arriving at its subject matter. Otherwise, as the Board explained in Modifying plant cells/MYCOGEN, no dominant patent could ever exist and each developer of a new method of arriving at that subject matter would be free of earlier patents. In many cases in the field of biotechnology, patent protection would then become illusory. 233. Thirdly, it is a general principle that the protection afforded by the claims must correspond to the technical contribution to the art made by the disclosure of the invention. The patentee is entitled to fair protection having regard to the nature and character of the invention he has described.”
“i) a principle of general application is simply an element of a claim which is stated in general terms; ii) a claim containing such an element is sufficiently enabled if the skilled person can reasonably expect the invention to work with anything which falls within the general term; and iii) a particular form of an element of a claim may improve the way the invention works and be inventive. However, the patent is not insufficient simply because the specification does not enable that improvement. It is still a way (albeit an improved way) of working the original invention.”
“A claim is not insufficient simply because it encompasses inventive improvements provided they embody the technical contribution the disclosure of the invention has made to the art.”
“This examination also indicated that a substantial proportion of the circulatory DNA had a molecular size >10 or even >23 kb (Fig. 1). The presence of such high-molecular-weight DNA species cannot be attributed to the plasma sample being contaminated by maternal cells because we took extreme care to obtain cell-free plasma samples.” 223.The authors also say (at page 1008): “With regard to the size distribution of total circulatory DNA, we determined that the pattern we had observed in pregnant women was very similar to that observed in samples taken from nonpregnant women as well as healthy male volunteers (Fig. 4). In none of these analyses were we able to detect large amounts of DNA with a molecular size greater than that indicated by the 23-kb molecular weight marker, in contrast to what we observed in our Southern blot analysis (Fig. 1). The reason for this anomaly may be that these large fragments are not easily eluted from the agarose gel under the conditions we are using, unlike in the Southern blotting, where the DNA is first treated with alkali to generate the small fragments required for efficient capillary transfer.”