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WO2012114075A1 - Procédé de traitement d'adn maternel et fœtal - Google Patents

Procédé de traitement d'adn maternel et fœtal Download PDF

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WO2012114075A1
WO2012114075A1 PCT/GB2012/000191 GB2012000191W WO2012114075A1 WO 2012114075 A1 WO2012114075 A1 WO 2012114075A1 GB 2012000191 W GB2012000191 W GB 2012000191W WO 2012114075 A1 WO2012114075 A1 WO 2012114075A1
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ffdna
dna
maternal
sample
amount
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Tracey Elizabeth Madgett
Neil David Avent
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Plymouth University
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Plymouth University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
    • C12Q2521/531—Glycosylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • 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.
  • ffDNA cell free fetal DNA
  • NIPD noninvasive prenatal diagnosis
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NIPD fetal genetic traits, including those involved in chromosomal aberrations, such as Down syndrome is possible.
  • this method involves two separate stages and therefore unnecessarily complicates and lengthens the diagnostic procedure.
  • 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.
  • 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.
  • WGA whole genome amplification
  • 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.
  • ffDNA is hypomethylated in comparison with maternal DNA
  • selective amplification of ffDNA specific sequences is achievable using a methylation sensitive enzyme.
  • 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.
  • the maternal DNA is amplified at a slower rate to the ffDNA, thereby increasing the concentration of ffDNA relative to maternal DNA.
  • 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.
  • 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.
  • 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.
  • the enriched ffDNA product may be assessed using known methods.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the amplification of the ffDNA may be conducted to selectively target and amplify selected regions of the fetal genome. It is
  • the simultaneous amplification of polymorphic regions of chromosomes 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.
  • 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.
  • 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.
  • 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.
  • one such compound is streptavidin.
  • 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.
  • 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.
  • the smaller ffDNA fragments remain unamplified, as it is unlikely that both PCR primers will find their target sites on the ffDNA fragments.
  • 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 enrichment of ffDNA relative to free maternal DNA in the sample may be employed as part of a diagnostic regime.
  • the present invention provides a non-invasive method for identifying genetic abnormalities in the genome of a fetus, the method comprising:
  • 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.
  • the present invention provides a ffDNA enriched plasma sample obtainable by the method as hereinbefore described.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • each 25uL PCR reaction consisted of: 1x Mastermix containing polymerase, dNTPs, buffer and MgCI 2 (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.
  • FIG. 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.
  • FIG. 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).
  • FIG. 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.
  • FIG. 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
  • 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.
  • 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:
  • 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
  • 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
  • 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
  • FIG. 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.
  • FIG 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.
  • FIG. 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.
  • FIG. 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.
  • '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.
  • FIG. 13 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
  • Figure 13b shows the traces following a PCR run with a denaturation temperature of 80 o C.
  • 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.
  • Chromosome 21 Similar work has been carried out for Chromosome 18 and Chromosome 13, with analogous results.
  • 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.
  • RHDEX7 GGGTGTTGTAACCGAC3TGCTGGG ( 3ATTCCCCACAGCTCCATCATGGGCTACAACTTCAGC : 6
  • RHCEE 7 GTGTGTTGTAACCGAGTGCTGGGGATTCACCACATCTCCGTCATGCACTCCATCTTCAGC : 6
  • RHCEEX7 AACGGCAATGGCAT : 134
  • 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.
  • 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.

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Abstract

Cette invention concerne un procédé de traitement d'un échantillon de plasma maternel comprenant de l'ADN maternel libre et de l'ADNff, le procédé comprenant l'enrichissement in situ de la quantité d'ADNff par rapport à la quantité d'ADN maternel libre. Dans un mode de réalisation, la quantité d'ADNff dans l'échantillon est augmentée, par exemple, par des procédés de PCR. Dans un autre mode de réalisation, la quantité d'ADN maternel libre dans l'échantillon est réduite, en particulier, par amplification sélective, par exemple, par des procédés de PCR, et déplétion sélective. Le procédé est particulièrement utile pour détecter des anomalies génétiques chez le fœtus, en particulier, le syndrome de Down et autres aneuploïdies.
PCT/GB2012/000191 2011-02-25 2012-02-24 Procédé de traitement d'adn maternel et fœtal Ceased WO2012114075A1 (fr)

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CN103215350B (zh) * 2013-03-26 2016-11-02 苏州贝康医疗器械有限公司 一种基于单核苷酸多态性位点的孕妇血浆中胎儿dna含量的测定方法
CN104846089A (zh) * 2015-05-06 2015-08-19 厦门万基生物科技有限公司 一种孕妇外周血中胎儿游离dna比例的定量方法
CN111433855A (zh) * 2017-07-18 2020-07-17 康捷尼科有限公司 筛查系统和方法
CN115992202A (zh) * 2021-10-18 2023-04-21 深圳华大基因股份有限公司 提高孕妇外周血中胎儿游离dna浓度的方法及其应用

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