“Overall, the skilled person would have taken the discussion and conclusions of the paper at face value, as little supporting data or relevant control information is provided.”
“As noted in my first report at paragraph 7.16, there is littlesupporting data and relevant control data in Kazakov. I therefore agree with Professor Lovett that Kazakov contains various statements that are not supported by data presented in the paper. I also agree with Professor Lovett that Figures 1 and 2 do not support the conclusions which Professor Lovett says in his report they are intended to support.”
“This achievement represented an important milestone in the prenatal diagnostic field, which had previously been focused on the analysis of foetal cells. Professor Lo’s finding that maternal serum contained cell-free foetal DNA in clinically significant quantities opened up an entirely new, and unexpected, avenue of enquiry with respect to non-invasive prenatal diagnostic testing.”
“The first publication claiming to have found foetal DNA in maternal serum and plasma was published by Dr Lo and colleagues in the Lancet in 1997. It was viewed as a very interesting development, and the Lo 1997 paper was widely cited and the discovery was widely reported.”
“It has now been discovered that foetal DNA is detectable in maternal serum or plasma samples. This is a surprising and unexpected finding; maternal plasma is the very material that is routinely discarded by investigators studying non-invasive prenatal diagnosis using foetal cells in maternal blood. The detection rate is much higher using serum or plasma than using nucleated blood cell DNA extracted from a comparable volume of whole blood, suggesting that there is enrichment of foetal DNA in maternal plasma and serum. In fact, the concentration of foetal DNA in maternal plasma expressed as a % of total DNA has been measured as from 0.39% (the lowest concentration measured in early pregnancy), to as high as 11.4% (in late pregnancy), compared to ratios of generally around 0.001% and up to only 0.025% for cellular fractions (Hamada et al 1993). It is important that foetal DNA is not an artefact of the clotting process.”
“The invention provides a detection method performed on a maternal serum or plasma sample from a pregnant female, which method comprises detecting the presence of a nucleic acid of foetal origin in the sample wherein said nucleic acid is a paternally inherited sequence which is not possessed by said pregnant female. The detection may comprise quantifying the nucleic acid or determining the sequence of the nucleic acid. The invention thus provides a method for prenatal diagnosis.”
“The invention provides a method of performing a prenatal diagnosis on a maternal blood sample, which method comprises obtaining a non-cellular fraction of the blood sample and performing nucleic acid analysis on the fraction to detect the presence of a nucleic acid of foetal origin in the sample, wherein said sequence is a paternally inherited sequence which is not possessed by the mother. A method of performing a prenatal diagnosis on a maternal blood sample according to the invention may comprise removing all or substantially all nucleated and anucleated cell populations from the blood sample and subjecting the remaining fluid to a test for foetal nucleic acid indicative of a maternal or foetal condition or characteristic.”
“[0076] These observations indicate that maternal plasma/serum DNA may be a useful source of material for the non-invasive prenatal diagnosis of certain genetic disorders. To demonstrate that clinical applications are possible, a number of important questions need to be answered. First, foetal DNA in maternal plasma and serum needs to be shown to be present in sufficient quantities for reliable molecular diagnosis to be carried out. Second, data on the variation of foetal DNA in maternal plasma and serum with regard to gestation age is required to determine the applicability of this technology to early prenatal diagnosis.”
“[0077] In this Example we have addressed both of these issues by developing a real time quantitative TaqMan polymerase chain reaction (PCR) assay (Heid et al. 1996) for measuring the copy numbers of foetal DNA molecules in maternal plasma and serum. … Our data show that foetal DNA is present in maternal plasma and serum at concentrations similar to those achieved by many foetal cell enrichment protocols. We have also investigated the changes of foetal DNA concentration in maternal serum at different gestational ages. Using this plasma or serum-based approach, we show that the reliable detection of foetal DNA is achievable and therefore useful for the non-invasive prenatal diagnosis of selected genetic disorders.”
“1. A detection method performed on a maternal serum or plasma sample from a pregnant female, which method comprises detecting the presence of a nucleic acid of foetal origin in the sample, wherein said nucleic acid is a paternally inherited sequence which is not possessed by said pregnant female. 4. A method according to any one of claims 1 to 3, wherein said detecting comprises amplifying said nucleic acid. 5. A method according to claim 4, wherein said amplification is by the polymerase chain reaction. 7. A method according to any one of the preceding claims, wherein the presence of a foetal nucleic acid sequence from the Y chromosome is detected. 8. A method according to claim 7, for determining the sex of the foetus.”
“… the law does not deem the skilled person to assume the prior art has any relevance to the problem he is addressing or require him to take it forward. Having considered it, he may conclude that it is simply not a worthwhile starting point and so put it to one side. ”
“EXTRACELLULAR DNA IN THE BLOOD OF PREGNANT WOMEN The level of extracellular DNA increases in the blood of women during pregnancy. By means of PCR, the full-size Alu repeats were observed among extracellular blood DNA repeats of pregnant women. Furthermore, with Tc65 type primer the PCR method allowed to observe in the blood DNA fragments flanked by inverted Alu repeats (inter-Alu repeats). The presence of such a type of inter Alu repeats was estimated in the blood of women being in the first trimester of pregnancy only, but was not estimated among blood DNA fragments of women of the last trimester of pregnancy. It is discussed which types of cells may serve as a source of extracellular blood DNA (either trophoblasts, lymphocytes, or decidual cells), the significance of such DNA for pregnancy being appreciated.”
“According to available data, cellular proliferation, differentiation, and cell death occurs in the uterus during pregnancy… It was anticipated that these processes exert an influence on the specifics of the nucleotide composition of the extracellular DNA in the blood of pregnant women.”
“[w]ith the help of the Tc65 primer, we detected inter-Alu repeats in the blood of women only in the first trimester of pregnancy (Fig. 2).”
“[i]t is important that inter-Alu repeats have been detected only in the blood of women in the first trimester of pregnancy. This fact most likely reflects the difference in content of the cellular processes that are characteristic of the early and late stages of pregnancy.”
“Thus, in the early stages of pregnancy in humans, cells of the foetus (trophoblast) and the mother (cells of the endometrium and lymphocytes) may excrete DNA … it can be conjectured that the inter-Alu repeats discovered by us in the blood serum of pregnant women may play some kind of regulatory role in the early stages of pregnancy …”
“I agree there is no evidence to show that there is an increase, and there never would have been the evidence, because he was using a qualitative technique.”
“The skilled person would have found the information presented in the abstract interesting from the perspective of prenatal screening and diagnosis, particularly the fact that the authors say that there is an increase in the levels of extracellular DNA in the blood of pregnant women during pregnancy and the suggestion that trophoblast (i.e. foetal) cells are a possible source of extracellular blood DNA in such women.”
“We should step back a moment and say that we are assuming this is good science and the skilled clinician would look at this and immediately say the science was bad. We would take it to a geneticist, who would also say the science is really bad. For us to say we can make any kind of statement from this paper, it is possible for us to speculate, but there is no data and no scientific support for anything that is in this paper.”
“Q. There is no basis in Kazakov from which a skilled person could conclude that if you carried out the test that you are suggesting he might have come up with to look for foetal DNA that it would actually work to find foetal DNA, that it was likely to work.” “A. But I would not have approached the experiment with a definite expectation. That is why you do experiments; to actually investigate things.”
“This invention relates to prenatal diagnosis using non-invasive techniques. In particular, it relates to prenatal diagnosis by detecting foetal nucleic acids in serum or plasma from a maternal blood sample. Conventional prenatal screening methods for detecting foetal abnormalities and for sex determination traditionally use foetal samples derived by invasive techniques such as amniocentesis and chorionic villus sampling. These techniques require careful handling and present a degree of risk to the mother and to the pregnancy.”
“It has now been discovered that foetal DNA is detectable in maternal serum or plasma samples. This is a surprising and unexpected finding; maternal plasma is the very material that is routinely discarded by investigators studying non-invasive prenatal diagnosis using foetal cells in maternal blood. The detection rate is much higher using serum or plasma than using nucleated blood cell DNA extracted from a comparable volume of whole blood, suggesting that there is enrichment of foetal DNA in maternal plasma and serum. It is important that foetal DNA is found in maternal plasma as well as serum because this indicates that the DNA is not an artefact of the clotting process.”
“This invention provides a method of performing a prenatal diagnosis on a maternal serum or plasma sample which method comprises detecting the presence of a nucleic acid sequence of foetal origin in the sample.”
“The term “prenatal diagnosis” as used herein covers determination of any maternal or foetal condition or characteristic which is related to either the foetal DNA itself or to the quantity or quality of the foetal DNA in the maternal serum or plasma. Included are sex determination, and determination of foetal abnormalities which may be for example chromosomal aneuploidies or simple mutations. Also included is detection and monitoring of pregnancy-associated conditions such as pre-eclampsia which may result in differing amounts of foetal DNA being present in the maternal serum or plasma. The nucleic acid detected in the method according to the invention may be of a type other than DNA e.g. mRNA.”
“The method according to the invention may be particularly useful for sex determination which may be carried out by detecting the presence of a Y chromosome. It is demonstrated herein that using only 10 µl of plasma or serum a detection rate of 80% for plasma and 70% for serum can be achieved. The use of just 1ml of maternal plasma or serum resulted in a 100-fold increase in the absolute amount of foetal genetic material available for analysis. This is expected to provide a very accurate system for detecting paternally-inherited foetal DNA sequences.”
“…whether the specification is sufficient or not is highly sensitive to the nature of the invention. The first step is to identify the nature of the invention and decide what it claims to enable the skilled man to do. Then one can ask whether the specification enables him to do it.”
“The law contemplates that patents will not lack sufficiency even though the claims cover inventive improvements. If the law were otherwise there would be no room for patents which disclosed a principle of general application unless the specification described how to carry out later inventions using the principle.”
“As for the point made by the Court of Appeal, it is of course correct so far as it goes. The choice of a particular form of an integer falling within the terms of the claim may improve the way the invention works and be in itself an inventive step. The specification is not insufficient merely because it does not enable the person skilled in the art to make such an invention. The use of the improvement is still a way of working the original invention. But TKT does not rely upon the fact that the use by TKT of an endogenous EPO gene was inventive. 152. …”
“In my opinion the facts did not support the application of this principle. Assuming the claims can be read, as the judge thought, to include any way of making EPO by recombinant DNA technology, the specification does not disclose a way of making it in sufficiently general terms to include the TKT process. It discloses only how to make EPO by introducing exogenous DNA coding for EPO into a host cell. The TKT method is not a version of this process which, although untried, could reasonably be expected to work as well. It is different.”
“Variants that are not foreseeable at the priority date may well, due to later developments, become an obvious variant at a later date. This may happen in case of a pioneer invention, where at the priority date the full breadth of the possible applications could or has not been fully recognised and therefore was not sufficiently taken into account when drafting a claim. Another possibility is that a new technique becomes available after the patent was granted, which makes available an obvious variant. It would be harsh and contrary to fair protection for the patentee to deny him the right to attack those, again provided such variant falls within the inventive concept and reasonable legal certainty is taken into account. So infringement by equivalence is not limited to foreseeable variants only.”
“ In appropriate cases such as the present it is only possible to define the invention … in a way which gives fair protection having regard to the nature of the invention which has been described by using functional terminology in the claims …” “ … the need for a fair protection governs both the consideration of the scope of claims and the requirement for sufficient disclosure. Unless variants of components are also embraced in the claims, which are, now or later on, equally suitable to achieve the same effect in a manner which could not have been envisaged without the invention, the protection provided by the patent would be ineffectual …”
“For the board it is a fundamental principle of patent law that a claim can validly cover broad subject matter, even though the description of the relevant patent does not enable every method of arriving at that subject matter to be carried out. Otherwise no dominant patent could 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. This is not to say that some claims might not be too broad in scope and not be enabled over their whole scope for the purposes of Article 83 EPC… The boards have considered this question of allowability of broad claims versus the requirements of Article 83 EPC, strictly on a case-by-case basis, influenced by the extent to which the information in the patent could be used to develop further embodiments without a major conceptual leap.”
“the Opponent considered that claim 1 as granted is broader than the disclosure of the priority document because the more general concept of “detection” (as opposed to prenatal diagnosis) would not be disclosed in the priority document. The Opposition Division considers that the passage on page 2 lines 5 and 6 of the priority document discloses explicitly said more general concept of “detection”.”
“An invention is a practical product or process, not information about the natural world.”
“The claim is not saved from unpatentability simply by the addition of the phrase ‘‘the use of’’. What matters is the substance of the claim rather than its form.”
“… the judges themselves should be astute to ensure that they decide cases openly, transparently and in public and that their reasons are published in full and made available as widely as can be.”
“41 …where litigation has taken place and judgment given, any disapplication of the principle of open justice must be rigidly contained, and even within the small number of permissible exceptions, it should be rare indeed for the court to order that any part of the reasoning in the judgment which has led it to its conclusion should be redacted. As a matter of principle it is an order to be made only in extreme circumstances.”
“During the oral proceedings, the Representative of the Proprietor admitted that she was not aware of a method allowing to detect sequences which do not differ from the maternal DNA.”
“a licence from the proprietor or applicant for a patent conferring on the licensee, or on him and persons authorised by him, to the exclusion of all other persons (including the proprietor or applicant), any right in respect of the invention to which the patent or application relates.”
““Affiliate” means, with respect to a Person, any other Person directly or indirectly controlling, controlled by, or under common control with, such Person at any time during the period for which the determination of affiliation is being made. For purposes of this definition, the term “control” means, with respect to any Person, the possession, directly or indirectly, of the power to direct or cause the direction of management policies of such Person, whether through the ownership of voting securities or by contract or otherwise.” ““Illumina Parties” means, collectively, Illumina and its Affiliates”
“The exclusive (even as to the Sequenom Parties), worldwide, sub-licensable right under the Pooled Patents to exploit NIPD IVD Products in the NIPD IVD Field.”
“On the terms and conditions of this Agreement, Sequenom, on behalf of itself and its Affiliates, hereby grants to Illumina and its Affiliates an exclusive, irrevocable and perpetual (subject to Section 2.3(b)), non-transferable and non-assignable license (except as permitted under Section 9.1) worldwide license, with the exclusive right to grant sublicenses, under the Sequenom Owned Patents and Isis Patents, to Exploit NIPD LDT Tests in the NIPD LDT Field and to Exploit NIPD IVD Products in the NIPD IVD Field …” 248.Section 2.3(b) provides: “The license rights set forth in Section 2.3(a) granted to any Affiliate of Illumina shall automatically terminate with respect to such Person when it ceases to be an Affiliate of Illumina. Persons that become Affiliates of Illumina after the Effective Date shall be licensed under the license rights set forth in Section 2.3(a) only for those licensed acts that occur on or after the date it becomes an Affiliate”
“The words ‘or on him and persons authorised by him’ seem to me to contemplate a licence which confers upon the exclusive licensee a power to sub-licence. The essential element of the transaction appears to be the exclusion of all other persons including the patentee or applicant.”
“25. These papers represented an exciting development in the non-invasive detection of foetal chromosomal aneuploidy, as they presented non-invasive prenatal diagnosis methods which allowed chromosomal aneuploidies to be detected for the first time without isolating foetal cells or discriminating between the maternal and foetal nucleic acid sequences. 26. I do not recall any discussion of the use of digital PCR or massively parallel sequencing for non-invasive prenatal diagnosis at any of the meetings or conferences that I attended prior to the publication of these papers by Professor Lo and Professor Quake. 27. Having read about the work of Professor Lo and Professor Quake, my group sought to build upon it by developing robust statistical models for use with digital PCR and massivelyparallel sequencing-based methods, as well as applying these techniques to the non-invasive prenatal diagnosis of recessive Mendelian disease in maternal cell-free DNA.”
“At the Quake priority date there was an appreciation that cffDNA [cell-free foetal DNA] should enable the detection of aneuploidies from a sample of maternal plasma or serum. As aneuploidy is characterised by a difference in the copy number of the aneuploid chromosome then, in theory, this difference could be distinguishable if a sufficiently accurate method could be used. However, there was thought to be a significant hurdle in reducing the theory into practice as the foetal fraction presented in maternal plasma was believed to be very low. That hurdle was, in essence, a problem in counting the changes in copy number of the foetal chromosome with sufficient accuracy, which was beyond the capabilities of qPCR machines available.”
“It has recently been shown that high-throughput whole genome DNA sequencing can be used for the detection of foetal aneuploidy (Chiu et al 2008; Chu et al 2009a; Fan et al 2008). This is an exciting development with great potential because it is a direct method that requires no gene or chromosome specific biomarkers and provides chromosome wide insight into karyotyping”
“Shotgun sequencing: a new era in non-invasive prenatal diagnosis? A recent paper by the group of Stephen Quake of Stanford University has provided a potentially novel approach for rapid determination of aneuploidy. The approach is to sequence a population of DNA molecules using so-called next-generation DNA sequencers. Using two such machines (a Solexa and a 454), Fan et al clearly demonstrate that populations of chromosome 21 and 18 derived DNAs are quantitatively different from other chromosomes in trisomy 21 and 18 maternal plasma sample respectively.”
“The latest breakthrough in this area has come from the group of Stephen Quake at Stanford University. They have reported non-invasive diagnosis of aneuploidy by shotgun sequencing of foetal DNA from maternal plasma using Solexa-Illumina and 454/Roche sequencers. This sequencing approach is polymorphism independent and therefore diagnostically universally applicable.”
“such a scheme would in particular eliminate the risk of iatrogenic foetal loss, which occurs at a small but nonnegligible rate among women investigated in this way.”
"It has been known for over a century that foetal cells migrate into the maternal blood stream. Much effort and large resources have been devoted to capturing these for NIPD, either by fluorescence in-situ hybridisation of intact foetal cell nuclei or using DNA extracted from intact foetal nuclei… Most research groups, however, have concluded that this approach is too labour intensive and not reliable enough for use in clinical practice."
“In the first instance this concerned the exploitation of DNA sequences of paternal origin. These were shown to allow foetal genetic sexing (of special relevance in X-linked disease), identification of rhesus-D-positive foetuses (of special importance when the mother is rhesus D negative, and also NIPD of some other genetic conditions.”
“The most common indication for prenatal diagnosis is, however, an increased risk for Down syndrome (DS). NIPD for DS presents another type of challenge, i.e. how best to identify (and quantify the amount of) foetal chromosome-21-derived material in maternal plasma so as to detect the three copies in a DS pregnancy in comparison to the normal two copies.”
“So far, three main approaches have been proposed. First, it should be possible to search for chromosome-21-specific polymorphic DNA sequences, such as single nucleotide polymorphisms (SNP) to allow NIPD of DS by measurement of allelic ratios in cff DNA (Chow et al., 2007; Dhallan et al., 2007). Second, the recent identification of chromosome 21 foetal specific mRNA in maternal plasma; this mRNA,originating exclusively from foetal cells in the placenta, provides yet another facility to be exploited for NIPD of foetal DS (Lo et al., 2007). Third, it is also clear that the possibility exists for using differences in DNA methylation between maternal and foetal DNA sequences so as to identify and quantify specific sequences in cff DNA (Chan et al., 2006; Tong and Lo, 2006; Tong et al., 2006).”
“The first method [SNPs] depends upon an enrichment method that is currently controversial… The second, mRNA based, method is promising, but in its current embodiment requires suitable allele combinations in parents and foetus… The third, epigenetic approach does not depend upon allele ratios of common polymorphisms in its simplest embodiment. It therefore has the advantage of applicability to a broad population. The epigenetic markers described herein have been obtained with this approach in mind.”
“It remains to be seen how many epigenetic biomarkers will be required for NIPD of Down syndrome to achieve comparable accuracy with that of invasive diagnosis by the ‘gold standard’ of quantitative fluorescence-PCR and/or karyotyping.”
“Q. So far as the work on maternal plasma is concerned, of which there is a published trail in the papers in this case, that falls, as we will see, into a number of different categories, as I think you have appreciated in your report, but they all have one thing in common: they seek specific markers for foetal DNA; correct? A. They do: foetal placentally-derived DNA, epigenetic markers, which we have heard about before, and single nucleotide polymorphisms inherited from the father, but there was a fourth number-crunching approach, the brute force approach, which was not in the literature, granted, but it was not a novel idea. Several of us had already considered this approach before the priority date.”
“Non-invasive prenatal diagnosis (NIPD) is entirely dependent on the efficacy in identification of foetal cells and/or foetal DNA in maternal blood samples. During the initial year of the SAFE project we have therefore focused attention on the integrated evaluation and development of foetal specific biomarkers. …”
“The application of foetal DNA for maternity screening (for increased risk of foetal aneuploidy such as trisomy 21 Down syndrome and/or pregnancy complications such as preeclampsia and preterm labour) as well as NIPD per se is crucially dependent on the extraction of optimal (as pure as possible) foetal DNA from maternal plasma.”
“The MLPA has also been applied to the analysis of single cells, in particular the detection of chromosomal copy number – e.g. trisomy 21. If this technical approach can be adapted forthe utilisation of maternal plasma samples, or foetal DNApurified from this source, then there will be widespreadintroduction of NIPD for trisomy 21.”
“The MLPA assay involves the simultaneous analysis of 8 chromosome 21 loci and control markers dispersed throughout other chromosomes. The single cells were isolated, and subjected to a pre-amplification stage and then MLPA analysis. The figure clearly shows that three copies of chromosome 21 can be defined by the assay- thus could be applied to purified cells isolated in the activities described in WP1, however WP3 activity will include the adaptation of the technique to purified foetal DNA isolated from maternal plasma.”
“It is hoped that, in not too distant a future, the same technology may be applied for ‘noninvasive’ prenatal diagnosis on foetal cells or DNA retrieved from maternal blood samples, leading to a reduced requirement for invasive procedures that carry a risk for associated foetal loss.”
“It has now been found that these problems can be solved by quantitative examination of large numbers of chromosome samples through the use of highly scalable techniques. This approach is termed here “digital analysis”, and involves the separation of the extracted genomic material into discrete units so that the detection of a target sequence (e.g., chromosome 21) may be simply quantified as binary (0,1) or simple multiples, 2, 3, etc. The primary example of a technique that can be used to yield such “digital” results is “digital PCR,” which allows efficient amplification from single molecules, followed by subsequent quantitative analysis. Digital PCR, as the term is used here, refers to a quantitative, limited dilution of a nucleic acid sample, such as into multiwell plates, then the amplification of a nucleic acid molecule in a well, which due to the dilution, should be either 0 or 1 molecule.”
“[0031] In one aspect, the present method of differential detection of target sequences may involve direct sequencing of target sequences the genetic material. Single molecule sequencing, as is known, is further described below. The method may also comprise sequencing of amplified derivatives of the target sequences clones or amplicons of the genetic material. That is, a target sequence in a discrete sample is amplified by PCR, i.e. as an amplicon, or cloned into a vector that is grown up and thereby amplified by obtaining multiple copies of the vector insert.”
“[0093] It should be appreciated that methods involving PCR or other amplification are not the only way to detect or enumerate the molecules in a given discrete reaction sample. It is possible to use single molecule flow cytometry to count single molecules that have been labeled with a sequencespecific fluorescent probe. It is also possible to sequence thetarget sequence in the reaction sample directly, either afteramplification or at the single molecule level.”
“[0096] A methodology useful in the present inventionplatform is based on massively parallel sequencing of millionsof fragments using attachment of randomly fragmentedgenomic DNA to a planar, optically transparent surface and solid phase amplification to create a high density sequencing flow cell with millions of clusters, each containing ~1,000 copies of template per sq. cm. These templates are sequenced using four-color DNA sequencing-by-synthesis technology..”. “Sequencing may be combined with amplification-based methods in a microfluidic chip having reaction chambers for both PCR and microscopic template-based sequencing. Onlyabout 30 bp of random sequence information are needed toidentify a sequence as belonging to a specific humanchromosome.”
“the statistical reliability of the present method can be dramatically improved simply by increasing the number of wells tested. Since about 240 genome equivalents is required per panel, and about 4,700 genome equivalents are found in a 20 ml sample, it is possible, given the present description, to simply run additional analyses to increase statistical significance.”
“A method of detection of foetal aneuploidy in a mixture of maternal and foetal genetic material, in a sample of maternal tissue, characterized by: (a) distributing the genetic material into reaction samples, wherein each sample contains on average not more than aboutone target sequence per sample, wherein DNA to be analyzed will be either present or absent in a reaction sample, due to random variations between reaction samples; (b) measuring the presence of different target sequences in the reaction samples by digital analysis to obtain binary results providing differential detection of the target sequences in a mixture of maternal and foetal genetic material, wherein said target sequences comprise sequences from two chromosomes, one of which is possibly aneuploid and one of which is presumed diploid; (c) analyzing the binary results from step (b) by counting the frequency of positive responses from target sequences followed by (d) statistical analysis of the results of step (c) whereby the frequency of positive responses from target sequences provides data sufficient to distinguish euploid from aneuploid target sequences, wherein the measuring step comprises directsequencing of the maternal and foetal genetic material.”
“A method for detecting a genetic abnormality that involves a quantitative difference between maternal and fetal genetic sequences by differential detection of target sequences in a mixture of maternal and fetal genetic material, comprising the steps of: a) distributing the genetic material into discrete samples, each sample containing on average not more than one target sequence per sample, wherein the discrete samples are in reaction samples where the target sequences can be analyzed; b) measuring the presence of different target sequences in the discrete samples, wherein the measuring comprises direct sequencing of the genetic material or sequencing of amplified derivatives of the target sequences in clones or amplicons of the genetic material; and, c) analyzing a number of the discrete samples, wherein the number of discrete samples analyzed and the results from the discrete samples provide data sufficient to obtain results distinguishing said different target sequences, wherein one of the different target sequences is diploid in the maternal genetic material and aneuploid in the fetal genetic material and another of the different target sequences is diploid in both the maternal and the fetal genetic material, thereby detecting a genetic abnormality that involves a quantitative difference between maternal and fetal genetic sequences.”
“Current methods for analysis of cellular genetic content include comparative genomic hybridization (CGH) (3), representational difference analysis (4), spectral karyotyping/M-FISH (5,6), microarrays (7-10), and traditional cytogenetics. Such techniques have aided in the identification of genetic aberrations in human malignancies and other diseases (11-14). However, methods employing metaphase chromosomes have a limited mapping resolution (about 20 Mb) (15) and therefore cannot be used to detect smaller alterations. Recent implementations of comparative genomic hybridization to microarrays containing genomic or transcript DNA sequence provide improved resolution, but are currently limited by the number of sequences that can be assessed (16) or by the difficulty of detecting certain alterations (9). There is a continuing need in the art for methods of analyzing and comparing genomics.” 348.Paragraph [0004] states that: “Because chromosomes are visualized on an optical microscope, the ability to resolve detailed mutations (involving only a small part of a chromosome) is limited. While more detailed karyotyping techniques, such as FISH (fluorescent in situ hybridization) are available, they rely on specific probes and it is not economically or technically feasible to perform FISH on the entire chromosome set (i.e., the complete genome).”
“In recent work, a method was provided for karyotyping a genome of a test eukaryotic cell by generating a population of sequence tags after restriction endonuclease digestion from defined portions of the genome of a test cell (17). This method is not optimal because a small number of areas of the genome are expected to have a lower density of restriction endonuclease cleavage sites and could be incompletely evaluated. The authors estimate these areas to encompass 5% of a genome. Furthermore, the resolution of the method is dependent on the restriction enzyme used and the method cannot reliably detect very small regions of the genome on the order of several thousand base pairs or less.”
“The current invention provides for a method of karyotyping a genome of a test cell (e.g., eukaryotic or prokaryotic) by generating a pool of fragments of genomic DNA by a random fragmentation method, determining the DNA sequence of at least 20 base pairs of each fragment, mapping the fragments to the genomic scaffold of the organism, and comparing the distribution of the fragments relative to a reference genome or relative to the distribution expected by chance. The number of a plurality of sequences mapping within a given window in the population is compared to the number of said plurality of sequences expected to have been sampled within that window or to the number determined to be present in a karyotypically normal genome of the species of the cell. A difference in the number of the plurality of sequences within the window present in the population from the number calculated to be present in the genome of the cell indicates a karyotypic abnormality.”
“Preferably, the test cell and the reference cell is from the same species. The cell is a eukaryotic cell or a prokaryotic cell. The eukaryotic cell a mammalian cell. The mammal is, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The cell is a cancer cell, an embryonic cell, or a foetal cell. The cell is isolated from amniotic fluid or is derived from in vitro fertilization. Optionally, the cell is from a subject with a hereditary disorder.”
“By mapping to a genomic scaffold is meant that the sequences are aligned along each chromosome. The test cell distribution (i.e. chromosome map density) is defined as the number of mapped sequences (i.e., fragments) by the number of possible map locations present in a given chromosome. The number of possible map locations is defined by the size of the observation window and the length of the chromosome. No particular length is implied by the terms observation window. For example, the observation window is 25 Mb, 10 Mb, 4 Mb, 2 Mb, 500 kb, 250 kb, 60 kb, 30 kb, or 10 kb or less in length.”
“Furthermore, chromosome abnormality includes any sort of genetic abnormality including those that are not normally visible on a traditional karyotype using optical microscopes, traditional staining, of FISH. One advantage of the present invention is that chromosomal abnormality previously undetectable by optical methods (e.g., abnormalities involving 4 Mb, 600 kb, 200 kb, 40 kb or smaller) can be detected.” 354. Paragraph [0060] explains that: “The method of the invention can be used to determine changes in copy number for portions of the genome on a genomic scale. Such changes include gain or loss of whole chromosomes or chromosome arms, interstitial amplifications or deletions, as well as insertions of foreign DNA. Rearrangements, such as translocations and inversions, can be detected by the method of the invention, e.g., where large fragments are generated and the ends sequenced, or where the scaffold-predicted ends are a different distance apart than the size of the fragment sampled.”
“The data shown herein demonstrate that the method of the invention, called Sequence-Based Karyotyping, can accurately identify regions whose copy number is abnormal, even in complex genomes such as the human genome. Advantageously, the method permits the identification of specific amplifications and deletions that had not been previously described by comparative genomic hybridization (CGH) or other methods in any human cancer. The approach is particularly applicable to the analysis of human cancers, wherein identification of homozygous deletions and amplifications has historically revealed genes important in tumor initiation and progression. The method of the invention can be used with a variety of other applications. For example, the approach could be used to identify previously undiscovered alterations in hereditary disorders. A potentially large number of such diseases are thought to be due to deletions or duplications too small to be detected by conventional approaches. These may be detected with Sequence-Based Karyotyping, even in the absence of any linkage or other positional information.”
“Complex sample sequencing in accordance with the invention can be used for detection of pathogens in blood, water, air, soil, food, and for the identification of all organisms in a sample without any prior knowledge. In accordance with this method, populations of organisms can be identified by preparing a mixed DNA and cDNA sample, sequencing random fragments from the DNA and RNA in the sample and mapping sequences to a hierarchical database of all known sequences… According to one embodiment, a sample (e.g. blood, water, air, food, or soil) can be used to generate 1 million sequence reads …. ”
“Section 76 did not prevent the granted patent claiming a different combination from that in the application if the amended claim had in it the essential elements required by both the application and the specification of the patent to achieve the objects of the invention. The section prevented disclosing either by deletion or addition any inventive concept which was not disclosed before, but did not prevent him from claiming the same invention in a different way”
“A methodology useful in the present invention platform is based on massively parallel sequencing of millions of fragments using attachment of randomly fragmented genomic DNA to a planar, optically transparent surface… These templates are sequenced using four-color DNA sequencing-bysynthesis technology. See, products offered by Illumina, Inc., San Diego California… ”
“Q. That is talking about random fragmentation; correct? A. Yes. Q. And that the randomly fragmented DNA is attached to the surface to create a sequencing flow cell? A. Yes, that is the kind of text I was referring to, where there is some text that looks as if it is lifted from an Illumina brochure and inserted into the text, but it seems to me that is referring to an Illumina technology. Q. Which was random shotgun sequencing? A. Yes.”
“Q. You would agree the natural reading, so far as the skilled person is concerned, of this sentence is that it is describing a shotgun sequencing method, because it is talking about attaching a randomly fragmented DNA to a planar optically transparent surface. There is no reference at all to any intermediate step of library creation or anything else. A. No, I agree with that, but I just wanted to point out that it does not exclude a certain kind of targeted library.”
“The presence or absence of different target sequences in the discrete samples is detected; and the results are analysed whereby the number of results from the discrete sample provide data sufficient to obtain results distinguishing different target sequences”
“"Nucleic Acid Sequencing" means all methods for determining the order of nucleotides in a nucleic acid molecule, which methods are capable of determining the nucleotide sequence of a nucleic acid having an unknown sequence…”
“It is therefore desirable that noninvasive tests have high sensitivity and specificity to minimize false negatives and false positives, respectively. However, foetal DNA is present in low absolute concentration and represent a minor portion of all DNA sequences in maternal plasma and serum. It is therefore also desirable to have methods that allow the noninvasive detection of foetal chromosomal aneuploidy by maximizing the amount of genetic information that could be inferred from the limited amount of foetal nucleic acids which exist as a minor population in a biological sample containing maternal background nucleic acids.”
“[0059] … The parameter may be, for example, a simple ratio of the first amount to the second amount, or the first amount to the second amount plus the first amount. In one aspect, each amount could be an argument to a function or separate functions, where a ratio may be then taken of these separate functions. One skilled in the art will appreciate the number of different suitable parameters.”
“Based on the comparison, a classification of whether a foetal chromosomal aneuploidy exists for the first chromosome is determined. In one embodiment, the classification is a definitive yes or no. In another embodiment, a classification may be unclassifiable or uncertain. In yet another embodiment, the classification may be a score that is to be interpreted at a later date, for example, by a doctor.”
“A method for performing prenatal diagnosis of a foetal chromosomal aneuploidy in a biological sample obtained from a female subject pregnant with a foetus, wherein the biological sample is maternal plasma or serum and wherein the sample includes cell-free nucleic acid molecules from the female subject and the foetus, the method comprising: performing a random sequencing on at least a portion of a plurality of the nucleic acid molecules contained in the biological sample to obtain a pre-determined number of sequences, wherein the sequences represent a fraction of the human genome; aligning, with a computer system, each sequence to a human genome; determining a first amount of sequences identified as being aligned to a first chromosome; determining a second amount of sequences identified as being aligned to one or more second chromosomes; determining a parameter from the first amount and the second amount; wherein the parameter represents a relative amount between the first and second amounts; and comparing the parameter to one or more cut-off values, to determine a classification of whether a foetal chromosomal aneuploidy exists for the first chromosome.”
“[0132] Additionally, there are now a number of alternative approaches to the manual setup of digital real-time PCR analysis as used in the current study for conducting digital PCR. These alternative approaches include microfluidics digital PCR chips (Warren, L et al. 2006 Proc Natl Acad Sci USA 103, 17807 - 17812; Ottesen EA et al. 2006 Science 314, 14641467) emulsion PCR (Dressman D et al. 2003 Proc Natl Acad Sci USA 100, 8817 - 8822), and massively parallel genomic sequencing (Margulies M et al 2005 Nature 437, 376 - 380), etc. With the use of these methods, digital RNA SNP and digital RCD could be performed rapidly on a large number of sample, thus enhancing the clinical feasibility of the methods proposed here for non-invasive prenatal diagnosis.” … “[0192] The variant of digital PCR is the performance of massively parallel genomic sequencing using emulsion PCR in a sequencing machine such as the Roche GS20 system (http://www.454.com/about-454/partners.asp) the Applied Biosystems 'supported oligo ligation detection' (SOLiD) and the Illumina Solexa sequencing technology. The general principle of this strategy is that if one is to do random sequencing of DNA fragments that are present in the plasma of a pregnant woman, then one would obtain genomic sequences which would originally have to come from either the foetus or the mother. A proportion of such sequences would be from the chromosome involved in an aneuploidy such as chromosome 21 in this illustrative example… ”
“The opposition division finds that in the cited paragraphs “random sequencing” is not used in a general context applicable to all embodiments of the invention, but in sections relating to specific embodiments, namely “digital (real-time) PCR” and “massively parallel genomic sequencing” (see paragraphs [0132] and [0192] of the priority document. The expression “the general principle of this strategy” in paragraph [0192] can only be understood in relation to “the variant of digital PCR [which] is the performance of massively parallel genomic sequencing using emulsion PCR” and would not be read by the skilled person to apply to the broad concept as in present claim 1.”
“The opposition division also considers the expression “fraction of the human genome" to lack priority because even if one would consider that the sequencing methods of [0192] inevitably yielded “a fraction of the human genome” the disclosure of [0192] would still remain limited to a specific embodiment of the invention, namely “Massive parallel genomic sequencing using emulsion PCR" (see heading of para. [0192])… ”
“2.4 University reserves the right to: 2.4.1 use and develop any of the Inventions and the Prospective Patent solely for academic research and publication purposes at all times provided that University provides a copy of any manuscript to Licensee, at the time of submission, for any publications submitted to a journal indexed by the Institute of Scientific Information; and to extend the academic research and publication rights, set forth above, to other collaborating academic organisations in whatever countries. 2.4.2 forthwith upon the request of the Hong Kong SAR Government (the “Government”) unconditionally grant to the Government an irrevocable, nonexclusive, perpetual world-wide, freely transferable, sub-licensable and royalty free license to use the Invention in such manner and for such purposes as may be decided by the Government. Such request will be made by the Government when in the opinion of the Government: (a) the public mission of the Commissioner for Innovation and Technology of the Government or any person authorized to act on his behalf needs to be fulfilled; or (b) it is in the public interest to do so.”
“Screening is the process of identifying healthy people who may be at increased risk of disease or condition. The screening provider then offers information, further tests and treatment. This is to reduce associated risks or complications.”
“Conventional prenatal diagnostic methods of a foetal chromosomal aneuploidy, e.g., trisomy 21, involve the sampling of foetal materials by invasive procedures such as amniocentesis or chorionic villus sampling, which pose a finite risk of foetal loss. Noninvasive procedures, such as screening by ultrasonography and biochemical markers, have been used to risk-stratify pregnant women prior to definitive invasive diagnostic procedures. However, these screening methods typically measure epiphenomena that are associated with the chromosomal aneuploidy, e.g., trisomy 21, instead of the core chromosomal abnormality, and thus have suboptimal diagnostic accuracy and other disadvantages, such as being highly influenced by gestational age.”
“As Aldous LJ said at [33]: "If the host computer is situated in Antigua and the terminal computer is in the United Kingdom, it is pertinent to ask who uses the claimed gaming system. The answer must be the punter. Where does he use it? There can be no doubt that he uses his terminal in the United Kingdom and it is not a misuse of language to say that he uses the host computer in the United Kingdom. It is the input to and output of the host computer that is important to the punter and in a real sense the punter uses the host computer in the United Kingdom even though it is situated in Antigua and operates in Antigua. In those circumstances it is not straining the word 'use' to conclude that the United Kingdom punter will use the claimed gaming system in the United Kingdom, even if the host computer is situated in, say, Antigua. Thus the supply of the CD in the United Kingdom to the United Kingdom punter will be intended to put the invention into effect in the United Kingdom." 156. I agree with RIM that asking and answering Aldous LJ's questions in this case leads to a different answer. Who uses the method of operating a messaging gateway system that has the claimed features? The answer is RIM. Where do they operate it? The answer is in Canada.”