PRIORITY CLAIM

This application claims priority to Australian provisional patent application 2009904275, filed 7 September 2009, the content of which is hereby incorporated by reference. FIELD

The present invention relates generally to methods for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid. The present invention also contemplates amplification of the extracted nucleic acids, particularly whole genome amplification of the extracted nucleic acids.

BACKGROUND

Whole Genome Amplification (WGA) may be used to amplify a genomic DNA template to generate amplicons that are representative of the genomic DNA template. In some instances, WGA is used to amplify rare and/or low concentration genomic DNA templates, thus enabling a range of genotyping techniques that could not otherwise be performed. For example, WGA may be used to amplify genomic DNA derived from single cells for comparative genomic hybridisation.

Many approaches to WGA have been described and can be generally classified as PCR-based or strand displacement amplification techniques.

Regardless of the methodology, for successful WGA, the amplicons generated by WGA must be representative of the original genomic DNA template. However, many WGA methodologies generate amplicons that have significant amplification bias and/or poorly represent or misrepresent the original genomic DNA. These problems limit the usefulness of WGA in methods such as comparative genomic hybridisation. Thus, it would be desirable to reduce amplification bias in WGA and/or to make WGA amplicons more representative of the original genomic DNA.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

SUMMARY

In a first aspect, the present invention provides a method for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid, the method comprising the steps of:

lysing the one or more cells in the sample to release the nucleic acid from the one or more cells into a supernatant; and

contacting the supernatant containing the nucleic acid with a protease under conditions suitable for the protease to digest one or more proteins associated with the nucleic acid.

Digestion of one or more proteins associated with the nucleic acid is beneficial for further processing of the extracted nucleic acid. For example, digestion of one or more proteins associated with the nucleic acid may lead to higher quality amplicons being generated from amplification of the extracted nucleic acid and/or improved hybridisation of the extracted nucleic acid to a probe or primer.

In some embodiments, the method of the present invention is adapted to the extraction of a nucleic acid from a single cell. In a second aspect, the present invention also provides a method for amplifying a nucleic acid, the method comprising:

extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid using the method of the first aspect of the invention; and amplifying the extracted nucleic acid.

In some embodiments, the amplification comprises whole genome amplification. In whole genome amplification methods, nucleic acids extracted according to the first aspect of the invention have been demonstrated to lead to the generation of amplicons having a smaller size and which are more representative of the original genomic DNA.

In some embodiments, the amplicons produced according to the second aspect of the invention may be used in a comparative genomic hybridisation method. The use of amplicons produced according to the second aspect of the invention in a method for comparative genomic hybridisation can increase the sensitivity, reproducibility and/or reliability of the results generated in the comparative genomic hybridisation method.

In a third aspect, the present invention also provides a kit for performing the method of the first aspect and/or second aspect of the invention, the kit comprising a protease together with instructions for performing the method of the first aspect and/or second aspect of the invention.

DESCRIPTION

It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description. In a first aspect, the present invention provides a method for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid, the method comprising the steps of:

lysing the one or more cells in the sample to release the nucleic acid from the one or more cells into a supernatant; and

contacting the supernatant containing the nucleic acid with a protease under conditions suitable for the protease to digest one or more proteins associated with the nucleic acid.

As described later, digestion of one or more proteins associated with the nucleic acid is beneficial for further processing of the extracted nucleic acid. For example, digestion of one or more proteins associated with the nucleic acid may lead to higher quality amplicons being generated from amplification of the extracted nucleic acid and/or improved hybridisation of the extracted nucleic acid to a probe or primer, relative to an extracted nucleic acid which has not been contacted with a protease under conditions suitable for the protease to digest one or more proteins associated with the nucleic acid. As set out above, the present invention contemplates a method for extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid. However, in some embodiments, the method of the present invention is adapted to the extraction of a nucleic acid from a single cell. As such, in some embodiments, the sample comprises a single cell.

The cell or cells for use in the method may be any suitable eukaryotic or prokaryotic cell. Examples of suitable eukaryotic cells include animal, plant or fungal cells, while examples of suitable prokaryotic cells include bacterial or archaeal cells.

In some embodiments, the one or more cells comprise an animal cell. In some embodiments, the one or more cells comprise a mammalian cell. In some embodiments the one or more cells comprise a human cell.

In some embodiments, the one or more cells may be derived from a multicellular organism. In some embodiments the one or more cells may be somatic cells derived from an adult form of the organism. Methods for the isolation of various types of somatic cells from an organism are well known in the art and the present invention contemplates any such methods. In some embodiments, the one or more cells may be derived from an immature form of the organism and/or may be a germ cell. For example, in some embodiments, the cell may be a foetal cell, a cell derived from an embryo (including a blastomere) or a germ cell (including oocytes and sperm). Examples of foetal cells include a foetal cell taken from the amniotic fluid surrounding the foetus, a foetal cell taken from the maternal circulation, or a foetal cell taken from the mother's reproductive tract (eg. cervical or vaginal lavage). Foetal blood cells, unlike mature blood cells, are nucleated and may be isolated from the maternal circulation on the basis of this nucleation.

In the case of an embryonic cell, a small number of cells (usually one or two cells) may be removed from an embryo. In this procedure, one or more cells in an embryo may be removed by cleavage stage embryo biopsy. This procedure is usually performed on day 3 of development, when the embryo is at the 6-8 cell stage. The biopsy consists of two stages. The first is to make a hole in the zona pellucida that surrounds the embryo at this time, usually using acid Tyrodes solution or a non-contact laser. Once the hole is made, the cell may then be removed from the embryo.

In the case of a germ cell, for example an oocyte or sperm cell, the germ cell may be analysed directly. Alternatively, in the case of screening for maternal abnormalities, a polar body from the oocyte may be isolated.

Examples of suitable methods for the isolation of embryonic cells, foetal cells or germ cells include:

isolation of foetal cells from cervical mucous, for example as described by

Katz-Jaffe et al. (BJOG 112: 595-600, 2005);

embryo biopsy, for example as described by Le Caignec et al. (Nucleic Acids Research 34(9): e68, 2006);

polar body biopsy, for example as described by Vialard et al. (Fertility and Serility 87(6): 1333-1339, 2007);

amniocentesis and/or chorionic villous sampling, for example as described by Nagel et al. (Prenatal Diagnosis 18: 465-475, 1998); and

isolation of foetal cells from maternal circulation, for example as described by Kolialexi et al. (Prenatal Diagnosis 27: 1228-1232, 2007).

As set out above, the method of the present invention contemplates lysing the one or more cells in the sample to release the nucleic acid from the one or more cells into a supernatant. The present invention contemplates any suitable method for the lysis step. Examples of suitable cell lysis or cell disruption methods include:

osmosis based methods wherein lowering the ionic strength of culture media causes cells to swell and burst;

enzymatic methods including digestion using enzymes such as lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase and the like;

mechanical cell disruption methods including, for example, beadbeating, rotor stator processors, valve type processors, French presses and the like;

ultrasound (typically 20-50 kHz) sonication;

detergent based cell lysis including the use of nonionic or zwitterionic detergents such as CHAPS, and the Triton X series of detergents, or the use of ionic detergents such as SDS;

use of high pressure (usually nitrogen or other inert gas up to about 25,000 psi) which is rapidly released;

and the like.

In some embodiments, the cells are lysed in the absence of added protease.

In some embodiments, lysing the one or more cells in the sample comprises alkaline lysis of the one or more cells in the sample.

As referred to herein "alkaline lysis" refers to lysis of a cell in a strongly alkaline solution. Reference herein to a "strongly alkaline" solution includes, for example, a solution having a pH of greater than 7, 8, 9, 10, 11, 12 or 13. Examples of strongly alkaline solutions may include solutions of bases such as sodium hydroxide, potassium hydroxide and the like.

In some embodiments, alkaline lysis may also be performed in the presence of a detergent or surfactant.

In some embodiments, alkaline lysis may also be performed in the presence of a deprotecting agent for thiolated DNA. An example of such a deprotecting agent includes dithiothreitol (DTT). In some embodiments, the alkaline lysis comprises lysis in a buffer comprising potassium hydroxide and DTT. In some embodiments the alkaline lysis buffer comprises potassium hydroxide at a concentration of about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM or about 400mM. In some embodiments, the alkaline lysis buffer comprises DTT at a concentration of about about 25 mM, about 50 mM, about 75 mM, or about 100 mM. In some embodiments wherein alkaline lysis is used, the method further comprises a neutralisation step prior to contacting the supernatant with the protease. A range of suitable neutralisation steps would be readily ascertained by those of skill in the art. Typically, a neutralisation step might include the addition of a neutralisation solution containing a buffer (eg. Tris) to the supernatant. In some embodiments, the neutralisation solution may also include an acid (eg. HC1).

As set out above, the present invention contemplates contacting the supernatant containing the nucleic acid with a protease in order to digest one or more proteins associated with the nucleic acid.

As referred to herein, a protease may be any enzyme that conducts proteolysis. In some embodiments, proteases begin protein catabolism by hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain.

In some embodiments, the protease comprises an endopeptidase. As referred to herein, an "endopeptidase" should be understood as any protease which hydrolyses a peptide bond between nonterminal amino acids within a polypeptide or protein.

In some embodiments, the protease comprises a serine protease (or serine endopeptidase). As referred to herein, a "serine protease" or "serine endopeptidase" should be understood to include any protease or endopeptidase in which one of the amino acids at the active site is serine. Serine proteases may be grouped into clans that share homology and may be further subgrouped into families with similar sequences. The major clans found in humans include the chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans.

In some embodiments, the protease comprises a serine protease of the chymotrypsin- like clan. Three exemplary groups of serine proteases of the chymotrypsin-like clan include the chymotrypsins, trypsins, and elastases. All of these enzymes are similar in structure, although they cleave polypeptides at different sites:

Chymotrypsins cleave peptide bonds following a bulky hydrophobic amino acid residue. Preferred residues include phenylalanine, tryptophan, and tyrosine, which fit into a hydrophobic pocket in the active site of the enzyme. Trypsins cleave peptide bonds following a positively-charged amino acid residue. Instead of having the hydrophobic pocket of a chymotrypsin, there exists an aspartic acid residue at the base of the pocket. This residue can interact with positively- charged residues such as arginine and lysine on the substrate peptide to be cleaved. Elastases cleave peptide bonds following a small neutral amino acid residue, such as alanine, glycine, and valine. The pocket that is in trypsin and chymotrypsin is now partially filled with valine and threonine, rendering it a mere depression, which can accommodate smaller amino acid residues. In some embodiments, the protease comprises a trypsin.

Trypsins predominantly cleave peptide chains at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. Trypsins have an optimal operating pH of about 8 and optimal operating temperature of about 37°C. A range of exemplary trypsin proteins are described under Enzyme Commission code EC 3.4.21.4 (eg. see http://www.expasy.Org/cgi-bin/nicezyme.pl73.4.21.4).

As set out above, the method of the first aspect of the invention contemplates contacting the supernatant containing the nucleic acid with a protease under conditions suitable for the protease to digest one or more proteins associated with the nucleic acid. "Conditions suitable for the protease to digest one or more proteins associated with the nucleic acid" will be ascertainable by a person skilled in the art. By way of example, in the case of trypsins, suitable conditions may include a pH of about 8, and a temperature of about 37°C. Suitable incubation times will also be ascertained by a person skilled in the art.

In some embodiments, the method further comprises the step of inhibiting or substantially inactivating the protease at a time after contacting the protease with the supernatant. Inhibition and/or substantial inactivation of the protease may be desirable where the extracted DNA is later used in a further enzymatic process, for example DNA amplification or restriction digestion.

Any suitable method for the inhibition and/or substantial inactivation of the protease may be used. For example, organic and/or inorganic inhibitors of the enzyme may be added. Alternatively, the conditions in the supernatant may be altered to effect inhibition and/or substantial inactivation of the protease, for example, the temperature, pH, salinity or the like may be adjusted to outside the active range of the protease. In some embodiments, inhibiting and/or substantially inactivating the protease comprises the addition of a divalent cation at a concentration sufficient to inhibit and/or substantially inactivate the protease. For example, suitable divalent cations may include Mg ²⁺ and Ca ²⁺ . In some embodiments, the divalent cation may be added as a salt which includes the divalent cation, for example, Mg ²⁺ may be added in the form a magnesium salt such as MgCh.

In some embodiments, inhibiting and/or substantially inactivating the protease comprises holding the supernatant for a time and at a temperature sufficient to inhibit and/or substantially inactivate the protease. Suitable times and temperatures sufficient to inhibit and/or substantially inactivate a particular protease would be readily ascertained by a person skilled in the art. However, by way of example, temperatures of at least 50°C, 55°C, 60°C or 65°C held for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes may be sufficient to inhibit and/or substantially inactivate proteases such as trypsin.

In a second aspect, the present invention also provides a method for amplifying a nucleic acid, the method comprising:

extracting a nucleic acid from a sample comprising one or more cells containing the nucleic acid using the method of the first aspect of the invention; and amplifying the extracted nucleic acid.

"Amplifying" a nucleic acid, as referred to herein, includes any in vitro use of an enzyme to replicate a specific target nucleic acid to a level at which it may be detected. Examples of amplification include polymerase chain reaction (PCR), transcription mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), random amplification (as discussed below), various whole genome amplification methods (as discussed below) and the like.

In some embodiments, the amplification comprises random amplification.

"Random amplification" as referred to herein may include randomly primed amplification using one or more primers including a sequence of one or more random nucleotides, the sequence of random nucleotides being sufficiently long so as to enable the primer to hybridize to the target nucleic acid under selected conditions at random positions and serve as a primer for extension by a polymerase. In some embodiments, the primer may be a primer including a stretch of six or more contiguous nucleotides of random sequence. In some embodiments, random amplification may also be achieved with one or more primers of defined sequence, but wherein at least some cycles of the amplification reaction have sufficiently low stringency to enable random binding of the one or more primers to the template. In some embodiments, a low number of cycles of amplifcation may be performed under low stringency conditions that allow the one or more primers to prime synthesis randomly throughout the target, followed by a second stage amplification performed under more stringent conditions for generally a larger number of cycles.

In some embodiments, the amplification comprises whole genome amplification.

"Whole genome amplification" as referred to herein should be understood to include the amplification of a genomic DNA template in an aspecific way, in order to generate amplicons that are representative of the original genomic DNA but with a higher copy number of one or more representative regions of the genomic DNA.

Examples of whole genome amplification methods include:

Primer Extension PCR (PEP) which involves a high number of PCR cycles using Taq polymerase and 15 base random primers that anneal at a low stringency temperature;

Degenerated Oligonucleotide Primed PCR (DOP-PCR) which uses Taq polymerase and semi-degenerate oligonucleotides that bind at a low annealing temperature at approximately one million sites in the human genome. The first cycles are followed by a large number of cycles with a higher annealing temperature, allowing only for the amplification of the fragments that were tagged in the first step;

Ligation Mediated PCR (LMP) which uses endonuclease or chemical cleavage to fragment a genomic DNA sample and uses linkers and primers for its amplification;

T7-based linear amplification of DNA (TLAD) which is a variant of a protocol originally designed to amplify mRNA that has been adapted for WGA. It uses Alu I restriction endonuclease digestion and a terminal transferase to add a polyT tail on the 3' terminus. A primer is then used with a 5' T7 promoter and a 3' polyA tract, and Taq polymerase is used to synthesise the second strand. Then the sample is submitted to in vitro transcription and posterior reverse transcription; and Multiple displacement amplification (MDA) is a non-PCR-based isothermal method based on the annealing of random hexamers to denatured DNA, followed by strand-displacement synthesis at constant temperature. As DNA is synthesized by strand displacement, a gradually increasing number of priming events occur, forming a network of hyper-branched DNA structures. The reaction can be catalysed by the Pfe ^' 29 DNA polymerase or by the large fragment of the Bst DNA polymerase.

In some embodiments, the amplification, or whole genome amplification comprises DOP-PCR. In some embodiments, the DOP-PCR comprises the use of an oligonucleotide primer comprising includes six or more contiguous nucleotides of random sequence. In some embodiments, the one or more primers comprise a primer with the following nucleotide sequence: C C GAC T C G AGNNNNN AT GT GG (SEQ ID NO: 1); wherein each N is independent and each may be any nucleotide. In whole genome amplification methods, template nucleic acids extracted according to the first aspect of the invention have been demonstrated to lead to the generation of amplicons having a smaller size than in equivalent nucleic acid extraction methods without a protease treatment. Without limiting the present invention to any specific mechanism or mode of action, it is thought that protease treatment of the nucleic acid digests one or more proteins associated with the nucleic acid and thus improves access for a nucleic acid primer and/or polymerase to the template nucleic acid. In this way, additional primer binding sites are made available to amplification primers and/or polymerase. The increased availability of primer binding sites in a genomic DNA template has the effect of making the primer binding sites generally closer together. This, in turn, has the effect of both decreasing the average size of the amplicons and making the amplicons generally more representative of the original genomic DNA template.

As would be appreciated, the second aspect of the invention should not be considered limited to random amplification and/or whole genome amplification. Rather, the second aspect of the invention contemplates any nucleic acid amplification wherein contacting the template nucleic acid with a protease under conditions suitable for the protease to digest one or more proteins associated with the nucleic acid would be desirable. As such, amplification may also include any nucleic acid amplification reaction, wherein the protease treatment of the template nucleic acid improves access of a polymerase and/or primer to the template nucleic acid. In some embodiments, the method of the second aspect of the invention may be useful where a nucleic acid template is recalcitrant to amplification due to the presence of protein associated with the nucleic acid.

The amplicons produced according to the second aspect of the invention may be used for any purpose.

In some embodiments, the amplicons produced according to the second aspect of the invention may be used in a comparative genomic hybridisation method. As such, the present invention provides a comparative genomic hybridisation method comprising the use of an amplicon produced according to the second aspect of the invention.

In comparative genomic hybridisation methods, the amplicons may be used as immobilised nucleic acids on a solid substrate and/or the amplicons may be labelled and hybridised to an immobilised nucleic acid. Methods for performing comparative genomic hybridisation are well known in the art and the present invention contemplates the use of the amplicons in any suitable method. However, an exemplary method for comparative genomic hybridisation is described in WO 2004/088310, the content of which is hereby incorporated by reference.

In some embodiments, and as described in the examples, the use of amplicons produced according to the second aspect of the invention in a method for comparative genomic hybridisation can increase the sensitivity, reproducibility and/or reliability of the results generated in a comparative genomic hybridisation method. For example (described in detail in Example 3) when a trypsin digestion step was used in the cell lysis step of a comparative genomic hybridisation method, the rate of false negative, false positive and Y diagnosis improved in the comparative genomic hybridisation. In addition, a decrease in average slide standard deviation (S.D.), which is an indicator of the quality of amplification and hybridisation, was also observed in a comparative genomic hybridisation which used trypsin digestion in the cell lysis step.

In a third aspect, the present invention also provides a kit for performing the method of the first aspect and/or second aspect of the invention, the kit comprising a protease together with instructions for performing the method of the first aspect and/or second aspect of the invention.

In some embodiments, the protease included in the kit may be a protease as described with reference to the first aspect of the invention. Thus, in some embodiments, the protease may comprise any of an endopeptidase, a serine protease, a serine protease of the chymotrypsin-like clan or trypsin.

In some embodiments, the kit further comprises a buffer suitable for alkaline lysis of a cell, as hereinbefore described. In some embodiments, the buffer comprises potassium hydroxide and DTT.

In some embodiments, the kit further comprises one or more reagents for the amplification of a nucleic acid. In some embodiments, the one or more reagents comprise a DNA polymerase. A range of DNA polymerases would be readily ascertained by a person of ordinary skill in the art and the kit of the present invention may include any suitable DNA polymerase. In some embodiments, the DNA polymerase included in the kit may be a DNA polymerase suitable for nucleic acid amplification, including random amplification and/or whole genome amplification as hereinbefore described. In some embodiments, the one or more reagents comprise a nucleic acid primer. In some embodiments, the primer(s) may be suitable for nucleic acid amplification, including random amplification and/or whole genome amplification as hereinbefore described.

In addition to reagents for the amplification of a nucleic acid such as DNA polymerases and/or nucleic acid primers, the kit may also include additional reagents for the amplification of a nucleic acid including, for example, buffers, dNTPs, one or more restriction endonucleases (eg. as used in some WGA methods), and the like.

In further embodiments, the kit may also comprise one or more reaction vessels in which the methods of the present invention may be performed. Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention, including DNA restriction and ligation for the generation of the various genetic constructs described herein. See, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition). Cold Spring Harbor Laboratory Press, 2001.

The present invention is further described by the following non-limiting examples: BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows DOP-PCR products amplified from DNA extracted from single human cells using the standard lysis method (lanes 2-17 of the top row), the extended lysis method (lanes 18 to 28 top row and lanes 3 to 6 bottom row) and an embodiment of the lysis method of the present invention utilizing a trypsin digestion step (lanes 7 to 22 of the bottom row). Lane 1 in each of the top and bottom rows shows an Sppl marker, while lane 29 in the top row and lane 24 in the bottom row shows a pUC19 marker.

Figure 2 shows the results of CGH using DNA extracted from single human cells using the control cell lysis method.

Figure 3 shows the results of CGH using DNA extracted from single human cells using the extended lysis cell lysis method. Figure 4 shows the results of CGH using DNA extracted from single human cells using a cell lysis method according to an embodiment of the present invention, which incorporates a trypsin digestion step.

EXAMPLE 1

Protocols RHS Protocol 010 - Single Cell, KOH-mediated lysis

Alkaline Lysis Buffer (200mM KOH, 50mM DTT) was added to a single cell in a PCR tube. 5μί of Alkaline Lysis Buffer was used for a final first round DOP-PCR reaction volume of 50 μί.

The mixture was then incubated at 65°C for 5 min followed by 6°C for 5 min. For the extended lysis extraction method, the mixture was incubated at 65°C for 10 min.

After incubation, Neutralization Buffer 3 (500mM Tris-HCl pH 8.3, 200mM HC1) was added. 5μί of Neutralization Buffer 3 was added for a final first round DOP-PCR reaction volume of 50μί. For the trypsin digestion method (but not the control or extended lysis extraction methods), Ιμί of 8mg/mL stock trypsin (Porcine trypsin 1:250 MP Biomedicals Cat # 103139) was added to the cell lysate after the addition of neutralisation buffer 3. This mixture was then incubated at 37°C for 10 minutes. After incubation, 3μί of 25mM MgCh is added to the lysate. The trypsin was then further inactivated by incubation at 65°C for 10 min followed by 6°C for 4 minutes.

RHS Protocol 011 - First Round DOP PCR with KAPA 2G Robust

For DNA extracted using the typsin digestion step, PCR reaction mixes were prepared as follows (/50μ1): Ultra-Pure H2O - 20.6μ1, 0.1% Gelatin - 5.0μ1, dNTPs (2.5mM each) - 5.0μ1, DOP PCR primer (20μΜ) - 5.0μ1, KAPA 2G Robust (5υ/μ1) - 0.4μ1, lysed single cell (template) - 14μ1.

For samples prepared using the control or extended lysis extraction methods, the following changes were made: 21.6μ1 of Ultra-Pure H2O was used, 3μ1 of MgCh was added and ΙΟμΙ of template was used.

PCR reactions were run according to the following program:

Cycles Temperature Duration

1 95°C 5min

8 94°C lmin

30°C lmin 30sec

ramp to 72°C l°C/4sec

72°C 3min

27 94°C lmin

58°C lmin

72°C 2min + 14sec/cycle

1 72°C 7min

15°C Soak 2μ1 of each PCR product was then applied to a 1% agarose gel. To load into the gel, 2μ1 of the PCR product was combined with ΙμΙ, 6X Loading Buffer and 3μί PCR water. Electrophoresis was then performed at 120 volts for 25 - 30 min.

RHS Protocol 012 - Labelling: Second Round DOP-PCR and PCR Product Purification

A PCR mastermix for labelling with DY547 was prepared as follows (/24μ1): 11.55μ1 of Ultra-Pure H2O; 5.0μ1 of 5X KAPA 2G buffer A; 2.5μ1 of 0.1% Gelatin; 2.5μ1 of DOP- PCR primer (20μΜ); 1.5μ1 of DY547 dNTP labelling mix (3:1); 0.2μ1 of K2GR (5υ/μ1); and 0.75 μΐ of DY547-dUTP.

A PCR mastermix for labelling with DY647 was prepared as follows (/24μ1): 11.70μ1 of Ultra-Pure H2O; 5.0μ1 of 5X KAPA 2G buffer A; 2.5μ1 of 0.1% Gelatin; 2.5μ1 of DOP- PCR primer (20μΜ); 1.5μ1 of DY647 dNTP labelling mix (4:1); 0.2μ1 of K2GR (5υ/μ1); and 0.6 μΐ of DY647-dUTP.

To each mastermix, template produced according to RHS protocol 011 (see above) was added at a rate of Ιμΐ template / 24μ1 of mastermix.

The mixtures were then subjected to a PCR program as set out below: cycles temperature duration

1 95°C 5min

15 94°C Imin

58°C Imin

72°C 2min + 14sec/cycle

1 72°C 7min

15°C soak The resultant labelled PCR products were then purified using a MoBio PCR purification kit.

RHS Protocol 014 - Array-CGH Hybridisation i) Preparation of the Probe Mixtures

Labelled DNA mixture for hybridization was prepared in a sterilized 0.5ml PCR tube (MBP tube) by combining the following: 35μί of human Cot-1 DNA (lmg/ml - final amount 35μg), 20μί of sheared salmon sperm DNA (lmg/ml - final amount 20μg), ΙΟμΙ, of DY547-labelled DNA (~l^g), ΙΟμΙ, of DY647-labelled DNA (~l^g), 7.5μί of NaAC, pH 5.2 (3M), 150μί of 100% ethanol.

The tube containing the DNA mixture was incubated at -20°C for 1 hour in the dark. DNA pellets were then collected by centrifugation at 10,000 g for 25 min at 4°C. The ethanol was then decanted. The DNA pellets were then washed with 200μ1 of 75% ethanol, followed by centrifugation at 10,000 g for 5 min at 4°C and removal of the ethanol supernatant. DNA pellets were then dried at 50°C for ~5 min in a heating block. The dried DNA pellets were then redissolved by adding Ιΐμΐ of hybridisation solution to each dried DNA pellet. This mixture was then incubated at 37°C for 5 min. After incubation the tubes were mixed and briefly centrifuged. The incubation, mix and brief centrifugation were repeated. The DNA probes were then denatured at 80°C for 10 min in a PCR machine followed by pre-annealing the probes at 37°C for 30 min (Corbett PC9606C; program 80). it) Blocking Slides

Array slides were immersed in blocking buffer (Blocklt; Arraylt Corporation, Sunnyvale, California, Cat # BKT) and left at room temperature for a minimum of lh and no longer than 24h (typically 2h), on an orbital mixer. The slides were then removed from the blocking buffer, and rinsed for 1 min under running RO H2O. The slides were then centrifuged (array side up) until dry in an Arraylt centrifuge. The dried slides were then stored at room temperature, in a slide box wrapped with aluminium foil until required. Blocked slides remained useable for several hours.

Hi) Hybridisation

A hybridisation chamber (Corning) was disassembled and the base, the cover, and metal clips were placed on a warming tray. An array slide (with the printed array slide up) and coverslips were also placed on the warming tray. The disassembled hybridisation chamber and the array slide were then incubated for 30-60 min on the warming tray.

After incubation, the array slide was overlaid on an array template. 10.5μ1 of pre- annealed probes were pipetted onto the array area of the array slide sitting on the warm tray and then covered with a coverslip. The array slide (DNA up) was then placed in the base of the hybridisation chamber. 10 μΐ of PCR water was then pipetted into the humidifying well at each end of the chamber base. The cover of the chamber was then placed over the base and secured with clips. The hybridisation chamber was then incubated at 37°C for 16-20 hrs. iv) Post-Hybridization Washing

Hybridised slides were removed from the hybridisation chamber and then sequentially immersed in the following: 50% formamide/2X SSC at room temperature in the dark until the coverslip slid off;

50% formamide/2 X SSC at 45°C for 10 min;

50% formamide/2 X SSC at 45°C for 10 min;

2 X SSC at 45°C for 5 min;

2 X SSC at 45°C for 5 min;

I X SSC at room temperature with agitation on an orbital mixer at 160 rpm for 10 min; and

H2O with agitation.

The slides were then dried using a microarray high-speed centrifuge (TeleChem International, Inc.). After drying, slides were either scanned immediately or stored in a slide box, wrapped in foil at room temperature in the dark for up to 2 months.

EXAMPLE 2

Evaluation of Pre-PCR lysis and Trypsin Digestion

Single human cells were selected for controls, extended lysis, or trypsin digestion.

The cells for the extended lysis test were processed first: cells were lysed as described above in RHS protocol 10, but with the lysis step extended to 10 minutes, before being neutralised and then stored at 4°C until PCR mastermix was added.

Control cells and cells for trypsin digestion were processed next: the cells were lysed for 5 minutes and then neutralised as described above in RHS protocol 010. The control cells were then stored at 4°C until mastermix was added. As described above, the cells for trypsin digestion were then treated with trypsin, incubated for 10 minutes at 37°C. Magnesium chloride was added to block/slow the action of trypsin and then these samples were incubated at 65°C for 10 minutes to further inactivate the trypsin, the samples were then cooled. The appropriate mastermix was then added to the cells and the samples were placed in the FlexCycler (0.5mL block - Analytik Jena AG) and amplified as described above under RHS protocol 011.

Two microlitres of unpurified DOP-PCR product was run on a 1% agarose gel for 30 minutes at 120V. The results are shown in Figure 1. As can be seen in Figure 1, for the control method and the extended lysis method, the DOP-PCR products were generally in the size range of ~5kb-250bp. For the DNA extraction method incorporating trypsin digestion, the DOP-PCR products fell into the size range of ~5kb-147bp. However, unlike the control and extended lysis methods, the bulk of the DOP-PCR products amplified from the extraction using trypsin digestion were generally smaller, in the size range of <2kb-147bp.

EXAMPLE 3

Comparative Genomic Hybridisation

DNA was extracted from single human cells of known karyotype using each of the control, extended lysis and trypsin digestion methods described above. The extracted DNA was then amplified and used for comparative genomic hybridisation (CGH). The CGH method was performed according to RHS protocol 014 as described above.

The results of CGH using DNA extracted using the control, extended lysis and trypsin digestion methods are shown in Figures 2, 3 and 4, respectively. A summary of these results is shown in Table 1, below:

TABLE 1: Summary of Results

Trypsin digestion

When a trypsin digestion step was used in the cell lysis, the rate of false negative and false positive improved when compared to the control method and the extended lysis method. The false positive rate was particularly promising for this method since the only autosomal false positive was observed on a slide that was poorly hybridised and 75% of the cells on this slide (007179) also gave more than one false ratio. Repeating the cells from this slide may elucidate if the false positives were an artifact of poor hybridisation/printing. The average slide standard deviation (S.D.) was also slightly lower for this treatment. As slide S.D. tends to indicate the quality of amplification and hybridisation, these results suggest that incorporation of a trypsin digestion step in cell lysis generally improves the quality of results generated in subsequent CGH. The improvement in slide S.D. may be a consequence of the trypsinised cells showing smaller 1 ^st round fragments, which may reduce preferential incorporation in the second round and/or improve the ability of the fragments to hybridise to the array.

Extended Lysis

The rate of false positive/negatives was decreased when compared to the control cells. The improvement in FP/FN rates was not as great as that seen with the trypsin digestion treatment.

Control Cells

These cells were generally the worst performing cells with more false negative and more false positives. From these results it appears that the incorporation of trypsin digestion into a cell lysis and nucleic acid extraction method may improve the trisomy detection and decrease the number of false positives in subsequent CGH.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.