WO2014023790A1 - Polymerase chain reaction method for amplifying nucleic acid - Google Patents

Abstract

The present invention relates to a method for amplifying nucleic acid present in a sample in which the nucleic acid is immobilised on a solid support in the presence of an inhibitor of the polymerase chain reaction. The inhibitor of the polymerase chain reaction is bound to an ion-exchange resin to prevent it interfering with the amplification process.

Description

Polymerase Chain Reaction Method for Amplifying Nucleic Acid

Field of the Invention The present invention relates to the field of nucleic acid amplification, particularly to the use of the polymerase chain reaction to amplify nucleic acids. The invention provides methods and kits which can be used to amplify nucleic acids by removing inhibitors of the polymerase chain reaction which are often found in biological samples and interfere with the amplification process. The invention has applications in the long-term storage, recovery and further processing of nucleic acids and is particularly useful in genotypying, diagnostics and forensics applications.

Background to the Invention

The polymerase chain reaction (PCR) is a common tool used in molecular biology for amplifying nucleic acids. US4683202 (Mullis, Cetus Corporation) describes a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof.

US 5593824 and US 5763157 (TremI) describe biological reagent spheres useful for the PCR. Additionally, this invention describes a convenient approach by means of excipient mixes comprising suitable carbohydrates, such as Ficoll and melezitose, useful for storage of reagents used in downstream genetic analysis such as PCR.

Long-term storage, transport and archiving of nucleic acids on filter paper or chemically modified matrices is a well-known technique for preserving genetic material before the DNA or RNA is extracted and isolated in a form for use in genetic analysis such as PCR. Thus, EP1563091 (Smith et al, Whatman) relates to methods for storing nucleic acids from a samples such as cells or cell lysates. The nucleic acid is isolated and stored for extended periods of time, at room temperature and humidity, on a wide variety of filters and other types of solid support or solid phase media. Moreover, the document describes methods for storing nucleic acid-containing samples on a wide range of solid support matrices in tubes, columns, or multiwell plates.

WO/9003959 (Burgoyne) describes a cellulose-based solid support for the storage of DNA, including blood DNA, comprising a solid matrix having a compound or composition which protects against degradation of DNA

incorporated into or absorbed on the matrix. This document also discloses methods for storage of DNA using the solid medium, and for recovery of or in situ use of DNA.

US5496562 (Burgoyne) describes a cellulose-based solid medium and method for DNA storage. Method for storage and transport of DNA on the solid medium, as well as methods which involve either (a) the recovery of the DNA from the solid medium or (b) the use of the DNA in situ on the solid medium (for example, DNA sequence amplification by PCR) are disclosed. Unfortunately the methods described suffer from the disadvantage in that they require the removal of phenolic or inhibitory compounds before the PCR is performed.

Various methods for PCR optimization have been developed by molecular biologists to improve PCR performance and minimize failure. However, PCR is an extremely sensitive technique requiring only a few nucleic acid molecules in a single reaction for amplification across many orders of magnitude. Therefore adequate measures to avoid contamination from inhibitors of the technique in the laboratory environment are required. Because PCR inhibitors are a common source of contamination, many molecular biology laboratories have implemented procedures that involve dividing the laboratory into separate areas. In this case, it is usual to have one laboratory area dedicated to the preparation and handling of pre-PCR reagents and the set-up of the PCR reaction, and another area for post- PCR processing, such as gel electrophoresis or PCR product purification. It is usual for the set-up of PCR for experiments to be carried out in a "clean-room" environment with reagent additions completed in a laminar flow hood work station. Polymerase chain reactions are also routinely assessed in the presence of positive and negative control reactions to assess the impact of inhibitors.

Accordingly, there are significant problems with inhibitory substances or inhibitors interfering with polymerase chain reactions on nucleic acids, including nucleic acids stored or immobilised on solid supports such as cellulose-derived filters. PCR inhibitors usually affect PCR through interaction with DNA or interference with the DNA polymerase. In a multiplex PCR reaction, it is possible for the different sequences to experience different inhibition effects to varying degrees, leading to a disparity in results. Alternatively, various inhibitors reduce the availability of cofactors (such as magnesium) or otherwise interfere with the interaction of PCR cofactors with the DNA polymerase. Current methods for inhibitor removal are often carried out during the DNA purification procedure by binding to single or double stranded DNA covalently linked to a support. However, this is a tedious process and prior art methods have a number of clear

disadvantages in terms of cost, complexity and in particular, user time. For example, column-based nucleic acid purification is a typical solid phase

extraction method to purify nucleic acids. This method relies on the nucleic acid binding through adsorption to silica or other support depending on the pH and the salt content of the buffer. Examples of suitable buffers include Tris-EDTA (TE) buffer or Phosphate buffer (used in DNA microarray experiments due to the reactive amines). The purification of nucleic acids on such spin columns includes a number of complex and tedious steps. Nucleic acid purification on spin columns typically involves three time-consuming and complex steps/stages: the sample containing nucleic acid is added to the column and the nucleic acid binds due to the lower pH (relative to the silanol groups on the column) and salt concentration of the binding solution, which may contain buffer, a denaturing agent (such as guanidine hydrochloride), Triton X-100, isopropanol and a pH indicator;

the column is washed with 5 mM KP04 pH 8.0 or similar, 80% EtOH); and the column is eluted with buffer or water.

Alternative methods involve the binding of nucleic acids in the presence of chaotropic agents such that DNA binds to silica or glass particles or glass beads. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. Typical chaotropic agents include guanidinium thiocyanate or guanidinium hydrochloride and recently glass beads have been substituted with glass containing minicolumns.

Some of the pitfalls of quantitative real-time reverse transcription polymerase chain reaction, including the effect of inhibitors, are described by Bustin & Nolan (J. Biomolecular Techniques, 2004, 15, 155-166).

Huggett et al (BMC Research Notes, 2008 1 : 70-79) describes differential susceptibility of PCR reaction to inhibitors including those from urine samples. US 6348336 (Alpha Therapeutics) describes a complex process for preparing samples from liquid blood, blood plasma, blood serum or liquid plasma proteins, including blood factor products, for PCR testing which minimizes contaminants which interfere with the PCR analysis. The process includes centrifugation of the initial sample to form a sample pellet, removing at least a portion of the

supernatant from the pellet, and washing the pellet with an aqueous buffer. The buffer and the washed pellet are then centrifuged, and a portion of the remaining supernatant is removed along with any contaminants contained therein. The purified, substantially contaminant-free pellet is then processed for PCR analysis. The process uses polyethylene glycol. US8012691 (Applied Biosystems) describes multiplex compositions and methods for quantification of human nuclear DNA and human male DNA, and for the detection of PCR inhibitors. US20120003645 (Life Technologies) describes compositions, methods and kits for nucleic acids synthesis, amplification and one or more reagents to increase tolerance to PCR inhibitors.

US201 100227832 (DNA Polymerase Technology) discloses the use of Taq polymerase mutant enzymes for nucleic acid amplification in the presence of PCR inhibitors.

Trace metal ion and heavy metal cation removal are described in the Chelex-100 and Chelex-20 chelating ion exchange instruction manual (http://www.bio- rad.com/webmaster/pdfs/9184 Chelex.PDF).

Ollikka et al (Analytical Biochemistry, 2009, 386, 20-29) discloses genotyping of celiac disease-related-risk haplotypes using a closed-tube polymerase chain reaction analysis of dried blood and saliva disk samples, with DNA extracted and inhibitors removed using a complex QIAamp Blood Mini Kit (Qiagen).

Walsh ef a/ (Biotechniques, 1991 , 10, 506-513) describes the use of Chelex-100 for the simple extraction of DNA for PCR-based typing from forensic material such as cloth, threads or from plastic wrap, but not from cellulose-derived paper materials.

Fa Yi et al, 2007, (http://www.ncbi.nlm.hih.gOv/pubmed/18175573) discloses a method of extracting DNA samples for aged bloodstains on filter paper using Chelex-100.

Chaorattanakawee et al (Am.J.Trop.Med.Hyg., 2003, 69(1 ), 42-44) describes a Chelex based method for extracting DNA from dried blood samples on filter papers. The method involves multiple washings to remove inhibitory reagents, including soaking the blood samples overnight in phosphate buffered saline, prior to PCR. The results indicated that the sensitivity of the PCR increased with length of storage of the dried blood samples, being lowest with samples stored for less than 4 years.

Belgrader et al (Biotechniques, 1995, 19, 426-432) describes the automated DNA purification and amplification from blood-stained cards using a robotic workstation. Furthermore, this publication describes the development of a method for the purification and amplification of DNA on blood-stained cards, followed by a complex solutions comprising of phenol, 75% (v/v) isopropanol, 25mM potassium acetate and an 80°C drying step. This method was

implemented into a high-throughput automated system using a Biomek 1000 robotic workstation. In addition, the processes of DNA purification and

amplification were coupled into a completely automated and uninterrupted prototype system, and the resultant PCR products generated by this system were subjected to an automated, multifacted desalting process to remove inhibitory substance using dialysis for further capillary electrophoresis analysis of DNA. A number of varying types of PCR inhibitors exist. Inhibitors may be present in the original sample, such as blood, fabrics, tissue and soil but may also be added as a result of the sample processing and the DNA extraction techniques used. Excess salts including potassium chloride and sodium chloride, etc., all contribute via various inhibitory mechanisms, to the reduction of PCR efficiency. PCR inhibitors have been an obstacle to success in diagnostics, molecular biology, and forensics. All users of the PCR are likely to be impacted by inhibitors at some time, but the wide range of forensic sample types and variety of sampling conditions encountered make forensic scientists particularly vulnerable. PCR inhibitors generally exert their effects through direct interaction with DNA or interference with thermostable DNA polymerases. Direct binding of agents to single stranded or double-stranded DNA can prevent amplification and facilitate co-purification of inhibitor and DNA. Inhibitors can also interact directly with a DNA polymerase to block enzyme activity. DNA polymerases have cofactor requirements that can be the target of inhibition; for example, magnesium is a critical cofactor, and agents that reduce Mg²⁺ availability or interfere with binding of Mg²⁺ to the DNA polymerase can inhibit PCR.

The presence of PCR inhibitors in samples, particularly biological samples, is well-known and a number of approaches are documented that describe their removal. However, none of these approaches are particularly efficient.

Moreover, the application of samples to solid supports, particularly cellulose- derived materials, increases the problems with PCR inhibition because of the inclusion of potent chemicals such as SDS which are typically used to stabilise nucleic acids on the solid support (see Burgoyne WO/9003959). Common sample types known to contain inhibitors include blood, fabrics, tissues and soil (Table 1 ). Other important sources of inhibitors are the materials and reagents that come into contact with samples during processing or nucleic acid purification. These include excess KCI, NaCI and other salts, ionic detergents such as sodium deoxycholate, sarkosyl and sodium dodecyl sulphate (SDS), ethanol and isopropanol, phenol (see Burgoyne above) and others. Table 1 lists examples of PCR inhibitors and sources of PCR inhibitors.

Lactoferrin Blood

Immunoglobulin G (IgG) Blood

Indigo dye Denim

Table 1. PCR Inhibitors

The assured manner to avoid PCR inhibition is to prevent the inhibitor from being processed with the sample. For inhibitors that are inherent to the sample, as is the case for blood and certain tissues, this is not possible. For forensic casework samples on other materials, such as blood on denim or saliva on food items, the inhibitor-containing substrate may be avoided by using swab-transfer methods rather than processing cuttings or pieces of stained or contacted material. DNA purification is the method used most often to remove inhibitors. A wide range of commercially available kits, such as the DNA IQ System (Promega), and in- house laboratory- derived methods are available to extract DNA, but only a few of these methods have been widely adopted in forensic laboratories because, in part, adoption of a new method requires labour intensive validation. Validation should evaluate the method's ability to efficiently extract inhibitor-free DNA from a wide range of sample types. Extraction methods that are proven to eliminate inhibitors from the purified template DNA should be favoured. There are several options to overcome the effects of inhibitors that are not eliminated during extraction. The choice of DNA polymerase can have a large impact on resistance to inhibition. AmpliTaq Gold DNA polymerase (Applied Biosystems), which is a common DNA polymerase for use with commercial multiplex forensic short tandem repeat (STR) kits, is among the most sensitive to inhibition. This underscores the importance of sample handling and extraction and highlights an opportunity for future improvement. Increasing the amount of DNA polymerase in the reaction or using additives such as bovine serum albumin (BSA), which provides some resistance to inhibitors in blood, are proven methods. BSA is included in the Promega PowerPlex Systems. However, users should be cautious about adding BSA to STR amplifications. BSA quality can vary greatly between sources, and material should be rigorously quality-tested; this can give variable results and can lead to lower product yield following PCR amplification. Finally, adding less DNA template to the amplification can often improve performance greatly, emphasizing STR kit sensitivity as a key advantage when generating profiles from templates that contain inhibitors. Inhibition of multiplex STR amplifications can result in reduced product yield or complete failure. When inhibited samples exhibit a partial profile, a specific pattern of locus dropout is common. Quite often, smaller loci in the kit are preferentially amplified. The same pattern is typical of highly degraded DNA templates, and very often, inhibited samples are mistakenly assumed to be degraded. Use of multiplex real-time PCR to quantitate DNA provides an opportunity to use an internal positive control (IPC) to detect PCR inhibitors. For example, the Quantifier System (Applied Biosystems) uses an IPC. Real-time PCR data can also be used to detect inhibitors by analysing target amplification efficiency. This IPC strategy has been used in combination with two autosomal targets of differing size to simultaneously assess both inhibitors and template degradation. While the additional information about inhibition and degradation obtained by real-time quantitation systems allows users to make better choices for sample processing and ultimately leads to higher amplification success rates, it is a complex approach.

Given the wide range of PCR inhibitor-laden sample types and the options available for handling them, a multifaceted approach is the best solution for amplification failure. The best defence against STR amplification failure in forensics applications is to combine sound sample handling and processing techniques with extraction systems proven to efficiently purify inhibitor-free DNA. Despite those efforts, inhibitors may still be present, underlining the value of using quantitation systems capable of detecting them, and more importantly, emphasizing the importance of using sensitive and robust multiplex STR amplification systems. In order to try to assess the extent of inhibition that occurs in a reaction, a control can be performed by adding a known amount of a template to the investigated reaction mixture (based on the sample under analysis). By comparing the amplification of this template in the mixture to the amplification observed in a separate experiment in which the same template is used in the absence of inhibitors, the extent of inhibition in the investigated reaction mixture can be concluded. If the inhibition occurring in the sample-derived reaction mixture is sequence-specific, then this method will yield an underestimate of the inhibition as it applies to the investigated sequence.

A number of techniques exist to overcome or mitigate inhibition. For example, the method of sample acquisition can be refined to avoid unnecessary collection of inhibitors. Furthermore, in forensics, swab-transfer of blood on fabric or saliva on food, may prevent or reduce contamination with inhibitors present in the fabric or food. Indeed, DNA purification techniques exist and kits are commercially available to enable extraction of DNA to the exclusion of some inhibitors. In addition to methods for the removal of inhibitors from samples before PCR, some DNA polymerases offer some resistance to different inhibitors and increasing the concentration of the chosen DNA polymerase also confers some resistance to polymerase-targeted inhibitors. For PCR methods involving blood samples, the addition of BSA reduces the effect of some inhibitors on PCR.

However, none of the methods described above are completely free of problems and none are totally efficient for the complete removal of inhibitors. The problem is particularly acute for nucleic acid samples stored or immobilised on solid supports such as cellulose-based matrices, where the nucleic acid is in the presence of the PCR inhibitor.

There is therefore a need for an improved and simplified process for removing inhibitors of the polymerase chain reaction from samples prior to nucleic acid amplification by PCR wherein the nucleic acid is immobilised on a solid support. The present invention addresses this problem and provides methods and kits which can be used for the removal of polymerase chain reaction inhibitors from solid supports, particularly cellulose-derived supports.

Summary of the Invention

The present invention provides a method for amplifying nucleic acid by prior removal of inhibitors that would otherwise interfere with the amplification process. According to a first aspect of the present invention, there is provided a

polymerase chain reaction method for amplifying nucleic acid present in a sample wherein the nucleic acid is immobilised on a solid support in the presence of an inhibitor of the polymerase chain reaction, the method comprising the steps of

(i) contacting a sample comprising an inhibitor of the polymerase

chain reaction and a nucleic acid immobilised on a solid support with an ion-exchange resin to bind the inhibitor;

eluting the nucleic acid from the solid support into a reaction vessel containing a polymerase chain reaction reagent mixture; and

(iii) amplifying the nucleic acid.

The method of the invention can be used either in single tube or a high- throughput 96-well format in combination with automated sample processing as described by Baron et al,( 201 1 , Forensics Science International: Genetics Supplement Series, 93, e560-e561 ). This approach would involve a minimal number of steps and increase sample throughput. The risk of operator-induced error, such as cross-contamination is also reduced since this procedure requires fewer manipulations compared to protocols associated with currently used, more labour intensive kits (e.g. QIAmp DNA blood mini kit, Qiagen). The risk of sample mix-up is also reduced since the procedure requires few manipulations.

Importantly, the method is readily transferable to a multi-well format for high- throughput screening. The present invention can thus improve sample storage and processing for carrying out PCR reactions to aid genetic interrogations. The invention can be conducted in a 96 well/high throughput format to facilitate sample handling and thus eliminate batch processing of samples.

The term "inhibitor" as used herein means naturally occurring or synthetic molecules or compounds which interfere or restrict or limit the amplification of nucleic acid by the polymerase chain reaction.

In one aspect, the method additionally comprises the step of applying the sample to the solid support prior to contacting the sample with the ion exchange resin. The sample containing the nucleic acid may be derived from any source. This includes, for example, physiological/pathological body fluids (e.g. secretions, excretions, exudates) or cell suspensions of humans and animals;

physiological/pathological liquids or cell suspensions of plants; liquid products, extracts or suspensions of bacteria, fungi, plasmids, viruses, prions, etc.;

liquid extracts or homogenates of human or animal body tissues (e.g., bone, liver, kidney, etc.); media from DNA or RNA synthesis, mixtures of chemically or biochemically synthesized DNA or RNA; and any other source in which DNA or RNA is or can be in a liquid medium. In another aspect, the sample is a cellular sample. The cellular sample may originate from a mammal, bird, fish or plant or a cell culture thereof. Preferably the cellular sample is mammalian in origin, most preferably human in origin.

In a further aspect, the method additionally comprises the step of lysing the sample. Cell lysis can be effected by a number or agents including surfactants or detergents. Sodium dodecyl sulphate (SDS) is an example of a detergent frequently used to lyse biological cells.

In one aspect, the nucleic acid is immobilised on the solid support for at least 24 hours. The nucleic acid may be immobilised on the solid support for longer periods, for example, for at least 7 days, for at least 30 days, for at least 90 days, for at least 180 days, for at least one year, and for at least 10 years. In this way the nucleic acid may be stored in a dried form which is suitable for subsequent analysis. Typically, samples are stored at temperatures from -200°C to 40°C. In addition, stored samples may be optionally stored in dry or desiccated conditions or under inert atmospheres.

In another aspect, the method additionally comprises the step of transferring a portion of the solid support comprising the nucleic acid and the inhibitor to the reaction vessel prior to contacting the sample with the ion exchange resin.

In one aspect, the portion is transferred to the reaction vessel by punching or cutting a disc from the solid support. Punching the portion or disc from the solid support can be effected by use of a punch, such as a Harris Micro Punch (Whatman Inc.; Sigma Aldrich)

In another aspect, the ion-exchange resin is selected from the group consisting of cation-exchange resin, LID chromatography resin, magnetic ion-exchange resin and functionalised ion-exchange neutral buoyancy resin. Examples of cation exchange resins include chelating-Sepharose (GE

Healthcare) or Chelex 100 (BioRad).

LID chromatography resin or beads are engineered to contain an ion exchange core (e.g. Sephacryl-based) with an outer inert filtration surface. Lid beads are a new type of restricted access chromatography bead with a charged inner core (WO/201 1/102790; GE Healthcare; see also Kepka et al, 2004, J.

Chromatography, 1057, 1 15-124).

Functionalised Sepharose with a magnetic core (e.g. Mag Sepharose; GE Healthcare) is an example of a magnetic ion-exchange resin. Functionalised ion-exchange neutral buoyancy beads are described in

US5679539 (Hudson & Cook).

In addition the surface of containers (including micro-titre plate wells and tubes) can be modified with appropriate ion exchange groups or resins that will facilitate inhibitor binding.

In a further aspect, the solid support is a cellulose-based matrix. Examples of cellulose-based matrices include FTA™ (data file 51668), 903 neonatal cards and 31 -ETF cards available from GE Healthcare.

In one aspect, the cellulose-based matrix comprises i) a weak base; ii) a chelating agent; iii) a surfactant or detergent; and iv) uric acid or a urate salt. Examples of such matrices are given in WO 96/39813 (Burgoyne).

In another aspect, the polymerase chain reaction reagent mixture is present in a dried form, such as a "Ready-to-Go™" (RTG) format. The advantage of dried or lyophilised formulations of the polymerase chain reaction reagents is that they can be easily solublised by the addition of water, thus saving operator time and facilitating operator usage. To minimise operator error, the dried reagent mixture can be pre-dispensed into the reaction vessel, such as the well of a multi-well plate. Examples of such an RTG mixture include "lllustra Ready-to-Go RT-PCR beads" available from GE Healthcare (product code: 27-9266-01 lllustra Ready- To-Go RT-PCR Beads). These freeze-dried beads that include the reagents necessary for one-step reverse transcription-PCR, can be pre-dispensed into a reaction vessel, such as the well of a multi-well plate, as a single dose ready for use. The preformulated, predispensed, ambient-temperature-stable beads thus ensure greater reproducibility between reactions, minimize pipetting steps, and reduce the potential for pipetting errors and contamination. In a further aspect, the nucleic acid is selected from the group consisting of DNA, RNA and oligonucleotide. The term "nucleic acid" is used herein synonymously with the term "nucleotides" and includes DNA, such as plasmid DNA and genomic DNA; RNA, such as mRNA, tRNA, sRNA and RNAi; and protein nucleic acid, PNA. In one aspect, the sample is a cellular sample selected from the group consisting of blood, saliva, urine, faeces, hair, skin and muscle.

In another aspect, the inhibitor is selected from the group consisting of bile salt, complex carbohydrate, haeme, melanin, eumelanin, myoglobulin, polysaccharide, proteinase, calcium ion, urea, haemoglobulin, lactoferrin and immunoglobulin.

In a further aspect, the reaction vessel is a well in a multi-well plate. Multi-well plates are available in a variety of formats, including 6, 12, 24, 96, 384 wells (e.g. Corning 384 well multi-well plate, Sigma Aldrich).

In one aspect, the method further comprises the step of detecting the amplified nucleic acid.

In another aspect, the method further comprises the step of quantifying the amplified nucleic acid. Reverse transcriptase polymerase chain reaction (RT- PCR) can be used to quantify the nucleic acid.

In one aspect, the method further comprises purifying the amplified nucleic acid. In another aspect, the method further comprises the step of cloning the amplified nucleic acid.

In a further aspect, the method is for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool and a forensics tool. According to a second aspect of the present invention, there is provided a kit for amplifying nucleic acid comprising a solid support and an ion-exchange resin. The solid support is preferably a cellulose-based matrix. Preferably, the cellulose-based matrix is selected from the group consisting of FTA card, 903 card and 31 ETF card.

Brief Description of the Figures Figure 1 presents the results from PCR amplification following extraction of dried blood spots on a cellulose-based solid support

Detailed Description of the Invention Chemicals and Materials Used

A list of the chemicals and their sources is given below:

FTA, 903, 31 -ETF papers for storing nucleic acid were obtained from GE

Healthcare UK Limited;

Normal human blood;

Chelex 100 Molecular Biology Grade Resin (Bio-Rad Catalog Code 142-1253);

Harris Uni-core punch (Sigma, Cat.Z708860-25ea, lot 31 10);

TaqMan RNase P detection reagents (Applied Biosystems, Product Code

4316831 ); and

TaqMan Universal Master Mix (Applied Biosystems, Product Code 4324018)

Experimental Results

RNase P measurement from dried blood spots from cellulose matrices using qPCR following extraction on a solid phase system. Aliquots (30μΙ) of normal human blood were applied to FTA, 903 and 31 -ETF papers and were allowed to dry. Punches (3mm diameter) were extracted from each paper type (tnormal human blood) using the Harris Uni-core punch. Chelex-100 resin was prepared as a 5% (w/v) suspension in water. A Chelex suspension (1200μΙ) was added (with constant stirring) to each paper type (tnormal human blood), vortexed briefly, and heated at 56°C for 30mins. Tubes containing sample were transferred to a second heating station, incubated at 98°C for 8mins and centrifuged at 1300rpm for 2mins. The supernatants were transferred to a second tube for storage, prior to genetic analysis and PCR.

RNase P standards were prepared as follows:

The 10ng/μΙ DNA control tube was removed from the RNase P detection kit, and placed on ice. Three 0.5ml tubes were labelled, 1 ng, 0.1 ng, and 0.01 ng and 90μΙ of sterile HPLC grade water was pipetted into each tube. The stock DNA control tube was vortex mixed and pulse centrifuged to pool the DNA solution at the bottom of the tube. 10μΙ of the thawed 10ng/μΙ DNA control was pipetted into the tube labelled 1 ng. The standard was vortex mixed and spun in a microcentrifuge. 10μΙ of the 1 ng solution was pipetted into the tube labelled 0.1 ng. The standard was vortex mixed and spun in a micro-centrifuge. 10μΙ of the 0.1 ng solution was pipetted into a tube labelled 0.01 ng. The standard was vortex mixed well and spun in a micro-centrifuge. The DNA standards were placed on ice. 2μΙ of each standard was added to each well containing 23μΙ master mix. The master mix for 32 samples was set up as follows:

400μΙ Taqman Universal Master Mix; 40μΙ RNase P probe; 296μΙ water, 736μΙ total volume.

PCR reaction was set up as follows:

25μΙ total volume per well; 12.5μΙ TaqMan Universal Master Mix; 1 .25μΙ RNase P Probe; 9.25μΙ water; 2μΙ Standard/Sample/No Template Control, in a total volume of 25μΙ per well. Standards and samples were added to the appropriate wells as above, plates centrifuged at 100rpm for 1 min and sealed. PCR was carried out on an Applied Biosystems 7900HT qPCR instrument following the manufactures' user instructions for the RNase P detection reagents (Applied Biosystems). The thermal cycling conditions were: 50°C for 2min, 95°C for 10min, followed by 40 cycles of: 15secs at 95°C and 1 min at 60°C. Following amplification, data analysis was carried out using SDS 2.4 software (Applied Biosystems). The results are shown in Tables 2-5 and presented graphically in Figure 1.

Figure 1 shows PCR results following extraction using a solid phase system (Chelex-100 treatment) of dried blood spots prepared on a cellulose derived matrix (a) FTA paper; (b) 903 paper; (c) 31 -ETF paper. Figure 1 presents RNase P levels obtained from dried blood spots using quantitative PCR. DNA samples were either prepared by extraction ("Extracted") using Chelx-100 or tested without extraction ("Crude") from crude samples directly from the paper samples. As can be seen, high yields of nucleic acid were obtained from the extracted samples but PCR was inhibited using crude samples directly in the test.

Table 2. Dose response RNase P Assay

Table 3. RNase P results from FTA

RNase P concentration (ng) Treated Untreated

903-1 0.001 1 12 0

903-1 0.003000 0

903-2 0.00101 1 0

903-2 0.002500 0

Table 4. RNase P results from 903

RNase P concentration (ng)

Treated Untreated

31 ETF-1 0.001 1 19 0

31 ETF-1 0.003554 0

31 ETF-2 0.001754 0

31 ETF-2 0.002200 0

Table 5. RNase P results from 31-ETF

While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practised by other than the described embodiments, which are presented for the purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow.

Claims

Claims

1. A polymerase chain reaction method for amplifying nucleic acid present in a sample wherein the nucleic acid is immobilised on a solid support in the presence of an inhibitor of the polymerase chain reaction, the method comprising the steps of

(i) contacting a sample comprising an inhibitor of the polymerase chain reaction and a nucleic acid immobilised on a solid support with an ion-exchange resin to bind said inhibitor;

(ii) eluting said nucleic acid from said solid support into a reaction vessel containing a polymerase chain reaction reagent mixture; and

(iii) amplifying the nucleic acid.

2. The method of claim 1 , additionally comprising the step of applying said sample to the solid support prior to contacting the sample with the ion exchange resin.

3. The method according to claim 1 or 2, wherein the sample is a cellular sample.

4. The method according to any of claims 1 to 3, the method additionally comprising the step of lysing the sample.

5. The method according to any preceding claim, wherein the nucleic acid has been immobilised on the solid support for at least 24 hours.

6. The method according to any preceding claim, additionally comprising the step of transferring a portion of the solid support comprising the nucleic acid and the inhibitor to said reaction vessel prior to contacting the sample with the ion exchange resin.

7. The method according to claim 6, wherein the portion is transferred to the reaction vessel by punching or cutting a disc from the solid support.

8. The method according to any preceding claim, wherein said ion-exchange resin is selected from the group consisting of cation-exchange resin, LID chromatography resin, magnetic ion-exchange resin and functionalised ion-exchange neutral buoyancy resin.

9. The method according to any preceding claim, wherein the solid support is a cellulose-based matrix.

10. The method of claim 9, wherein said cellulose-based matrix comprises i) a weak base; ii) a chelating agent; iii) a surfactant or detergent; and iv) uric acid or a urate salt.

1 1 .The method according to any preceding claim, wherein said polymerase chain reaction reagent mixture is present in a dried form, such as a RTG format.

12. The method according to any preceding claim, wherein the nucleic acid is selected from the group consisting of DNA, RNA and oligonucleotide.

13. The method according to any preceding claim, wherein said sample is a cellular sample selected from the group consisting of blood, saliva, urine, faeces, hair, skin and muscle.

14. The method according to any preceding claim, wherein said inhibitor is selected from the group consisting of bile salt, complex carbohydrate, haeme, melanin, eumelanin, myoglobulin, polysaccharide, proteinase, calcium ion, urea, haemoglobulin, lactoferrin and immunoglobulin.

15. The method according to any preceding claim, wherein the reaction vessel is a well in a multi-well plate.

16. The method according to any preceding claim, further comprising the step of detecting the amplified nucleic acid.

17. The method according to any preceding claim, further comprising the step of quantifying the amplified nucleic acid.

18. The method according to claim 17, wherein reverse transcriptase

polymerase chain reaction (RT-PCR) is used to quantify the nucleic acid.

19. The method according to any preceding claim, further comprising purifying the amplified nucleic acid.

20. The method according to any preceding claim, further comprising the step of cloning the amplified nucleic acid.

21 .The method according to any preceding claim, for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool and a forensics tool.

22. A kit for amplifying nucleic acid comprising a solid support and an ion- exchange resin.

23. The kit of claim 22, wherein said solid support is cellulose-based matrix.

24. The kit according to claim 23, wherein said cellulose-based matrix is

selected from the group consisting of FTA card, 903 card and 31 ETF card.