Patent Application
20130102490
The present invention relates to a method for counting the copy number of a nucleic acid sequence in a cell, for example a single cell. The method may be used for counting the copy number of a chromatid in a cell. The ploidy status of the cell may be investigated by counting the copy number of chromatids for each chromosome in the cell.
In vitro fertilisation (IVF) is a process by which egg cells are fertilised by sperm in vitro and the resultant zygote transferred to the patient's uterus with the intent to establish a successful pregnancy. The first human baby resulting from an IVF procedure was born in 1978, and since then IVF has become a major treatment for infertility when other methods of assisted reproductive technology have failed.
Despite the fact that IVF procedures are now relatively routine in many countries, clinical pregnancy rates and baby take home rates after IVF are still poor. Chromosomal abnormalities, which usually cause miscarriage, result predominantly from anomalies during female meiosis. A major factor is advanced maternal age and its impact on the quality of the oocyte. It is known that the decreasing fertility of older women is mainly caused by age-dependent increases of aneuploidies in oocytes (and embryos). Selection of euploid oocytes is thus an attractive strategy to increase the number of live births following IVF procedures.
The ploidy status of oocytes can be indirectly investigated by analysing the chromosome content in polar bodies (PB) I and II. Polar bodies are results of the first and second meiotic division before and after fertilisation (see
Errors in meiotic divisions occur frequently and increase with maternal age; mechanisms are chromosome non-disjunction and early sister chromatid separation with higher frequency in meiosis I. Depending on the mechanism of malsegregation various chromosomal constellations can occur in oocyte and PB as exemplified for meiosis I (see
At a slightly lower frequency, errors occur also during meiosis II due to non-disjunction and chromatid malsegregation. In order to provide a true picture of the chromosome content of the ooctye, ideally one would need to investigate the chromosome content of PB I and II for all chromosomes at the resolution of chromatids.
Although preimplantation genetic diagnostic (PGD) procedures are known, all are associated with shortcomings. Fluorescence in situ hybridisation (FISH) is sometimes used with different colour fluorescence for each chromosome. So far, this technique has been used with a maximum of 12 chromosomes. As only a subset of chromosomes is investigated, this leaves non-stained chromosome aneuploidies undetected. Array-based methods have also been used, but they have a sensitivity which does not always resolve below the chromosome level, meaning that they may not detect sister chromatid malsegregation which can occur in both meioisis I and II leading to aneuploid embryos. Moreover the array-based methods take at least 48 hours, thus making embryo freezing and implantation in a consecutive cycle necessary.
There is thus a need for improved methods for investigating the ploidy status of oocytes.
The present inventors have developed a method which determines the absolute copy numbers of nucleic acid sequences, such as genomic markers, within a single cell. The copy numbers of nucleic acid sequences may, for example, represent the total number of each type of chromatid in the cell.
The method has been validated by chromatid counting in a haploid polar body and a diploid fibroblast at telophase, to assess the number of chromatids and through this the ploidy status of such single cells.
Thus in a first aspect, the present invention provides a method for counting the absolute copy number of a nucleic acid sequence in a cell, which comprises the following steps:
In step (i), the lysate may be divided into at least 8 aliquots per cell used to make the lysate. Where the cell is diploid, the lysate may be divided into at least 16 aliquots per cell.
Where a sample of the cell is used in step (i) it may comprise 10 cells or fewer. In order to work out the copy number of the nucleic acid, it is necessary to know the exact number of cells. In one embodiment, a single cell is lysed to provide the lysate of step (i). An advantage of using a single cell is that it avoids any inaccuracy associated with obtaining the cell number. Page: 3 Another advantage is that it determines copy-number unambiguously for that cell; with two or more cells, the total number of copies may be known, but there is no guarantee that all the cells have the same copy-number.
In a second aspect, the present invention provides a method for counting the absolute copy number of a chromatid in a cell by counting the copy number of one or more nucleic acid marker(s) unique to the chromatid using a method according to any preceding claim.
The copy number of a plurality of nucleic acid markers from the chromatid may be determined in order to analyse multiple loci on each chromatid. The plurality of nucleic acid markers may comprise one or more pairs or multiples of markers which occur in close proximity on the chromatid. This helps to monitor for PCR failure due to “allele dropout” (see below).
It is theoretically possible for sister chromatids to be apportioned to the same aliquot (co-segregate) which may lead to an underestimation of the chromatid number. Such errors can be overcome by analysing a plurality of markers for a given chromosome. Since the chromosomes break upon isolation, the markers segregate independently, so it is unlikely that co-segregation of one marker will occur at the same time as co-segregation of another marker, provided that the markers are far apart on the chromosome. In connection with this embodiment, the plurality of nucleic acid markers may comprise markers which occur far apart on the chromatid.
Where the method comprises analysis of a plurality of markers, the highest number indicated gives the absolute copy number of the nucleic acid in the cell. Markers which give a number lower than this maximum may represent an underestimate due to co-segregation and/or allele drop-out. These lower numbers can therefore be ignored.
The most frequent aneuploidies in humans are trisomy 21, 18 and 13. Hence, the method of the invention may involve counting the copy number of chromatids from one or more chromosomes 21, 18 or 13.
The method may count the absolute copy number of a plurality of chromatids in the cell, for example it may count the chromatids from at least 3 chromosomes such as chromosomes 21, 18 and/or 13.
In a third aspect, the present invention provides a method for investigating the ploidy status of a cell, by counting the absolute copy number of chromatids for each chromosome in the cell by a method according to the second aspect of the invention.
The “cell” may be a cell structure such as a polar body.
The cell may be derived from a cleavage stage embryo.
The cell may be a trophectoderm cell of a blastocyst.
The cell may be a fetal cell, for example from an amniotic fluid or a chorionic villus sample.
The cell may be in telophase.
In a fourth aspect, the present invention provides a method for counting the copy number of a chromatid in an oocyte, which comprises the step of counting the copy number of the chromatid in the oocyte-associated cell body by a method according to the second aspect of the invention and directly deducing the copy number of the chromatid in the oocyte.
In a fifth aspect, the present invention provides a method for investigating the ploidy status of an oocyte by investigating the ploidy status of the oocyte-associated polar body by a method according to the third aspect of the invention and directly deducing the ploidy status of the oocyte.
The oocyte may be from a human subject of 35 years or older. The oocyte may be from a human subject (of any age) who has fertility problems or has or carries an inheritable disease. The oocyte may be from a human subject undergoing IVF treatment.
In a sixth aspect, the present invention provides a method for in vitro fertilisation of an oocyte, which comprises the step of selecting an oocyte determined to be euploid by a method according to the fifth aspect of the invention.
The ploidy status of both polar body I and polar body II may be investigated.
In a seventh aspect the present invention provides a method for investigating the ploidy status of an embryo by investigating the ploidy status of an embryo-derived cell(s) by a method according to the fifth aspect of the invention.
In an eighth aspect, the present invention provides a primer set for use in a method according to the second aspect of the invention, which comprises a plurality of primers capable of amplifying a plurality of nucleic acid markers from a chromatid.
The set may comprise primers capable of amplifying one or more nucleic acid markers from a chromatid from each chromosome in the cell.
The set may comprise primers to amplify at least four nucleic acid markers per chromatid.
The set may comprise one or more primer(s) capable of amplifying or detecting a disease-specific gene, allele or mutation.
The set may comprise primers capable of amplifying one or more pairs or multiples of nucleic acid markers which occur in close proximity on the or each chromatid and/or primers capable of amplifying one or more pairs or multiples of nucleic acid markers which occur far apart on the or each chromatid.
As the method of the invention counts chromatids directly, this system is the only technique to date that allows detection of all kinds of malsegregation of chromosomal material for all chromosomes. It is thus the only technique which provides full and accurate information on the ploidy status of a cell.
Other major advantages of the method include the following:
After homologous chromosome synapsis and initiation of recombination, meiosis arrests in the first meiotic prophase and is only resumed at ovulation. After completion of meiosis I the oocyte undergoes meiosis II and arrests in metaphase. If no fertilisation takes place the oocyte is degraded; if fertilised meiosis II is completed.
A normal meiotic division results in the segregation of two homologous chromosomes with 2 chromatids each (euploidy). In the case of chromosome non-disjunction both homologous chromosomes segregate to the same pole leading to either quatrosomy or nullisomy in the oocyte. The other frequent mechanism is early sister-chromatid separation leading to either trisomy or monosomy in the oocyte.
PB I is lysed and the cell lysate is dispensed over 8 PCR reaction wells (aliquots), leading to single DNA molecules at limiting dilution with 0.25 genomes per PCR well in the case of euploidy. After 2 rounds of specific PCR amplifications the number of chromatids per chromosome is analysed by simply counting the numbers of positive PCR reactions representing target sequences on all chromosomes. In this example, the DNA content is divided into only 8 aliquots, raising the possibility that two chromatids may occasionally co-segregate (ie, be apportioned to the same aliquot) and be mis-counted as one. Such errors can be overcome either by dividing the sample into more aliquots (reducing the chances of co-segregation), or by analysing multiple markers scattered along each chromosome (since the chromosomes break upon isolation, so that the markers segregate independently and hence co-segregation of two copies of one marker will not occur at the same time as co-segregation of two copies of another marker).
PB I is expected to contain 2 copies for all chromosomes and was diluted into 8 aliquots which equals an average DNA content of 0.25 genomes per aliquot. The 4 markers analysed per chromosome were not linked but rather in distances of several megabases. As the primer panel used for this experiment had not been optimised there are several markers which did not work at all or were not robust in consecutive analyses; they are indicated by omission of the primer name. In cases of a missing result in the presence of the proper primer name allele drop out has occurred which is the case for markers 7, 19, 28, 30, 37, 38, 39, 45, 57, 69, 76 and 82. Markers 93-96 cannot be judged as no Y chromosome is present in polar bodies.
The cell was expected to contain 4 copies for all autosomes and 2 copies for chromosomes X and Y and was diluted into 16 aliquots which equals 0.25 genomes per aliquot for the autosomes and 0.125 genomes for the sex chromosomes. The markers used here were linked with 24 markers per chromosome, the chromosomes being chromosomes 10, 21, X and Y. The furthest column to the right gives the counts of positive PCRs per marker, green fields being in accordance with the expected numbers of positives. Again this marker panel was not optimised but demonstrates that the presence of chromatids can be verified. The shift of counts from 4 to 2 nicely reflects the reduction of chromatids from 4 to 2 as from autosomes to sex chromosomes. Moreover linkage can be observed along the markers showing that the DNA strands are intact over several kilobases. Use of a robust primer set with closely linked markers allows one to estimate how much allele drop out occurs, by observing linkage.
(a). Examples of euploid chromosomes.
(b). Example of euploid chromosome 14 and aneuploid chromosome 15 due to a meiosis II error.
(c). Meiosis I error resulting in a trisomy of the zygote.
(d). Repair of a meiosis I error with resulting euploidy.
In this scheme a PB1 has been analysed with markers on selected chromosomes. Markers are composed of 2×4 clustered markers per chromosome thus analysing 2 independent regions per chromosome at a redundacy of 4. Blue boxes indicate the PCR aliquot with a positive PCR, numbers within the boxes are the melting temperatures of the PCR products which are specific for each marker. With our lysis protocol DNA molecules have a length of several kb thus resulting in good linkage patterns. PCR products marked orange are judged as false positives as DNA from external contamination is more fragmented therefore giving the random odd additional signal. In this analysis there is only one marker with a false too low result—the forth marker on Xp. The linkage pattern clearly indicates that it has to be ADO as all other markers give 2 signals in identical PCR aliquots.
A combination of independent and linked markers distributed along all chromosomes should provide sufficient redundancy to compensate for signal loss due to DNA fragmentation, ADO and cosegregation. Each block of markers (brown and yellow) represents linked markers with distances of 500-1000 bp interrogating 6 independent regions with 2 (brown) and 4 (yellow) markers per region, each marker confirming the result of the other markers per region.
In a first aspect, the present invention provides a method for counting the copy number of a nucleic acid sequence in a cell.
The copy number is the number of copies of the nucleic acid sequence in the genome of the cell.
The method comprises the steps of
The number of aliquots which test positive give an absolute number for the copy number of nucleic acids in the cell. For example, if a single cell is lysed and the lysate split into multiple aliquots, two of which test positive by polymerase chain reaction (PCR-see below), it can be directly deduced that the cell contained two copies of the nucleic acid. For a single cell, the number of positive wells equates with the copy number of the nucleic acid, assuming there is no co-segregation, which is explained in more detail below.
It is possible to perform the method using more than one cell, as long as the exact number of cells in the sample is known or can be derived. For example, if two cells are lysed and the lysate split into multiple aliquots, four of which test positive by PCR, it can be directly deduced that the cells each contain two copies of the nucleic acid. The copy number of the nucleic acid per cell may be directly calculated by dividing the number of aliquots which test positive with the number of cells in the sample.
WO 2007/129000 describes a method of measuring the copy number frequency of one or more nucleic acids in a sample by comparing the frequency with which PCR amplification occurs of a) a test marker and b) a reference marker at limiting dilution.
In the method of WO 2007/129000 the objective is to discover the average number of copies of a given marker in a population of cells (typically at least ten cells). Using this method, one arrives at an estimate of mean copy-number by statistical methods. The amount of DNA per aliquot is chosen such that a large proportion (typically 50%) of aliquots are positive for the marker sequence leading to a high rate of co-segregation, and the results are deconvoluted statistically. In the method of present invention, on the other hand, the amount of DNA per aliquot is ideally small enough that co-segregation is rare; and rather than derive a statistical estimate of copy-number, the method provides an exact copy-number for a given nucleic acid in a cell.
The method of WO 2007/129000 uses processed genomic DNA, produced by a method involving cleaning steps. By contrast, in the method of the present invention, the total cell content plus lysis buffer is put into the PCR reaction as any cleaning step would be likely to cause a loss of material, i.e. loss of DNA.
In the method of the present invention it is possible that two copies of a given target sequence (“marker”) may happen to fall into the same aliquot as the DNA is divided (ie, they may “co-segregate”). Since PCR detects only the presence or absence of the marker in an aliquot, such instances lead to an under-counting of the copies of that marker. Such co-segregation, and hence under-counting, is statistically simple to predict and to take into account.
Errors arising from co-segregation can be reduced by splitting the DNA into more aliquots, so that co-segregation becomes less likely.
The cell lysate may be split into at least 5, 10, 15, 20 or more aliquots.
Each aliquot may have an average of 0.25 genomes per aliquot or less, for example 0.20, 0.15 or 0.1 genomes per aliquot or less.
Alternatively, or in addition, errors arising from co-segregation can be reduced by analysing multiple markers within the same nucleic acid sequence.
For example, where the method of the present invention is used for chromatid counting, chromatids break upon extraction, so that if multiple markers are used, they behave independently especially if they are far enough apart on the chromatid. Thus, whilst two copies of one chromatid marker may co-segregate and lead to an underestimate of chromatid number in that cell, two copies of another marker on the same chromosome may not. Where multiple markers are used in this way, the true chromatid number of the cell is the highest number indicated by any of the markers.
Errors may also arise due to PCR failure (“allele dropout”). This can be addressed by selecting markers known to amplify efficiently, by using multiple markers on each chromosome, and/or by using pairs of markers which are nearly adjacent on the chromosome. In this last case, one would expect both members of a pair to co-segregate (since the DNA is unlikely to break in the very small interval between them); failure of co-segregation of such paired markers would be indicative of PCR failure. The same approach can be extended to use triplets (or more) of markers in the same way.
It is difficult to rule out undesired co-segregation and allele dropout completely. However, they can be kept within manageable limits, and their frequency can be either predicted (co-segregation) or monitored (allele dropout). By analysing multiple loci on each chromosome, one can obtain a nucleic acid copy number and a measure of confidence in that number.
The term “nucleic acid” as used herein refers to a deoxyribonucleotide or ribonucleotide in either single or double-stranded form.
The nucleic acid may be genomic DNA.
The nucleic acid may be part of a chromatid or a chromosome.
A chromatid is one of the two identical copies of DNA making up a chromosome, which are joined at their centromeres. When the centromeres separate (during anaphase of mitosis and anaphase 2 of meiosis), the two strands are called sister chromatids.
The chromatid may be from a chromosome which is commonly associated with aneuploidy, such as chromosomes 21, 18 and 13.
In addition to counting chromatids, the method of the invention may be used for many other applications which involve a copy number change, for example nonreciprocal translocations, deletions or trinucleotide repeat disorders. It is even possible to detect reciprocal translocations and inversions by using linked markers spanning the breakpoints.
The cell under investigation using the method of the present invention may be a haploid or diploid cell.
The cell may be derivable from a cell sample such as a blood, plasma, serum, saliva, urine, tears, tissue, lymph, or tumour sample.
The cell may be a gamete such as an oocyte or a sperm cell.
The “cell” may be a cell structure such as a polar body.
Asymmetrical cell division (cytokinesis) leads to the production of polar bodies during oogenesis.
There may be one or two polar bodies in the oocyte. The first polar body is one of the two products after completion of meiosis I and may be considered haploid, with 23 duplicated chromosomes in humans (one of each pair of homologous chromosomes). The second polar body is also haploid, with 23 unduplicated chromosomes. Both are relatively small and contain little cytoplasm.
Polar bodies are the by-products of the egg's division during meiosis. As an egg matures, it goes through a two-step division process, dividing once at the time when ovulation would occur and again at the time of fertilization. The two haploid polar bodies are the by-products of this division, and are essentially discarded by the egg. By analyzing the polar bodies, it is possible to infer the genetic status of the egg, as shown in
The cell may be derivable from a pre-implantation embryo. For example, the cell may be derivable from a cleavage stage embryo or from a blastocyst. The cell may be a trophectoderm cell from a blastocyst.
The cell may be derivable from a post-implantation embryo. For example, the cell may be an embryonic cell derivable from an ongoing pregnancy, such as a cell from an amniotic fluid or chorionic villus sample.
The oocyte or embryo may be from or for a female subject who has one or more of the following:
The female subject may be about to undergo IVF treatment or may have an ongoing pregnancy as a result of IVF treatment. The IVF treatment may involve single embryo transfer.
The cell may be at telophase. Telophase is the final stage of both mitosis and meiosis, when a new nuclear envelope forms around each set of chromosomes and both sets of chromosomes unfold back into chromatin. The distinguished shape of cells in telophase allows for the selection of single cells at a defined chromosome status, i.e. all chromosome pairs in metaphase with 2 chromatids each, giving 4 copies.
As mentioned above, it is possible to perform the method of the invention with a plurality of cells, as long as the number of cells is known or can be derived.
The cell sample may have 10 or fewer, 5 or fewer, 3 or 2 cells.
The number of cells in the cell sample may be counted or derived by methods known in the art. For example FACS sorting may be used, or cell may be collected, for example with a micropipette, and directly counted under a microscope using visual control.
The method of the invention may also be used to investigate single gene defects and for mutation screening in the cell. The method of the invention is highly flexible when it comes to the composition of amplification primers, and so primers may be included which amplify disease specific genes or alleles to allow assessment of disease risk. A non-exhaustive list of such single gene disorders is given in Table I.
Disease risk of the maternal genomic content may be investigated in the case of PB diagnosis, whereas that of both maternal and paternal genomic content may be investigated if embryo or trophectoderm biopsies are performed.
As used herein, “amplification” refers to any process for multiplying strands of nucleic acid, such as genomic DNA, in vitro.
Amplification techniques include thermal cycling amplification methods, such as ligase chain reaction; and isothermal amplification methods, such as Strand Displacement Amplification (SDA), Q-beta replicase, nucleic acid-based Sequence Amplification (NASBA); and Self-Sustained Sequence Replication.
The amplification method may be polymerase chain reaction (PCR). PCR involves using paired sets of oligonucleotides of predetermined sequence that hybridise to opposite strands of DNA and define the limits of the sequence to be amplified. The oligonucleotides prime multiple sequential rounds of DNA synthesis catalysed by a thermostable DNA polymerase. Each round of synthesis is typically separated by a melting and re-annealing step, allowing a given DNA sequence to be amplified several hundred-fold in less than an hour.
The amplification step may be automated, making the method suitable for use in high-throughput screening techniques.
The nucleic acid sequence whose copy number is being determined may be a “marker” for a longer nucleic acid sequence. For example, it may be a marker for a section of genomic DNA, a chromatid or a chromosome.
For chromatid counting, the method may be used to count the number of a plurality of markers for each chromosome. This provides an internal cross-reference for the correct copy number for the chromatid. For the reasons explained above (co-segregation and allele drop-out), a given marker may produce an underestimation for the copy number. If a plurality of markers is used, this can be checked. The marker(s) giving the highest copy number (assuming there is no PCR contamination) can be assumed to give the correct number.
To check for and take steps to avoid errors due to co-segregation, markers may be chosen which are spaced far apart on the chromatid. For example, the markers may be separated by at least 500 kb, at least 1 Mb, at least 3 Mb or at least 5 Mb.
To check for and take steps to avoid errors due to allele drop out, markers may be chosen which amplify nucleic acids in close proximity on the chromatid. For example, the nucleic acids may be spaced by less than 2 kb, for example between 50 and 500 bp.
The marker nucleic acid sequence may be any length that is amplifiable by the chosen method. A disadvantage of using very long marker sequences is that the likelihood of allele drop out is increased. Typically marker sequences are chosen which are 75-130 bp in length.
Ploidy corresponds to the number of chromosomes in a cell. In humans, somatic cells are diploid, containing two complete sets of chromosomes, one set derived from each parent; and gametes are haploid.
The number of chromosomes in a single non-homologous set is called the monoploid number (x). The haploid number (n) is the number of chromosomes in a gamete of an individual. Both of these numbers apply to every cell of a given organism. For humans, x=n=23; a diploid human cell contains 46 chromosomes: 2 complete haploid sets, or 23 homologous chromosome pairs (for a female; a male has 22 homologous chromosome pairs, one X and one Y chromosome).
Euploidy is the state of a cell or organism having an integral multiple of the monoploid number. For example, a human cell has 46 chromosomes, which is an integer multiple of the monoploid number, 23. Aneuploidy is the state of not having euploidy. In humans, examples include having a single extra chromosome (such as Down syndrome), or missing a chromosome (such as Turner syndrome).
During oocyte maturation, normal division in meiosis I results in the segregation of two homologous chromosomes, one remaining in the oocyte and one extruded to the polar body, so that both the polar body and the oocyte have two chromatids each (euploidy). If an error occurs, the sharing of chromatids between oocyte and polar body may be unequal, leading to aneuploidy in both the polar body and the oocyte (see
Using the method of the invention, it is possible to investigate the ploidy status of a cell or polar body for one or more chromosomes. The method may be used for all 22 chromosomes together with X and (if appropriate) Y, producing a complete picture of the ploidy status of the cell.
The fifth aspect of the present invention relates to a primer set which comprises primers capable of amplifying a nucleic acid in accordance with step (ii) of the method of the first aspect of the invention.
The term “primer” is used herein interchangeably with “oligonucleotide” to mean a short length of nucleic acid which hybridises specifically to a target sequence enabling the nucleic acid sequence whose copy number is to be determined (i.e. the marker sequence) to be amplified.
The primers may be capable of hybridising at flanking regions of the nucleic acid marker sequence. The primers are chosen to have at least substantial complementarity with the different strands of the nucleic acid being amplified.
The primer must have sufficient length so that it is capable of priming the synthesis of extension products. The length and composition of the primer depends on many factors including, for example, the temperature at which the annealing reaction is conducted, concentration of primer and the particular nucleic acid composition of the primer. Typically the primer has 15-30 nucleotides, such as 18-20 bp.
The term “hybridise specifically” refers to hybridisation of the primer to the target sequence under stringent conditions, that is conditions under which a primer will hybridise preferentially to its target sequence and to a lesser extent to, or not at all to, other sequences.
The primer set may comprise two primers for each marker sequence: one “forward” and one “reverse” primer. Alternatively the primer set may comprise three primers in a hemi-nested configuration.
The set may comprise primers capable of amplifying one or more nucleic acid markers from a chromatid. The set may comprise primers capable of amplifying a plurality of nucleic acid markers from a chromatid. For example, the set may comprise primers capable of amplifying at least 4, 6, 8, 10, 15, 20, 25 or more markers for the chromatid or for each chromatid.
The set may comprise primers capable of amplifying one or more nucleic acid markers from a plurality of chromatids in the cell. For example, the set may comprise primers capable of amplifying markers from at least 3, 5, 8, 12 or 15 chromosomes. The set may comprise primers capable of amplifying markers from each chromosome in the cell.
The set may comprise one or more primer(s) capable of amplifying or detecting a disease-specific gene, allele or mutation.
The set may comprise primers capable of amplifying one or more pairs or multiples of nucleic acid markers which occur in close proximity on the or each chromatid and/or primers capable of amplifying one or more pairs or multiples of nucleic acid markers which occur far apart on the or each chromatid.
The primer set may be provided as part of a PCR kit, which may also contain deoxynucleotide triphosphates and/or Taq polymerase.
The kit may also comprise one or more container(s) and instructions for use.
As the method is highly suited for automated methods, such as high-throughput screening, the primer set may be provided as part of a multi-well plate, such as a 96-well plate, each well being ready to receive and aliquot of lysate.
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.
The ploidy status of an oocyte was ascertained by investigating the ploidy status of polar body I (PBI) using the chromatid counting method of the invention with four markers per chromosome.
The polar body was lysed and dispensed into 8 aliquots. PBI is expected to contain 2 copies for all chromosomes, so each aliquot comprises an average DNA content of 0.25 genomes per aliquot.
As shown in
A diploid fibroblast at telophase is expected to contain 4 copies of each autosome and 4 copies of X in females; or 2 copies of X and two copies of Y in males.
A fibroblast at telophase was selected due to its distinguished shape, lysed and divided into 16 aliquots. As for example 1, this gives an average of 0.25 genomes/aliquot for the autosomes and X in the female fibroblast and 0.125 genomes/aliquot for X and Y in the male fibrobast. Linked markes are used for four chromosomes: namely chromosomes 10, 21, X and Y.
As shown in
This is the first time that the chromosome content of a single cell has been resolved at the chromatid level allowing one to detect directly not only chromosome disjunctions for all chromosomes but also early sister-chromatid separation.
After correct meiosis I and II polar body I (PB1, PB2) contains 2 copies for all chromosomes while PB2 contains I copy. This is shown for chromosomes 17, 18 and 21 with a set of 12 markers per chromosomes with 2 linked groups of 6 markers (
It was shown that while meiosis I and II (MI and MII) were correct for chromosome 14, a MIT error occurred after correct MI for chromosome 15 (
Due to premature sister chromatid separation at meiosis I, only one chromatid of chromosome 17 segregated into PB1 (
(d) Repair of a Meiosis I Error with Resulting Euploidy.
No mistake was detected for chromosome 10 where PB1 has 2 positive PCRs for 4 markers and PB2 1 positive PCR. For chromosome 16 it was found that the opposite is the case: only 1 signal in PB1 and 2 signals in PB2 (
The polar body is deposited in 30 μl of distilled water, frozen and kept until analysis at −20° C. or lower. The first step for single cell MCC is cell lysis and DNA preparation in a system approximating a closed system such that no material is taken from the original vial in which the PB is stored. 10 μl cell lysis buffer is added to the tube containing Triton X-100 (2%, 0.1% final concentration) Tween 20 (2%, 0.1% final concentration) and Proteinase K (20 μg/μl, final concentration 0.25 μg/μl), briefly mixed, overlayed with oil and incubated at 50° C. over night. Cell lysats (40 μl) are dispensed into 8×5μl aliquots, overlayed with oil and proteinase K is heat inactivated by incubation at 95° C. for 5 minutes.
Amplification with Seminested PCR
The protocol is similar to the one described in WO2007/129000 for MCC with genomic DNA. This method has been proven to be robust and to allow multiplexing at very high levels. The following represents a typical protocol; precise conditions (number of multiplexed markers; precise volumes and thermocycling conditions, etc) may be varied as appropriate.
The first round of PCR analysis is a multiplexed amplification step for each PCR well (i.e. aliquot) with all pooled outer primers in each PCR well, so that all copies of any target sequence are amplified to some extent. 5μl mastermix for the multiplex first round PCR is added and thermocycling is carried out with hot start at 93° C. for 9 min, followed by 25 to 50 cycles of 20s at 94° C., 30s at 50° C. and 1 min at 72° C.
The second round of PCR uses the product of the phase 1 multiplex PCR at a dilution of 1:100 in water as a template to amplify individual marker sequences on each chromosome as semi-nested PCR with internal forward and reverse primers in a volume of 10 μl. Thermocycling under oil is carried out with hot start at 93° C. for 9 min, followed by 33 cycles of 20s at 94° C., 30s at 52° C. and 1 min at 72° C. Prior to PCR analysis on 108-well horizontal 6% polyacrylamide gels 8 μl 2× loading buffer (15% w/v Ficoll, 0.1 mg/ml bromophenol blue, 4×SyBr Green I) is added and gels are run at 10V/cm for 10 min digital PCR analysis is performed by scoring presence or absence of PCR product in each sample.
The second round of PCR and digital PCR read out has been automated as melting curve analysis on the BioMark system of Fluidigm company. This system has proven most suitable and convenient as it provides the following set up:
(i) PCRs are run on a 96×96 well chip, which allows amplification of 96 DNA templates with 96 primer pairs. PCR run time is short (2.5 hours) and need of reagents is minute as PCRs are run in a 5 nanoliter scale;
(ii) digital PCR read out can be performed by melting curve analysis on the chip on the same platform within 45 minutes and results can be exported into excel databases which can be easily analysed; and
(iii) the automation procedure meets one important requirement for PB diagnosis, which is that the time for analysis should be as short as possible.
Primers are selected using various criteria after masking repetitive elements from the human genomic sequence (Ensembl database, NCBI release 37, retrieval of masked sequence; http://www.ensembl.org). Amplicon length of the external products is a maximum of 120 bp and the internal product between 75 and 100 bp. Amplicons were located such that they build two triplets (see above, under “Statistical considerations and error avoidance”) of linked markers per chromosome; on metacentric chromosomes 1 cluster on the short arm and 1 cluster on the long arm of the chromosome, in the case of acrocentric chromosomes the clusters were situated proximal and distal to the centromere. All primer sets were checked electronically against the reference genome to ensure that they were predicted to give unique products (http://www.ncbi.nlm.nih.gov/projects/e-per). Typically primer length is 18-20 bp with melting temperature of 52-60° C. Design requires at least two guanine or cytosine bases at the 3′ end and at least one at the 5′ end.
As many as 1200 primers have been multiplexed with robust results (Eichinger et al. (2005) Nature. May 5; 435(7038):43-57) therefore a marker set for an all-chromosomes-screen can easily enlarged by addition of more primers for disease specific sequences and mutations.
The primers used in this study are listed in Table 2 (see Appendix I). In this Table: Fex=external forward primer; Fin=internal forward primer; and Rvs=reverse primer.
The fibroblasts were remaining amniocytes after karyotyping. Fibroblasts at telophase were picked with a micropipette under a light microscope with 200× magnification.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1007522.4 | May 2010 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2011/051979 | 5/4/2011 | WO | 00 | 1/9/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61395414 | May 2010 | US |
