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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

• 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.
• PCR polymerase chain reaction
• 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
• 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.
• 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.
• PCR inhibitors usually affect PCR through interaction with DNA or interference with the DNA polymerase.
• 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
• column-based nucleic acid purification is a typical solid phase
• 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.
• suitable buffers include Tris-EDTA (TE) buffer or Phosphate buffer (used in DNA microarray experiments due to the reactive amines).
• TE Tris-EDTA
• Phosphate buffer used in DNA microarray experiments due to the reactive amines.
• 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;
• a denaturing agent such as guanidine hydrochloride
• Triton X-100 Triton X-100
• isopropanol 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.
• 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.
• US201 100227832 discloses the use of Taq polymerase mutant enzymes for nucleic acid amplification in the presence of PCR inhibitors.
• 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.
• PCR inhibitors A number of varying types 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.
• 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 Mg 2+ availability or interfere with binding of Mg 2+ to the DNA polymerase can inhibit PCR.
• the assured manner to avoid PCR inhibition is to prevent the inhibitor from being processed with the sample.
• 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 bovine serum albumin
• 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.
• IPC internal positive control
• 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.
• 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.
• 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.
• a control can be performed by adding a known amount of a template to the investigated reaction mixture (based on the sample under analysis).
• the method of sample acquisition can be refined to avoid unnecessary collection of inhibitors.
• swab-transfer of blood on fabric or saliva on food may prevent or reduce contamination with inhibitors present in the fabric or food.
• DNA purification techniques exist and kits are commercially available to enable extraction of DNA to the exclusion of some inhibitors.
• 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.
• BSA reduces the effect of some inhibitors on PCR.
• 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.
• 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
• 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.
• 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.
• inhibitor means naturally occurring or synthetic molecules or compounds which interfere or restrict or limit the amplification of nucleic acid by the polymerase chain reaction.
• 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;
• liquids or cell suspensions of plants liquid products, extracts or suspensions of bacteria, fungi, plasmids, viruses, prions, etc.;
• the sample is a cellular sample.
• the cellular sample may originate from a mammal, bird, fish or plant or a cell culture thereof.
• the cellular sample is mammalian in origin, most preferably human in origin.
• 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.
• 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.
• the nucleic acid may be stored in a dried form which is suitable for subsequent analysis.
• samples are stored at temperatures from -200°C to 40°C.
• stored samples may be optionally stored in dry or desiccated conditions or under inert atmospheres.
• 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.
• 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)
• 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.
• cation exchange resins include chelating-Sepharose (GE)
• 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.
• Functionalised Sepharose with a magnetic core is an example of a magnetic ion-exchange resin.
• Functionalised ion-exchange neutral buoyancy beads are described in
• containers including micro-titre plate wells and tubes
• surface of containers can be modified with appropriate ion exchange groups or resins that will facilitate inhibitor binding.
• the solid support is a cellulose-based matrix.
• cellulose-based matrices include FTATM (data file 51668), 903 neonatal cards and 31 -ETF cards available from GE Healthcare.
• 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.
• a weak base ii) a chelating agent; iii) a surfactant or detergent; and iv) uric acid or a urate salt.
• the polymerase chain reaction reagent mixture is present in a dried form, such as a "Ready-to-GoTM” (RTG) format.
• RTG Ready-to-GoTM
• 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.
• 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).
• 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.
• the nucleic acid is selected from the group consisting of DNA, RNA and oligonucleotide.
• 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.
• the sample is a cellular sample selected from the group consisting of blood, saliva, urine, faeces, hair, skin and muscle.
• 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.
• 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).
• the method further comprises the step of detecting the amplified nucleic acid.
• the method further comprises the step of quantifying the amplified nucleic acid.
• Reverse transcriptase polymerase chain reaction RT- PCR
• RT- PCR Reverse transcriptase polymerase chain reaction
• the method further comprises purifying the amplified nucleic acid. In another aspect, the method further comprises the step of cloning the amplified nucleic acid.
• 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.
• a kit for amplifying nucleic acid comprising a solid support and an ion-exchange resin.
• the solid support is preferably a cellulose-based matrix.
• the cellulose-based matrix is selected from the group consisting of FTA card, 903 card and 31 ETF card.
• Figure 1 presents the results from PCR amplification following extraction of dried blood spots on a cellulose-based solid support
• RNase P standards were prepared as follows:
• the 10ng/ ⁇ 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 90 ⁇ 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. 10 ⁇ of the thawed 10ng/ ⁇ DNA control was pipetted into the tube labelled 1 ng. The standard was vortex mixed and spun in a microcentrifuge. 10 ⁇ 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.
• 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.

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