WO2012114075A1

WO2012114075A1 - Method for processing maternal and fetal dna

Info

Publication number: WO2012114075A1
Application number: PCT/GB2012/000191
Authority: WO
Inventors: Tracey Elizabeth Madgett; Neil David Avent
Original assignee: Plymouth University
Current assignee: Plymouth University
Priority date: 2011-02-25
Filing date: 2012-02-24
Publication date: 2012-08-30
Anticipated expiration: 2013-08-25
Also published as: GB2488358A; GB201103277D0

Abstract

A method for processing a sample of maternal plasma comprising maternal free DNA and ffDNA is provided, the method comprising the in situ enrichment of the amount of ffDNA relative to the amount of maternal free DNA. In one embodiment, the amount of ffDNA in the sample is increased, for example by PCR methods. In a further embodiment, the amount of maternal free DNA in the sample is decreased, in particular by selective amplification, for example by PCR methods, and selective depletion. The method is particularly useful in the detection of genetic abnormalities in the fetus, in particular Down Syndrome and other aneuploidies.

Description

METHOD FOR PROCESSING MATERNAL AND FETAL DNA

The present invention relates to a method for processing maternal and fetal DNA, in particular cell free fetal DNA (ffDNA), and its use in assisting with noninvasive prenatal diagnosis (NIPD) of fetal genetic traits.

Conventional methods of prenatal diagnosis for detecting genetically inherited conditions involve the use of invasive technologies. These include the likes of amniocentesis and chorionic villus sampling. Invasive methods such as these are known to carry a 1-2% risk of miscarriage. Despite the potential risks to both the fetus and the mother, a significant number of pregnant women opt for invasive prenatal diagnosis methods. This is due to the fact that, currently, prenatal diagnosis (as opposed to screening) for conditions such as Down Syndrome, (also known as Trisomy 21), is at present only possible via invasive techniques such as those mentioned above. The principle method for the detection of Trisomy 21 , after having obtained ffDNA via amniocentesis and/or chorionic villus sampling, is to assess the number and appearance of the fetal chromosomes (also known as karyotyping). In this way it is possible to detect whether there is an elevated amount of chromosome 21 , which is indicative of Down syndrome.

EP 1329517 provides an example of how fetal DNA sampled invasively via amniocentesis and/or chorionic villus sampling may be used in real time Polymerase Chain Reaction (real time PCR) in order to determine gross chromosomal abnormalities, in particular Trisomy 21. This method is extremely sensitive and readily amenable to automation and high-throughput screening. In this method DNA or RNA is to be obtained from both the genetic test locus and a particular control locus. This method detects specific nucleic acid amplification products as they accumulate in real-time by a sequence specific fluorescently labelled oligonucleotide probe. RT-PCR therefore addresses the problem of end point analysis commonly observed in traditional PCR assays where excessive amplification can impede the quantification of the amount of starting nucleic acid material. Alternative non-invasive methods, such as screening by ultrasonography and/or biochemical measurement of certain proteins, combined with maternal age, have typically been used as a first indicator to identify high risk pregnancies. In this way, pregnant women may deliberate whether to continue with more definitive, albeit riskier, invasive diagnostic procedures. Unfortunately these screening tests are prone to false positive results and detect only phenotypic features as opposed to the underlying genetic pathology giving rise to the particular condition. For example, screening can identify certain Trisomy 21 epiphenomena, such as thicker nuchal translucency, but cannot identify the core pathology of Trisomy 21. There is therefore a significant need for a method for the direct non-invasive detection of fetal genetic traits. It is of particular importance, however, to ensure that any such direct noninvasive procedure provides results to the same or better accuracy than those provided under current invasive prenatal diagnostic techniques, as mentioned above. Since the discovery in 1997 (Lo Y.M et a!,, 'Presence of fetal DNA in maternal plasma and serum', Lancet, Vol. 350, 1997, pages 485 to 487) of the presence of ffDNA within the maternal bloodstream, attempts have been made to replace the invasive procedures discussed above with a simple non-invasive blood test using ffDNA. The presence of ffDNA within the maternal bloodstream has been of limited use in clinical situations, principally used at present where the detection of paternally inherited conditions and/or fetal RhD blood group status in RhD negative mothers is required. In such cases, the amplification, by polymerase chain reaction (PCR) and/or real time PCR, of fetal genetic loci which are completely absent from the maternal genome and thus easily distinguishable as fetal specific has been a relatively simple exercise.

It has proved to be a much more complicated task to extend NIPD to cover the analysis of ffDNA for the determination of more complex fetal genetic traits, such as abnormal fetal chromosome copy numbers (aneuploidy), maternally inherited conditions and autosomal recessive monogenetic diseases. This is due in most part to the low concentration of ffDNA relative to the maternal free DNA. In particular, the low concentration of ffDNA makes it difficult to accurately detect single nucleotide polymorphisms when using standard techniques such as real-time PCR. Although increasing with gestational age of the fetus, it has been reported by Lo, Y.M et al., 'Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis' Am J Hum Genet 1998, 62, pages 768-775, that cell free fetal DNA represents at best 3-6% by mass of the free DNA in maternal plasma, the remainder being derived from maternal sources. As discussed above, the high background of maternal DNA can often interfere with the analysis of the ffDNA. A way of overcoming this effect is therefore required, before the NIPD methods such as real-time PCR can be utilised and offered on a large scale. It has been acknowledged by Chiu R.W. et al., ' on invasive prenatal diagnosis by single molecule counting technologies'; Trends Genet 2009; 25, pages 324-31, that fetal specific molecular markers such as placenta-specific methylation patterns and placentally derived mRNA may be used as an alternative in NIPD of fetal aneuploidies. This uses the same approach as outlined above, whereby distinguishable fetal specific nucleic acid markers are exploited in NIPD. While possible in principal, the so called 'epigenetic allelic ratio approach' is severely constrained in practice by the low abundance of ffDNA and the reliance on allelic heterozygosity between the fetus and the mother, as indicated by Voelkerding, K.V. et al., 'Digital Fetal Aneuploidy Diagnosis by Next Generation Sequencing', Clinical Chemistry 56, 2010, pages 336 to 338. An alternative and more preferred approach for the NIPD of Down syndrome is to provide a mechanism whereby it is possible to show the presence of an elevated amount of chromosome 21. Although feasible in theory, this approach is limited, once again, by a low concentration of ffDNA within the maternal plasma.

Lo, Y.M.D. et al., 'Digital PCR for the molecular detection of fetal chromosomal aneuploidy', PNAS, August 7, 2007, vol. 104, no. 32, pages 13116 to 13121 , suggests the detection of ratios of fetal chromosomes by digital counting techniques. In particular, there is disclosed the use of PCR to determine whether there is an overrepresentation of chromosome 2 in the maternal plasma of women carrying trisomy 21 fetuses. This polymorphism independent method, so called the 'digital relative chromosome dosage' (RCD) method is specifically intended to overcome the shortcomings of using foetal-specific molecular markers, which as discussed above are informative only for heterozygous foetuses. Digital PCR comprises the dilution and compartmentalisation of maternal plasma sample so that individual fetal and maternal target loci may be amplified in different wells. In this way it is possible to directly count the number of positive wells in which the target amplicon has been amplified without interference between the maternal free DNA and the ffDNA. By quantitatively comparing the amount of amplified products from the target locus with that of a reference chromosome it is possible to deduce whether there is an imbalance in chromosome copy number. The effectiveness of digital PCR, however, is once again constrained by the low percentage of ffDNA present within the maternal plasma. Digital PCR is therefore a lengthy process, which requires many reaction runs in order to generate reliable results.

In order to achieve increased ffDNA fractional concentrations, methods for selective enrichment of ffDNA and/or depletion of maternal free DNA are needed. Apart from treating the plasma samples with formaldehyde, (the effect of which has not been universally accepted as suggested in Chiu R.W.K et al., 'Non invasive prenatal diagnosis by single molecule counting technologies'; Trends Genet 2009; 25, pages 324-31 and/or targeting fetal specific molecular markers (which as discussed above minimises background maternal DNA interference) enrichment of ffDNA may be achieved via physical separation methods which exploit the difference in size between the ffDNA and the maternal free DNA.

Significant research by Li, Y., et al., 'Size separation of circulatory DNA in maternal plasma permits ready detection of fetal DNA polymorphisms' Clin Chem 2004, 50, 1002-101 1 , has revealed that ffDNA and maternal free DNA are

fragmented in nature, although the fragments of ffDNA tend to be considerably shorter in length than the fragments of maternal free DNA. Specifically ffDNA comprises 300 base pairs or less, as opposed to more than 500 base pairs for free maternal DNA. Indeed, in some circumstances free DNA smaller than 500 base pairs appears to be almost entirely derived from the fetus. This is thought to be due to the fact that the ffDNA is derived from apoptotic synctiotrophoblasts.

US 2005/0164241 discloses a method for the non-invasive detection of fetal genetic traits, which exploits this observation. This method comprises a first stage wherein a sample of blood plasma or serum from a pregnant woman is physically separated into ffDNA and maternal free DNA via size discrimination. Various types of chromatography and electrophoresis techniques are employed in order to obtain a fraction of said sample in which the extracellular DNA present therein substantially consists of DNA comprising 500 base pairs or less. Once the enriched sample- fraction is obtained, determination of the fetal genetic traits can be effected by methods such as PCR, ligase chain reaction, probe hybridisation techniques, nucleic acid arrays and the like. In this way, NIPD of fetal genetic traits, including those involved in chromosomal aberrations, such as Down syndrome is possible. However, this method involves two separate stages and therefore unnecessarily complicates and lengthens the diagnostic procedure.

Perhaps the most recently proposed diagnostic method to have exploited this observation has applied Digital PCR with high throughput next generation

sequencing (NGS)-based plasma diagnostics. Chiu, R.W et al., ' on invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study, 2011; 342:c7401doi:10. 136/bmj.c740V discuss how NGS could be used for the measuring of small increments in chromosome 21 DNA concentration. NGS-based NIPD however is known to incur high equipment and reagent costs and requires substantial technical and bioinformatic input and analysis. It is for these reasons that the implementation of NGS-based NIPD will only be considered as an alternative to current screening techniques once these issues have been resolved.

US 2010/0285537 discloses a method for selectively depleting a nucleic acid sample of non-target nucleic acids. The method employs at least two target specific primers or primer pairs, wherein the primer pairs comprise an inner primer or primer pair for amplifying a target nucleotide sequence on long and short nucleic acids. Each inner primer or primer pair comprises a 5' nucleotide tag. The method further employs an outer primer or primer pair for amplifying the target nucleotide sequence on long nucleic acids but not on short nucleic acids. Amplification by PCR produces short tagged target nucleotide sequences and longer non- tagged nucleotide sequences comprising the target nucleotide sequences. The shorter tagged target nucleotide sequences are exonuclease protected. Accordingly, only the longer non- tagged maternal DNA is depleted. This method requires the shorter ffDNA to be amplified as well as the longer maternal DNA. In addition and perhaps more importantly, the method purposefully interferes with the targeted short nucleotide sequences by tagging the same.

Accordingly, an artefact is necessarily introduced into the amplified ffDNA.

Notwithstanding the above, it is submitted that the method disclosed is unduly complicated, imposing undue burden on the user and is therefore unlikely to work in practice. US 2010/0285537 further discloses a method for enriching ffDNA by reducing the denaturation temperature of the PCR reaction, but at later stages of the PCR reaction.

Similarly, an alternative method for selectively amplifying ffDNA, takes advantage of sequence differences between ffDNA and maternal DNA. This is disclosed in the Chinese Medical Journal, (2010); Vol 123, pp 3343-3346, "Non invasive prenatal molecular detection of fetal point mutation for congenital adrenal hyperplasia using co-amplification at lower denaturation temperature PCR", by Du et al. Accordingly, this method, although suitable for detecting mutant sequences, would not be suitable for detecting common aneuploidies such as Down's Syndrome.

Another method for enriching ffDNA, taking advantage of sequence differences between ffDNA and maternal DNA, is disclosed in a Report entitled "Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma", (2008) Proc. Natl. Acad. Sci. USA; Vol 105, pp 19920 - 19925, by Lun et al. As above, this method is only suitable for detecting inherited monogenic diseases.

WO 2005/035725 relates to an alternative method for enriching cell free fetal DNA relative to maternal DNA. It has been hypothesised that ffDNA circulates in the mother's plasma within membrane bound vesicles formed as a result of the mechanism of programmed cell death. In light of this, the method involves treating the total maternal plasma (containing both maternal and fetal DNA fragments), with DNase for a certain period of time. According to the inventors, DNase treatment depletes only the unpackaged maternally derived sequences. The remaining ffDNA is then amplified according to a modified version of the whole genome amplification (WGA) protocol. Therefore the method of the invention disclosed in WO 2005/035725 does not selectively amplify ffDNA or selectively deplete maternal DNA in order to increase the concentration of ffDNA relative to maternal DNA.

WO 2007/103910 concerns a method for selectively amplifying ffDNA sequences from a mixed fetal-maternal source. The method takes advantage of differences in DNA methylation between ffDNA and maternal DNA. As ffDNA is hypomethylated in comparison with maternal DNA, selective amplification of ffDNA specific sequences is achievable using a methylation sensitive enzyme. Similarly, WO 2005/035725 uses a methylation sensitive enzyme for exploiting differences in DNA methylation states between ffDNA and maternal DNA to substantially reduce or destroy completely the maternal DNA. Such methods are therefore heavily restricted in terms of what disorders they may detect. Only certain regions of DNA, in particular the promoter regions, are noticeably methylated. In addition, regions such as these are typically irrelevant for the targeted amplification and detection of ffDNA abnormalities.

WO 2009/032781 relates to a method for amplifying both ffDNA and maternal DNA, using target and non target binding inside primers and non target binding outside primers, wherein the concentration of the outer primer is greater than that of the inside primer. In this way, the maternal DNA is amplified at a slower rate to the ffDNA, thereby increasing the concentration of ffDNA relative to maternal DNA. When subsequent isolation and extraction of the outside primer bound nucleic acid is preferred, the outside primers are modified with a tag facilitating isolation and/or extraction of the non target nucleic acid sequences. Therefore the method of the invention disclosed in WO 2009/032781 does not selectively enrich ffDNA.

There remains a need therefore for an improved NIPD method, which is not limited in the way that the known methods discussed above are constrained. In particular it would be advantageous if the NIPD method could overcome the problems associated with the low fractional concentration of ffDNA present within the maternal plasma sample, without imposing undue burden on the user.

It has now been found possible to significantly improve the efficiency and accuracy of the analysis of ffDNA as part of a non-invasive diagnostic regime for fetuses by the in situ enrichment of the ffDNA content of the maternal plasma or serum.

In a first, general aspect, the present invention provides a method for processing maternal plasma comprising maternal free DNA and ffDNA, the method comprising the in situ enrichment of the amount of ffDNA relative to the amount of maternal free DNA.

In the present specification, the term 'in situ enrichment' is a reference to a method of increasing the relative amount of the ffDNA to the materna free DNA in a maternal plasma sample with both DNA components being present, that is without physically separating or removing one or other of the DNA components from the plasma. The in situ enrichment may be performed by selectively increasing the amount of ffDNA in the plasma and/or by selectively decreasing the amount of free maternal DNA in the plasma.

The present invention provides the selective enriching of the amount of ffDNA present in the maternal plasma, without contamination from maternal free DNA. In this way, the enriched ffDNA product may be assessed using known methods. In particular, the enriched product is particularly suitable for use in conventionally applied, simple analytical methods, such as real time PCR and multiplex ligation- dependent probe amplification ( LPA). These methods are known and are being used routinely in the analysis of fetal material sampled by invasive methods. It is therefore an advantage of the method of the present invention that it can be used in conjunction with the known and routinely used techniques for diagnosis. The present invention therefore provides significant advantages over other methods such as costly digital PCR and NGS discussed above, which have yet to achieve widespread acceptance and use in routine diagnostic practice. It will be appreciated that the method of the present invention may be used to provide ffDNA enriched material for further use in all prenatal diagnosis procedures, including those for the assessment of abnormal fetal chromosome copy numbers (aneuploidy), inherited disorders such as haemoglobinopathy and cystic fibrosis, without the need for invasive measures. In addition, the method of the present invention can improve the efficiency and accuracy of the existing methods for detecting genetic traits, such as RhD blood group and sex linked conditions, which as discussed previously are already detectable by NIPD techniques. The method of the present invention has particular application in the assessment of Down syndrome and other aneuploidies.

The method of the present invention may be used alone, that is to produce a ffDNA enriched product, which may then be subjected to one or more diagnostic analyses. Alternatively, the method may be incorporated into a dedicated diagnostic regime for a particular disorder.

The method of the present invention comprises the in situ enrichment of the ffDNA in the sample, relative to the free maternal DNA. In this respect, the method does not physically separate DNA material from the sample. Rather, the ffDNA is enriched relative to the free maternal DNA by either increasing the amount of ffDNA present or by depleting the amount of free maternal DNA present, or by a

combination of both, either simultaneously or successively. Any suitable technique for increasing the amount of ffDNA present in the sample may be employed. A preferred method for enriching the ffDNA is by selectively amplifying the shorter ffDNA from the admixture of ffDNA and maternal free DNA. Techniques for amplifying DNA are known and will be readily understood by the person skilled in the art. The method may comprise amplifying all of the ffDNA material present in the sample. More preferably, the method is employed to amplify only one or more selected regions of the genome from the ffDNA material. In particular, the method may be employed to selectively amplify those regions of the fetal genome that are relevant to the diagnosis being conducted. For example, in the case of the diagnosis of Down syndrome and other aneuploidies, the method is applied to selectively amplify polymorphic regions of chromosome 21 of the ffDNA material, without amplifying free maternal DNA material.

Suitable techniques for amplification of the ffDNA include PCR, including real time PCR, digital PCR and multiplex PCR, and MLPA. In the case of PCR, the method of the present invention employs a limited denaturation of the DNA admix using conditions which result in essentially only the shorter DNA fragments present in the sample material being denatured. As noted above, ffDNA is present only in the shorter DNA material. Accordingly, selective amplification of the short DNA fragments present in the admixture of DNA will enrich the sample in ffDNA. The selective amplification is preferably conducted to target free DNA having less than 500 base pairs, more preferably less than 400 base pairs. It is especially preferred to selectively amplify DNA fragments having less than 300 base pairs, in order to provide increased efficiency in the enrichment of the ffDNA content and minimise the amplification of maternal DNA fragments.

The selective amplification of the smaller DNA fragments may be achieved using PCR techniques, operated under conditions that favour amplification of DNA fragments of the target size. In particular, it has been found that the smaller DNA fragments may be selectively amplified by appropriate selection of the denaturation temperature employed. PCR consists essentially of three steps: strand separation; hybridization of primers; and extension of primers by DNA synthesis. In a typical PCR, the first step is performed at a suitable denaturation temperature, in order to separate essentially all of the double stranded DNA fragments. The denaturation temperature is typically about 94 to 96°C. In the method of the present invention, however, the denaturation temperature is lowered, so that only the shorter DNA fragments and ffDNA are denatured and subsequently amplified. The specific critical denaturation temperature will vary according to the length of the DNA fragments being targeted and the composition of the DNA fragment (i.e. the GC content).

In a preferred embodiment, the method of the present invention involves the selective operation of the PCR in order to denature and subsequently amplify only those fragments of targeted fetal genes which are of use when diagnosing a particular disorder.

The critical denaturation temperature of the PCR employed may vary, according to the genetic material being targeted for amplification. In particular, depending on the region which is to be targeted the critical denaturation temperature employed will vary according to such factors as the exact number and composition of nucleotides present within the targeted sequence. It is for this reason that, for each gene target, the critical denaturation temperature and/or primers may need to be optimised prior to use. It is a particular advantage of this method that optimisation of PCR conditions for multiple gene targets on different chromosomes will enable multiplex PCR to use similar conditions.

In order to selectively amplify the selected gene target, the denaturation temperature of the PCR will typically be less than 94°C, more preferably less than 90°C, still more preferably less than 85°C. Critical denaturation temperatures of less than 80°C may be employed in some embodiments, again depending upon the genetic target selected. The critical denaturation temperature will generally be greater than 65°C, more preferably greater than 70°C. Critical denaturation temperatures of greater than 75°C may be employed, as required to selectively amplify the target sequence. A critical denaturation temperature of 80°C has been found to be appropriate for amplifying a specific region within Chromosome 21 of the ffDNA. A critical denaturation temperature of 80°C has been found to be appropriate for amplifying a specific region within Chromosome 18 of the ffDNA. A critical denaturation temperature of 75.5°C has been found to be appropriate for amplifying a specific region within Chromosome 13 of the ffDNA.

Use of the appropriate denaturation temperature will target the smaller DNA fragments in the sample, in particular the ffDNA, the strands of which are selectively separated. The strands of the longer, free maternal DNA fragments are not separated at the temperature selected. By use of appropriate primers, the amplification of ffDNA is achieved.

As noted above, the amplification of the ffDNA may be conducted to selectively target and amplify selected regions of the fetal genome. It is

advantageous if an amplification technique is used which itself also provides an indication of the presence of genetic abnormalities. For example, real time PCR may be employed with a binding die, in known manner, to provide a measurable indication of the amount of amplified DNA produced in the method. In this way, enrichment of the ffDNA is accompanied by an indicator of the presence of the targeted portion of the fetal genome. In the case of Down Syndrome and other aneuploidies, the simultaneous amplification of polymorphic regions of chromosomes, such as one of chromosomes 13, 18 or 21 , (depending on the exact aneuploidy) may be assessed to copy number of maternal and paternal alleles and compared with a reference chromosome, such as another of chromosome 13, 8 or 21 or a chromosome which is not commonly implicated in aneuploidies.

As noted above, the ffDNA in the sample plasma may also be enriched relative to the free maternal DNA by the in situ reduction of the amount of free maternal DNA. In an alternative embodiment, the present invention provides a method for enriching the ffDNA by selectively depleting the larger maternal free DNA from an admixture of ffDNA and maternal free DNA.

A preferred means to reduce the free maternal DNA present in the sample is by selective amplification of the free maternal DNA material, whilst incorporating a moiety in the synthesised DNA to render it susceptible to degradation by one or more enzymes. The free maternal DNA may be amplified, for example by PCR techniques, while incorporating uracil in the synthesised DNA. The thus synthesised DNA may be degraded using a suitable enzyme, in particular uracil deglycosylase (UNG).

In addition or as an alternative, the amplified maternal DNA is labelled with a compound which renders it susceptible to purification methods known in the art. The free maternal DNA may be amplified, for example by PCR techniques, to incorporate a label. A most suitable label is biotin. A compound having an affinity for the label, such as biotin, may then be used in order to remove the amplified maternal DNA. In the case of biotin, one such compound is streptavidin. In one embodiment, labelled magnetic beads, for example streptavidin-labelled magnetic beads, may then used to remove the labelled DNA from non-labelled DNA, in particular ffDNA. The selective amplification of the free maternal DNA is achieved using specific primers for targeting the amplification of DNA fragments greater than a specified length. In particular, PCR is applied with appropriate primers to selectively amplify DNA fragments having more than 300 base pairs, preferably more than 400 base pairs, more preferably at least 500 base pairs. In this way, the smaller ffDNA fragments remain unamplified, as it is unlikely that both PCR primers will find their target sites on the ffDNA fragments. More importantly, by using specific primers for targeting the amplification of maternal DNA only, this alternative embodiment for the method of the present invention does not interfere with and/or modify the ffDNA, in particular does not result in the ffDNA containing artefacts.

The resulting product is subjected to enzyme treatment and the result is the depletion of free maternal DNA fragments larger than the target fragment length, leaving the ffDNA present in combination with only shorter fragments of maternal DNA and remnants of the degraded DNA material. The method may be used to deplete a portion, more preferably substantially all, of the free maternal DNA fragments above the threshold number of base pairs.

As noted, the enrichment of ffDNA relative to free maternal DNA in the sample may be employed as part of a diagnostic regime.

Accordingly, in a further aspect, the present invention provides a non-invasive method for identifying genetic abnormalities in the genome of a fetus, the method comprising:

providing a sample of maternal plasma;

enriching the ffDNA content of the plasma by a method as hereinbefore described; and

analysing the ffDNA in the enriched sample to identify any genetic abnormalities in the fetus.

The ffDNA may be analysed to identify any abnormalities in the genetic material originating from the fetus, including sequence errors or chromosomal aneuploidies. The ffDNA in the enriched sample may be analysed using any technique, including those known in the prior art and discussed hereinbefore. It is particularly advantageous that known and currently used techniques relying on samples obtained by invasive techniques can be used to analyse the enriched sample. In a still further aspect, the present invention provides a ffDNA enriched plasma sample obtainable by the method as hereinbefore described.

Further, the present invention also provides a method of diagnosing a condition in a fetus arising from a genetic abnormality, the method comprising enriching and analysing a sample of maternal plasma as hereinbefore described. Conditions that may be diagnosed using the method of this aspect of the invention are as hereinbefore described, in particular Down Syndrome and other aneuploidies. Embodiments of the present invention will now be described, for illustrative purposes only, by way of the following Examples.

EXAMPLE 1

Experiments were conducted using small synthetic stretches of DNA, to simulate ffDNA and circulating DNA from blood donors to act as maternal free DNA. This example describes experiments conducted to determine the PCR critical denaturation temperature of the simulated ffDNA material.

A portion of the CCR5 gene (as annotated below) was employed to generate synthetic ffDNA, as follows:

The CCR5 gene is located on chromosome 3 and is a member of the beta chemokine receptor family. A real time PCR assay targeting CCR5 is generally used as a control alongside the real time PCR assays in order to determine the RHD status of fetuses from RHD negative mothers. A real time PCR for CCR5 will detect CCR5 products in maternal and fetal DNA in such assays. A portion of the CCR5 gene is set out below. The primers (bold) and probe

(italics) used in the CCR5 real time PCR assay are shown. The size of the PCR amplicon was 91 bp. ACTCACTGGTGTTCATCTTTGGTTTTGTGGGCAACATGCTGGTCATCCTCATCCT GATAAactgcaaaaggctgaagagcATGACTGACATCTACCTGCTCAACCTGGCCATCT CTGACCJGUTTTCCTTCTTACTGTCCCCTTCTGGGCTCACTKTGCTGCCGCCCA GTG GG AC TTTG G A AATAC AATGTGTC AACTCTTG AC AGG G CTCTATTTTATAGG CTTCTTCTCTGGAATCTTCTTCATCATCCTCCTGACAATCGATAGGTACCTGGCT GTCGTCCATGCTGTGTTTGCTTTAAAAGCCAGGACGGTCACCTTTGGGGTGGTG AC AAGTGTG ATC ACTTG G GTG GTG G CTGTGTTTG cgtctctcccagg aatcatcTTTACC A GATCTCAAAAAGAAGGTCTTCATTACA Primers were designed so that a PCR amplicon of approximately 250 to 350 base pairs could be generated with the region required for the real time PCR assay in the middle of the amplicon. This fragment was used as a synthetic ffDNA fragment having from 250 to 350 base pairs. The gene sequence was obtained from the National Center for Biotechnology

Information. Primer3 software was used to search for primers in the relevant area of the gene. Several sets of primers were chosen by Primer3 and these were all checked using BLAST software to ensure that there was a full match only to the gene of interest. One forward primer was ordered and two reverse primers.

The primers were tested in combination (forward with reverse A and forward with reverse B) using the appropriate annealing temperatures and male genomic DNA (ex. Promega, U.K.) as a template. Following optimization of the PCR conditions, the PCR amplicon was purified using agarose gel electrophoresis and the QIAquick Gel Extraction Kit (ex. Qiagen, U.K.). The extracted DNA was quantified using the NanoVue Plus (ex. GE Healthcare Life Sciences, U.K.). The positions of the working primers are shown in the aforementioned sequence by the lower case letters. The amplicon size was 326bp. A portion of the SRY gene (as annotated below) was employed to generate synthetic ffDNA, as follows:

A real time PCR assay for the single copy SRY gene was established by Lo et al. 'Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum: implications for non-invasive pre-natal diagnosis', Am. J. Hum. Genet., 1998, 62, pages 768 to 775, in order to determine fetal sex from ffDNA. The SRY gene is located on the Y chromosome. A portion of the SRY gene is set out below. The primers (bold) and probe

(italics) used in the SRY real time PCR assay are indicated. The size of the PCR amplicon is 137bp.

AGCTTTG I I I I I I l AAAGATAACATACACA ATATTGATAATGATAAACAATTCATA TAgctttttgtgtcctctcgttttGTGACATAAAAGGTCAATGAAAAAATTGGCGATTAAGTCA AAnCGCATTYTTCAGGACAGCAGTAGAGCAGTCAGGGAGGCAGATCAGCAGG GCAAGTAGTCMCGTTACTG ITTACCATGTTTTGCTTGAGAATGAATACATTGT CAGGGTACTAGGGGGTAGGCTGGTTGGGCGGGGTTGAGGGGgtgttgagggcggag aaatGCAAGTTTCATTACAAAAGTTAACGTAACAAAGAATCTGGTAGAAGTGAGTT TTGGATAGT

Primers were designed in a similar manner as described above and two forward primers and one reverse primer were ordered. The primers were tested in combination (forward A with reverse and forward B with reverse) using the appropriate annealing temperatures and male genomic DNA as a template.

Following optimization of the PCR conditions, the PCR amplicon was purified using agarose gel electrophoresis and the QIAquick Gel Extraction Kit. The extracted DNA was quantified using the NanoVue Plus. The positions of the working primers are shown in the figure above by the lower case letters. The amplicon size was 230bp.

To establish that ffDNA may be selectively amplified using PCR, the primers from the real time PCR assays for CCR5 and SRY were used to run gradient end point PCRs to determine the lowest denaturation temperature for each assay at which a product was no longer achieved using genomic DNA as a template, but was still achieved using the synthetic ffDNA fragments as templates. The products from the end point PCRs were separated using agarose gels. For CCR5, each 25uL PCR reaction consisted of: 1x Mastermix containing polymerase, dNTPs, buffer and MgCI₂ (TaqMan® Fast Universal PCR Mastermix, Applied Biosystems), primers as shown in bold above at 200nM (HPLC purified, Eurofins MWG Biotech, Germany), 5uL of genomic DNA template or synthetic ffDNA template at 0.02ng/uL concentration.

The cycling conditions were as follows. 50°C for 2mins, 95°C for lOmins, 50°C for 1min; 45 cycles of selected denaturation temperatures for 15secs; and 56°C for 1 min.

Similar conditions were used for SRY with the appropriate primers shown in bold above.

The gradient PCRs were run with six different temperatures at any one time and for CCR5 the initial denaturation temperature range was from 80^oC-95^oC.

Several PCRs were run whereby the temperature range was narrowed and decreased to the extent that the critical denaturation temperature was found to be 81 °C for this assay. At this temperature no amplicon was produced with genomic DNA as a template but an amplicon was produced with the synthetic ffDNA as a template.

A similar process was employed for determining the critical denaturation temperature for the SRY assay and for this assay 79°C was found to be the critical temperature. Real time PCR assays were then conducted for both CCR5 and SRY with the probes (5' FAM label, 3' BHQ1 quencher for CCR5 and 5' Yakima Yellow, 3' BHQ1 quencher for SRY) detailed in italics above. Two plates were used in each case, with one plate used under conditions with a denaturation temperature of 95°C and the other plate used under conditions with a denaturation temperature of 81 °C for CCR5 and 79°C for SRY.

The results are set out in Figures 1 to 4. Referring to the results shown in Figure 1 , there is shown the real time PCR CCR5 assay trace showing the increase in fluorescence with cycle number at a denaturation temperature of 95°C where the templates were either genomic DNA or synthetic ffDNA. The threshold cycle was determined using StepOne Software automatically. With synthetic ffDNA as a starting template, the mean Ct value was 10.13 and with genomic DNA as a starting template, the mean Ct value was 35.3.

Referring to the results shown in Figure 2, there is shown the real time PCR CCR5 assay trace showing the increase in fluorescence with cycle number at a denaturation temperature of 81 °C where the templates were either genomic DNA or synthetic ffDNA. The threshold cycle was determined by StepOne Software automatically. With synthetic ffDNA as a starting template, the mean Ct value was 1 .29 and with genomic DNA as a starting template, the mean Ct value was approximately 38 (one replicate undetermined).

Referring to Figure 3, there is shown the real time PCR SRY assay trace showing the increase in fluorescence with cycle number at a denaturation temperature of 95°C where the templates were either genomic DNA or synthetic ffDNA. The threshold cycle was determined by the StepOne Software automatically. With synthetic ffDNA as a starting template, the mean Ct value was 10.51 and with genomic DNA as a starting template, the mean Ct value was 36.04.

Referring to Figure 4, there is shown the real time PCR SRY assay trace showing the increase in fluorescence with cycle number at a denaturation temperature of 79°C where the templates were either genomic DNA or synthetic ffDNA. The threshold cycle was determined by the StepOne Software automatically. With synthetic ffDNA as a starting template, the mean Ct value was 20.41 and with genomic DNA as a starting template, the mean Ct value was undetermined

(indicating no amplification).

The results indicate that, at the critical denaturation temperatures, only the synthetic ffDNA template and not the genomic DNA templates are amplified by the PCR. EXAMPLE 2

Circulating free DNA was isolated from donor blood samples using the QIAamp Circulating Nucleic Acid Kit (ex. Qiagen, U.K). This type of DNA was used to simulate maternal free DNA from maternal plasma samples. Genomic DNA was also extracted from the buffy coat of the same blood samples using the QIAamp DNA Blood Mini Kit (ex. Qiagen, U.K). This DNA was used to determine the sex of the blood donors, using a real time PCR assay targeting the multi copy DYS14 sequence present on the Y chromosome, as described in Zimmermann et at., 'Optimised Real Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma', Clin. Chem. 2005, 51 , pages 1598 to 1604.

1 genome equivalent or copy number equals 6.6pg of DNA. Using this formula, the number of copies of DNA present in the circulating DNA samples was calculated, using a real time PCR assay for CCR5 and a standard curve with genomic DNA as a template.

Experiments to simulate the mixture of ffDNA and maternal free DNA found in maternal plasma were then performed, as follows:

The synthetic ffDNA template was mixed with circulating DNA or genomic DNA to simulate the distribution of ffDNA and maternal free DNA when obtained from maternal plasma or serum. Two types of mixture were generated as follows:

Female circulating DNA at 99% and synthetic ffDNA at 1 %;

Female circulating DNA at 99.9% and synthetic ffDNA at 0.1 %;

The percentages were calculated using copy number equivalents.

In addition to the mixed samples, samples of each DNA material at relevant concentrations, that is for circulating DNA at 99% and 99.9% and synthetic ffDNA at 1 % and 0.1%, water negative controls and standard curves were included on the real time PCR plates. The standard curves comprised circulating/genomic DNA in the range 250 copies/uL to 0.1 copy/uL. Experiments were carried out with both the CCR5 and SRY assays. Two identical real time PCR plates were used, with one run with a denaturation temperature of 95°C and one run with a denaturation temperature of 81 °C. Real time PCR results are shown in Figures 5 and 6. Results are shown for only the CCR5 assay.

Referring to Figure 5, there is shown the Real time PCR CCR5 assay trace showing the increase in fluorescence with cycle number at a denaturation

temperature of 95°C where the templates were either genomic DNA, synthetic ffDNA or mixed samples. The threshold cycle was determined by the StepOne Software automatically. With synthetic ffDNA at 1% concentration with genomic DNA at 99% as a starting template, the mean Ct value was 10.67 and with synthetic ffDNA at 0.1 % concentration with genomic DNA at 99.9% as a starting template, the mean Ct value was 14.27. Referring to Figure 6, there is shown the real time PCR CCR5 assay trace showing the increase in fluorescence with cycle number at a denaturation

temperature of 81 °C where the templates were either genomic DNA, synthetic ffDNA, or mixed samples. The threshold cycle was determined by the StepOne Software automatically. With synthetic ffDNA at 1 % concentration with genomic DNA at 99% as a starting template, the mean Ct value was 12.54 and with synthetic ffDNA at 0.1 % concentration with genomic DNA at 99.9% as a starting template, the mean Ct value was 16.42

Mean Ct values for the various samples at the two different denaturation temperatures are as follows:

95°C

Genomic DNA at 99% - mean Ct value 28.61 (CCR5 signal from genomic DNA) Genomic DNA at 99.9% - mean Ct value 32.38 (CCR5 signal from genomic DNA) Synthetic ffDNA at 1 % - mean Ct value 10.64 (CCR5 signal from synthetic ffDNA) Synthetic ffDNA at 0.1% - mean Ct value 14.40 (CCR5 signal from synthetic ffDNA) Synthetic ffDNA at 1% and genomic DNA at 99% - mean Ct value 10.67 (CCR5 signal from both genomic DNA and synthetic ffDNA)

Synthetic ffDNA at 0.1% and genomic DNA at 99.9% - mean Ct value 14.27 (CCR5 signal from both genomic DNA and synthetic ffDNA)

81°C

Genomic DNA at 99% - mean Ct value 32.91 (reduced CCR5 signal from genomic DNA)

Genomic DNA at 99.9% - mean Ct value 35.96 (reduced CCR5 signal from genomic DNA)

Synthetic ffDNA at 1% - mean Ct value 13.52 (still strong CCR5 signal from synthetic ffDNA)

Synthetic ffDNA at 0.1 % - mean Ct value 17.74 (still strong CCR5 signal from synthetic ffDNA)

Synthetic ffDNA at 1% and genomic DNA at 99% - mean Ct value 12.54 {CCR5 signal mainly from synthetic ffDNA, reduced contribution from genomic DNA) Synthetic ffDNA at 0.1% and genomic DNA at 99.9% - mean Ct value 16.42 {CCR5 signal mainly from synthetic ffDNA, reduced contribution from genomic DNA)

Having established the critical denaturation temperatures of the PCR enriched synthetic ffDNA for CCR5 and SRY, assays for chromosomes implicated in aneuploidies, principally chromosomes 21 (Down Syndrome), 18 (Edwards

Syndrome) and 13 (Patau Syndrome) were designed.

EXAMPLE 3 Experiments have been carried out using small synthetic stretches of DNA to simulate ffDNA and genomic DNA from blood donors to simulate maternal free DNA. The experiments have been focussed on three different regions of the genome - one for Chromosome 21 , one for Chromosome 18 and one for Chromosome 13. Short tandem repeat (STR) regions were assessed on each of these chromosomes and CA repeat regions chosen with high levels of heterozygosity. For each of the

chromosomes, primers were designed to be able to amplify a ~250bp region of DNA surrounding the CA repeat region by PCR. This fragment simulates the synthetic ffDNA. Primers were also designed to amplify a region internal to the ~250bp region by real time PCR. Primer3 software was used to search for primers in the relevant area of the gene (http://frodo.wi.mit.edu/prirner3 )· Several sets of primers were chosen by Primer3 and were all checked on BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to ensure that there was a 100% match only to the region of interest.

The regions of interest for Chromosomes 21 , 18 and 13 are shown in Figures 7, 8 and 9 respectively.

Referring to Figure 7, there is shown the region of interest for Chromosome 21. The STR chosen was D21 S1890 due to the high level of heterozygosity. The PCR primers are shown in lower case. The real time PCR primers are shown in bold and the probe sequence in italics. The CA repeat region is underlined. The PCR product is 229bp and the real time PCR product is 101 bp, with the caveat that both of these product sizes are dependent on the number of CA repeats present in an individual.

Referring to Figure 8, there is shown the region of interest for Chromosome 18. The STR chosen was D18S1 144 due to the high level of heterozygosity. The PCR primers are shown in lower case. The real time PCR primers are shown in bold. The CA repeat region is underlined. The PCR product is 259bp and the real time PCR product is 123bp, with the caveat that both of these product sizes are dependent on the number of CA repeats present in an individual.

Referring to Figure 9, there is shown the region of interest for Chromosome 13. The STR chosen was D13S 174 due to the high level of heterozygosity. The PCR primers are shown in lower case. The real time PCR primers are shown in bold. The CA repeat region is underlined. The PCR product is 230bp and the real time PCR product is 102bp, with the caveat that both of these product sizes are dependent on the number of CA repeats present in an individual. The experiments conducted in relation to Chromosomes 13 and 18 use SYBR Green real time PCR rather than double-dye probes. It is for this reason that no probe positions are identifiable in Figures 8 and 9.

Synthetic ffDNA fragments were generated by PCR in a similar manner to that described above. The PCR products were analysed using agarose gel electrophoresis and the relevant bands excised and subjected to the QIAquick Gel Extraction kit.

Real time PCR experiments were performed, using the synthetic ffDNA and genomic DNA as templates, in order to assess the critical denaturation temperature for this region of Chromosome 21. The following critical denaturation temperatures were tried: 84°C-83⁰C-82^oC-8rC-81.8^oC-81.6^oC-81.4⁰C-81.2°C and 80°C. It has been found that the critical denaturation temperature for chromosome 21 is 80°C, as shown in Figure 10a and 10b.

Having reference to Figure 10a, there is shown the real time PCR of

Chromosome 21 assay traces showing the increase in fluorescence with cycle number at a denaturation temperature of 95°C where the templates were either genomic DNA or synthetic ffDNA. Having reference to figure 10b, there is shown the increase in fluorescence with cycle number at a denaturation temperature of 80°C where the template was synthetic ffDNA but no amplification was seen from the genomic DNA template. In this experiment, female genomic DNA was used as both the maternal DNA template and as a template for generating the synthetic ffDNA. It was necessary to use templates where the sample acting as maternal DNA comprised different CA repeat alleles to the sample acting as ffDNA. Accordingly, a large number of genomic DNA samples were used as templates in order to generate the ~250bp PCR products there from. These PCR products were generated using a 5'FA labelled forward primer and the products were then run against a LIZ size standard on an Applied Biosystems 3130 Genetic Analyser for fragment analysis, as shown in Figure 11. Referring to Figure 11 , there is shown fragment analysis traces for the chosen synthetic ffDNA and maternal DNA samples. The y axis shows relative fluorescence units and the x axis shows DNA size in base pairs. Sample 6534H and sample female have been labelled as maternal DNA and synthetic ffDNA respectively. The amplicons run on the sequencer were amplified using the primers that generate a ~250bp product.

Figure 11 shows the two most preferred DNA templates. The fetal (sample female) and maternal (sample 6534H) samples have peaks on the left that coincide. The fetal sample peaks progressively get much stronger and then there is a region on the right where the maternal sample peaks are seen without any peaks from the fetal sample. Once templates had been decided upon, 'spike' experiments were completed using a mix of synthetic ffDNA and maternal DNA samples. The aim of the spike experiments was to test if the synthetic ffDNA would denature in the presence of maternal DNA but without the maternal DNA denaturing at the critical denaturation temperature (80°C). Results were assessed against experiments also carried out at the usual denaturation temperature of 95°C. Real time PCR assays were completed and also end point PCR assays were completed with the 5'FAM labelled forward primer for fragment analysis.

The templates used for the PCR and real time PCR assays were as follows;

No template control (water)

Standard curve - 4 concentrations of DNA - maternal DNA used at 95°C and synthetic ffDNA used at 80°C.

Maternal DNA alone

Synthetic ffDNA alone

Mix of maternal DNA and synthetic ffDNA (90% maternal, 10% synthetic ffDNA)

The results obtained from each of the above mentioned templates used for the PCR and real time PCR are shown in Figures 12a to 12f and 13a and 13b: With reference to Figure 12a, there is shown the real time PCR Chromosome 21 assay traces from the spike experiment showing the increase in fluorescence with cycle number at a denaturation temperature of 95°C where the template was maternal DNA. With reference to Figure 12b, no increase in fluorescence with cycle number at a denaturation temperature of 80°C is shown where the template was maternal DNA. With reference to Figure 12c, there is shown an increase in fluorescence with cycle number at a denaturation temperature of 95°C where the template was synthetic ffDNA. With reference to Figure 12d, there is shown an increase in fluorescence with cycle number at a denaturation temperature of 80°C where the template was synthetic ffDNA. With reference to Figure 12e, there is shown an increase in fluorescence with cycle number at a denaturation temperature of 95°C, where the template was a mix of maternal DNA and synthetic ffDNA. With reference to Figure 2f, there is shown an increase in fluorescence with cycle number at a denaturation temperature of 80°C, where the template was a mix of maternal DNA and synthetic ffDNA. The results shown in Figures 12e and 12f mirror the results shown in 12c and 12d.

Where a mix of maternal DNA and synthetic ffDNA was used as a template, the results mirrored the results seen by the synthetic ffDNA template alone, with this being particularly true at 80°C.

With reference to Figure 13, there is shown the fragment analysis traces from the Chromosome 21 spike experiment. The y axis shows relative fluorescence units and the x axis shows DNA size in base pairs. The traces for the maternal sample, the synthetic ffDNA sample and the mixed sample are overlaid. Figure 13a shows the traces following a PCR run with a denaturation temperature of 95°C and Figure 13b shows the traces following a PCR run with a denaturation temperature of 80^oC. The maternal DNA starting template trace, the synthetic ffDNA starting template trace and the mixed sample template trace are identified in Figures 13a and 13b. The amplicons run on the sequencer were amplified using the primers that generate a ~100bp product. Where a mix of maternal DNA and synthetic ffDNA was used as a template, the results mirrored the results seen by the synthetic ffDNA template alone, with this being particularly true at 80°C. At 80°C the maternal DNA peaks around 100bp are dramatically diminished.

The experiment described above relates to the D21 S1890 region of

Chromosome 21. However, similar work has been carried out for Chromosome 18 and Chromosome 13, with analogous results.

EXAMPLE 4

Experiments were conducted to enrich maternal plasma in ffDNA by the selective depletion of free maternal DNA.

The region selected to target was exon 7 of the RHD gene. RHD exon 7 is one of the exons detected in the real time PCR assay to determine the RHD status of fetuses from D negative mothers using ffDNA from maternal plasma. The assay is described in Finning et al. , 'Effect of High Throughput RHD Typing of Fetal DNA in Maternal Plasma on use of Anti-RHD Immunoglobulin in RHD-negative pregnant women: prospective feasibility study', BMJ, 2008, 336, pages 816 to 818.

The position of the RHD primers (in bold) and probe (in italics) are shown below. The RHD and RHCE genes show a high degree of sequence homology. The size of the amplicon for the real time PCR assay is 75bp.

The alignment of RHD and RHCE exon 7 sequences with real time primers and probe annotated is as follows:

* 20 * 40 * 60

RHDEX7 : GGGTGTTGTAACCGAC3TGCTGGG⁽3ATTCCCCACAGCTCCATCATGGGCTACAACTTCAGC : 6 RHCEE 7 : GTGTGTTGTAACCGAGTGCTGGGGATTCACCACATCTCCGTCATGCACTCCATCTTCAGC : 6

G GTGTTGTAACCGAGTGCTGGGGATTC CCACA CTCC TCATG CT CA CTTCAGC

80 100 120 RHDEX7 : TTGCTGGGTCTGCTTGGAGAGATCATCTACATTGTGCTGCTGGTGCTTGATACCGTCGGA : 120 RHCEEX7 : TTGCTGGGTCTGCTTGGAGAGATCACCTACATTGTGCTGCTGGTGCTTCATACTGTCTGG : 120

TTGCTGGGTCTGCTTGGAGAGATCA CTACATTGTGCTGCTGGTGCTT ATAC GTC G

RHDEX7 : GCCGGCAATGGCAT : 134

RHCEEX7 : AACGGCAATGGCAT : 134

CGGCAATGGCAT The portion of the RHD gene sequence around exon 7 with primers annotated is as follows:

CAGCAGCATTGGCATCACCTGGGACCTTGTTAGAAATGCTGTTAGACCCC ACCCCACATCCACTAAAGCCAGCTCTTCATTTCAACAAACTCCCCGATGA TGTGAGTGCACATTCAAGTCTGAGAAGGGCTTCTTTGAGGTGAGCCTTAG TGCCCATCCCCCTTTGGTGGCCCCGGATACCAAGGGTGTGTGAAAGGGGT GGGTAGGGAATATGGGTCTCACCTGCCAATCTGCTTATaataacacttgt

ccacagggGTGTTGTAACCGAGTGCTGGGGATTCCCCACAGCTCCATCAT

GGGCTACAACTTC^GC7TGCrGGG cr<3C7^"7GG^G^GyA7^"CATCTACATTG TGCTGCTGGTGCTTGATACCGTCGGAGCCGGCAATGGCATGTGGGTCACT GGGCTTACCCCCCATCCCCTTAACACTCCCCTCCAACTCAGGAAGAAATG TGTGCAGAGTCCTTAGCTGGGGCGTGTGCACTCGGGGCcaggtgctcagt aggcttcgGTGAATATTTGTTGGCTGATTTATTCAGAAATTCTGTCCAGC

CCCTACCTTGGATGGATTTATCACCTCTCCAGGCCACCTCTTCTTTCCAA ATAGGGCCACCTAGGTATAGACCAAAGACACGAAATCTTTTGTGATCCCA CAAACACAG AG CAGGTCAAATAG G C CC AAG C C AATTG AG ACTGTG GTTC A GGTCGTGATGCAGAGCTTTGCTGTGGACGTGCTCCCACTGCGTACTAGCT

Primers were designed to generate a ~250bp PCR amplicon to simulate ffDNA for RHD exon 7 with this published assay region in the middle of the amplicon (final choice of primers shown in lowercase letters above). Primer design was carried out in a similar manner to that for CCR5 and SRY described in Examples 1 and 2 above.

Primers were designed to generate a ~500bp PCR amplicon to simulate maternal DNA for RHD exon 7 with the ~250bp PCR amplicon in the middle of the larger amplicon. The primers were designed in a similar manner to those described above. The primers were consensus primers for RHD/RHCE and are shown underlined above (choice of two pairs). These primers were used to generate ~500bp PCR amplicons with the following templates:

a) RHD positive genomic DNA

b) RHD negative genomic DNA

c) RHD positive genomic DNA spiked at 5% with RHD ~250bp

PCR amplicon (synthetic ffDNA)

d) RHD negative genomic DNA spiked at 5% with RHD ~250bp PCR amplicon (synthetic ffDNA)

The amplification results are shown in Figure 14. In terms of nucleotides, dUTP was used instead of dTTP in the reactions.

In theory, the signal is coming from the following:

a) RHD and RHCE genes (lane 4)

b) RHCE gene (lane 6)

c) RHD and RHCE genes (lane 8)

d) RHCE gene (lane 10)

Following 25 cycles of PCR, UNG treatment was applied to half of the tubes. 0.25ul of UNG was added to 25uL PCR reactions and then incubated at 50°C for 5 mins, followed by 95°C for 0 mins. The results of UNG treatment are shown in lanes 5, 7, 9 and 11 of Figure 14. There appears to be complete degradation of the amplified ~500bp region of DNA in these samples. The intact nature of the synthetic ffDNA in the mixture can also be assessed by the RHD exon 7 real time PCR assay.

Despite trying different concentrations of dUTP, different amounts of UNG and different lengths of time of UNG treatment, UNG was not found to be 100% effective in degrading the maternal DNA fragments. It is thought that the original starting template of genomic DNA that does not contain dUTP was causing a problem with the real time PCR assays. Accordingly, mung bean nuclease has been used as an alternative enzyme for degrading this single stranded DNA. However, the experiments conducted were not conclusive as to whether the degradation was 100% effective or not. In a preferred alternative approach, the primers used to amplify the ~500bp of synthetic maternal DNA are biotin-labelled. Streptavidin-labelled magnetic beads were then used to remove all biotin-labelled products. This approach has been found to be much more effective than UNG and/or mung bean nuclease.

The examples have applied the method of the present invention to the CCR5, SRY, and RHD genes. However, it will be appreciated that the method is equally applicable to any other region of the fetal genome of interest, for example

polymorphic regions of chromosome 21 , as demonstrated in Example 3 above.

Claims

1. A method for processing a sample of maternal plasma comprising maternal free DNA and ffDNA, the method comprising the in situ enrichment of the amount of ffDNA relative to the amount of maternal free DNA.

2. The method according to claim 1 wherein the sample of maternal plasma is obtained non-invasively.

3. The method according to either of claims 1 or 2, wherein the amount of ffDNA in the sample is increased.

4. The method according to claim 3, wherein the amount of ffDNA is increased by the selective amplification of one or more ffDNA fragments.

5. The method according to claim 4, wherein the amount of ffDNA is increased by the selective amplification of DNA fragments in the sample having less than 500 base pairs.

6. The method according to claim 5, wherein the amount of ffDNA is increased by the selective amplification of DNA fragments in the sample having less than 400 base pairs.

7. The method according to claim 6, wherein the amount of ffDNA is increased by the selective amplification of DNA fragments in the sample having less than 300 base pairs.

8. The method according to any of claims 4 to 7, wherein the ffDNA is selectively amplified using PCR.

9. The method according to claim 8, wherein the ffDNA is selectively amplified using real time PCR, digital PCR, multiplex PCR and/or MLPA.

10. The method according to any of claims 3 to 9, wherein one or more target regions of the fetal genome are selectively amplified by PCR employing a critical denaturation temperature which is specific to the one or more target regions.

1 1. The method according to claim 10, wherein the critical denaturation temperature is less than 90°C.

12. The method according to claim 11 , wherein the critical denaturation temperature is less than 85°C.

13. The method according to claim 12, wherein the critical denaturation temperature is less than 80°C.

14. The method according to any of claims 10 to 13, wherein the critical denaturation temperature is greater than 70°C.

15. The method according to claim 14, wherein the critical denaturation temperature is greater than 75°C.

16. The method according to either of claims 1 or 2, wherein the amount of maternal free DNA in the sample is reduced.

17. The method according to claim 16, wherein the amount of maternal free DNA is first increased, the maternal free DNA in the sample thereafter being depleted.

18. The method according to claim 17, wherein the amount of maternal free DNA is increased by the selective amplification of DNA fragments having more than 300 base pairs.

19. The method according to claim 18, wherein the amount of maternal free DNA is increased by the selective amplification of DNA fragments having more than 400 base pairs.

20. The method according to claim 19, wherein the amount of maternal free DNA is increased by the selective amplification of DNA fragments having more than 500 base pairs.

21. The method according to any of claims 17 to 20, wherein the amount of maternal free DNA is increased using PCR.

22. The method according to claim 21 , wherein the amount of maternal free DNA is increased using real time PCR, digital PCR, multiplex PCR and/or MLPA.

23. The method according to any of claims 17 to 22, wherein increasing the amount of maternal free DNA includes incorporating a moiety in the synthesised DNA to render it susceptible to degradation.

24. The method according to claim 23, wherein the moiety incorporated into the synthesised DNA renders it susceptible to degradation by an enzyme.

25. The method according to claim 24, wherein the moiety is uracil.

26. The method according to claim 25, wherein the enzyme is uracil

deglycosylase (UNG).

27. The method according to any of claims 17 to 26, wherein increasing the amount of maternal free DNA includes incorporating a compound which renders the synthesised DNA susceptible to purification.

28. The method according to claim 27, wherein the compound incorporated into the synthesised DNA labels the DNA and renders it susceptible to purification by binding to a compound which has an affinity for the compound incorporated into the synthesised DNA.

29. The method according to claim 28, wherein the compound incorporated into the synthesised DNA is biotin.

30. The method according to claim 29, wherein the compound having an affinity for the compound incorporated into the synthesised DNA is streptavidin.

31. The method according to claim 30, wherein streptavidin forms part of a streptavidin-labelled magnetic bead.

32. The method according to any preceding claim, further comprising analysing the enriched ffDNA to identify abnormalities in the fetal genome.

33. A non-invasive method for identifying genetic abnormalities in the genome of a fetus, the method comprising.

providing a sample of maternal plasma;

enriching the ffDNA content of the plasma in situ; and

34. The method according to claim 33, wherein the method for enriching the ffDNA content of the plasma is as claimed in any of claims 1 to 32.

35. The method according to either of claims 33 or 34, wherein the ffDNA is analysed to identify sequence errors or chromosomal aneuploidies.

36. An ffDNA enriched plasma sample obtainable by the method according to any of clams 1 to 32.

37. A method of diagnosing a condition in a fetus arising from a genetic abnormality, the method comprising enriching a sample of maternal plasma by a method according to any of claims 1 to 32.

38. The method according to claim 37, wherein the condition is Down Syndrome or other aneuploidies.

39. A method for processing a sample of maternal plasma substantially as hereinbefore described, having reference to any one of Figures 1 to 14.