Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

• the present invention relates to the field of nucleic acid amplification, particularly to the use of a polymerase chain reaction to amplify nucleic acids.
• the invention provides methods and kits which can be used to amplify nucleic acids by combining a solid support such as an FT ATM paper with PCR reagents for one step amplification of nucleic acid samples.
• the invention has applications in the long term storage and easy processing of nucleic acids and is particularly useful in genotyping, diagnostics and forensics.
• PCR polymerase chain reaction
• EP 1563091 (Smith et al, Whatman) relates to methods for storing nucleic acids from 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 90/03959 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.
• EP 2290099 B 1 (Qiagen) describes again a method for processing and amplifying DNA.
• the method includes the steps of contacting the sample containing DNA to a solid support wherein a lysis reagent is bound to the solid support.
• the DNA is subsequently treated with a DNA purifying reagent and is purified.
• the application does not include a sequestrant on the solid support and requires a separate step for the removal of the lysis reagent and purification of the DNA before amplification.
• WO 96/39813 (Burgoyne) describes a solid medium for storing a sample of genetic material and subsequent analysis; the solid medium comprising a protein denaturing agent and a chelating agent.
• the method described is for chelating agents which are any compound capable of complexing multivalent ions including Group II and Group III multivalent metal ions and transition metal ions.
• the invention does not specifically mention cyclodextrin as a chelating agent, nor does it suggest the PCR analysis could be performed in a single step.
• US 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant.
• the advantage of this system is that conventional detergent removal requires a separation step however with the addition of cyclodextrin to neutralize the detergent it would remove the separation step needed and reduce chance of contamination.
• GB 2346370 (Cambridge Molecular Technologies Ltd) describes applying a sample comprising cells containing nucleic acid to a filter, the cells are retained by the filter and contaminants are not. The cells are lysed on the filter and retained alongside the nucleic acid. Subsequent steps filter out the cell lysate while retaining the nucleic acid.
• WO 96/18731 (Deggerdal) describes a method of isolating nucleic acid whereby the sample is bound to a solid support and sample is contacted with a detergent and subsequent steps performed to isolate the nucleic acid.
• WO 00/53807 (Smith, Whatman) describes a medium for the storage and lysis of samples containing genetic material which can be eluted and analyzed.
• the medium is coated with a lysis reagent.
• the medium could be coated with a weak base, a chelating agent, a surfactant and optionally uric acid.
• WO 99/38962 (Health, Centra Systems Inc.) describes a solid support with a bound lysis reagent.
• the lysis reagent can comprise of a detergent, a chelating agent, water and optionally an RNA digesting enzyme.
• the solid support does not contain cyclodextrin and requires further steps for purification of the nucleic acid for amplification analysis.
• 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).
• TE Tris-EDTA
• 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;
• a denaturing agent such as guanidine hydrochloride
• Triton X-100 Triton X-100
• isopropanol a pH indicator
• the column is eluted with buffer or water.
• chaotropic salts 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 salts include guanidinium thiocyanate or guanidinium hydrochloride and recently glass beads have been substituted with glass containing minicolumns.
• the best defense against PCR amplification failure in forensics applications is to combine sound sample handling and processing techniques with extraction systems proven to efficiently purify DNA.
• the purification steps involved in the standard FTA elute card protocol can be cumbersome and purification can lead to a loss in DNA quality.
• the present invention addresses this problem and provides methods and kits which can be used for single step amplification of nucleic acid from solid supports, particularly cellulose- derived supports.
• the present invention provides methods and kits which can be used to amplify nucleic acids by contacting a solid support with nucleic acid and amplifying the nucleic acid in the presence of said solid support for easy amplification of DNA samples.
• a method for amplification of nucleic acid comprising the steps
• the method further includes using Short Tandem Repeat (STR) profiling to produce an STR profile.
• STR Short Tandem Repeat
• the high pH solution is a solution having a pH of between about 10 to about 14. In a preferred embodiment, the high pH solution is a solution having a pH of between about 11 to about 13.5. In a more preferred embodiment, the high H solution is a solution having a pH of between about 12 to about 13.
• the eluted and denatured nucleic acid is contained in a minimum volume (e.g., 5-200 microliters), below which the eluted chemistry and other cellular components causes an inhibition of subsequent analytical procedures.
• a minimum volume e.g., 5-200 microliters
• the method of the invention further comprises the addition of a neutralizing solution such that a minimum volume so formed permits downstream molecular biology applications.
• the eluted nucleic acid comprises a DNA or RNA species.
• the nucleic acid is amplified in the presence of an RNase inhibitor and alpha-cyclodextrin such that amplification occurs efficiently and without loss or inhibition
• the eluted nucleic acid is mRNA, miRNA, rRNA, piRNA, or siRNA and the method further comprises gene expression analysis
• the proposed method of amplification comprises reverse transcription polymerase chain reaction, isothermal amplification or quantitative polymerase chain reaction.
• the nucleic acid amplification reagent solution comprises a polymerase, deoxyribonucleotide triphosphate (dNTP), a reaction buffer and at least one primer, wherein the primer is optionally labelled with a dye.
• dNTP deoxyribonucleotide triphosphate
• the composition of the solid support comprises guanidine thiocyanate, guanidine chloride, guanidine hydrochloride, sodium dodecyl sulphate, uric acid, EDTA or Tris buffer.
• the solid support is washed with an aqueous solution before step i).
• an embodiment of the invention comprises a solid support selected from the group consisting of a glass or silica-based solid phase medium, a plastics based solid phase medium, a cellulose-based solid phase medium, glass fiber, glass microfiber, silica gel, silica oxide, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetraflurorethylene, polyvinylidinefluoride, wool and porous ceramics.
• the solid support is a cellulose based matrix.
• the cellulose based matrix is in the form of a pre punched disc.
• a still further embodiment provides that the cellulose based matrix is in the form of an FTATM or FT ATM Elute card.
• the nucleic acid is stored on the solid support prior to step i).
• the nucleic acid is stored on the solid support for at least 30 minute.
• the nucleic acid may be immobilised on the solid support for longer periods, for example, for at least 24 hours, 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.
• amplification of nucleic acid comprising the steps:
• the advantage of amplifying the nucleic acid in the presence of the solid support is to reduce the number of steps required for nucleic acid amplification, thus saving operator time and facilitating operator usage. Another advantage is to avoid any loss of target nucleic acid, particularly when the amount of sample is low.
• the solid support is already in the reaction vessel prior to the addition of said cellular sample.
• the method of amplification is a polymerase chain reaction.
• the method of amplification comprises reverse transcription polymerase chain reaction, isothermal amplification or quantitative polymerase chain reaction.
• the nucleic acid amplification reagent solution comprises a polymerase, deoxyribonucleotide triphosphate (dNTP), a reaction buffer and at least one primer, wherein said primer is optionally labeled with a dye.
• dyes may include fluorescence dye FAMTM or CyDye DIGE FluorTM from GE Healthcare.
• the nucleic acid amplification reagent solution can be present in a dried form, such as a Ready-to-GoTM (RTG) format.
• RTG Ready-to-GoTM
• the advantage of dried or lyophilized formulations of the polymerase chain reaction reagents is that they can be easily solublized 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 Illustra Ready-to-Go RT- PCR beads available from GE Healthcare.
• nucleic acid is selected from the group consisting of DNA,
• 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 chaotropic salt is a guanidine salt.
• said guanidine salt is selected from the group consisting of guanidine thiocyanate, guanidine chloride and guanidine hydrochloride.
• the chaotropic salt is sodium salt such as sodium iodide.
• the solid support is washed with an aqueous solution following step i).
• the solid support is selected from the group consisting of a glass or silica-based solid phase medium, a plastics-based solid phase medium, a cellulose-based solid phase medium, glass fiber, glass microfiber, silica gel, silica oxide, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetraflurorethylene, polyvinylidinefluoride, wool and porous ceramics.
• said cellulose based matrix is in the form of a pre punched disc.
• said cellulose based matrix is in the form of an FT ATM Elute card.
• said cellulose based matrix is in the form of an indicating FT ATM Elute (iFTAe) Card wherein the dye indicates the presence of a biological sample.
• iFTAe FT ATM Elute
• the amplified nucleic acid is quantified using a PCR imaging system.
• the cellular sample is selected from a group consisting of eukaryotic or prokaryotic cell, virus, bacteria, plant and tissue culture cells.
• said cellular sample is selected from the group consisting of blood, serum, semen, cerebral spinal fluid, synovial fluid, lymphatic fluid, saliva, buccal, cervical cell, vaginal cell, urine, faeces, hair, skin and muscle.
• 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 sample containing the nucleic acid may be derived from any source. This includes, for example, physiological/pathological body fluids (e.g.
• the method is for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool, a forensics tool, STR profiling tool and DNA profiling.
• the nucleic acid is stored on the solid support prior to step ii). In one aspect, the nucleic acid is stored on the solid support for at least 30 minute. The nucleic acid may be immobilised on the solid support for longer periods, for example, for at least 24 hours, 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.
• the method of the invention can be used either in single tube or a high-throughput
• the method is readily transferable to a multi-well format for high-throughput screening.
• the present invention can thus improve sample 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 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.
• the sample 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)
• steps i) to v) are carried out in the presence of the solid support.
• the solid support is already in the reaction vessel prior to the addition of said cellular sample.
• the method of amplification is a polymerase chain reaction.
• the lysis reagent is selected from the group consisting of a surfactant, detergent and chaotropic salt.
• the lysis reagent is selected from the group consisting of sodium dodecyl sulfate, guanidine thiocynate, guanidine chloride, guanidine hydrochloride and sodium iodide.
• the solid support is impregnated with sodium dodecyl sulfate (SDS), ethylenediaminetetracetic acid (EDTA) and uric acid.
• SDS sodium dodecyl sulfate
• EDTA ethylenediaminetetracetic acid
• uric acid uric acid
• the solid support is in the form of an FT ATM pre punched disc.
• the cellulose based matrix is in the form of an indicating FT ATM (iFTA) Card wherein the dye indicates the presence of a biological sample.
• iFTA indicating FT ATM
• the solid support is selected from the group consisting of a glass or silica-based solid phase medium, a plastics-based solid phase medium or a cellulose- based solid phase medium, glass fiber, glass microfiber, silica gel, silica oxide, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetraflurorethylene, polyvinylidinefluoride, wool or porous ceramics.
• the solid support is washed with an aqueous solution following step i).
• the amplified nucleic acid is quantified using a PCR imaging system.
• the cellular sample is selected from a group consisting of eukaryotic or prokaryotic cell, virus, bacteria, plant and tissue culture cells.
• the cellular sample is selected from the group consisting of blood, serum, semen, cerebral spinal fluid, synovial fluid, lymphatic fluid, saliva, buccal, cervical and vaginal cells, urine, faeces, hair, skin and muscle.
• 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 sample containing the nucleic acid may be derived from any source. This includes, for example, physiological/pathological body fluids (e.g.
• the method is for use as a tool selected from the group consisting of a molecular diagnostics tool, a human identification tool, a forensics tool, STR profiling tool and DNA profiling.
• the nucleic acid is stored on the solid support prior to step ii). In one aspect, the nucleic acid is stored on the solid support for at least 30 minute. The nucleic acid may be immobilised on the solid support for longer periods, for example, for at least 24 hours, 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.
• 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., (2011, 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 labor 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 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 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.
• the sample 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)
• a kit for amplifying nucleic acid as herein before described and instructions for use thereof is provided.
• Figure 1 presents the STR profile from the PCR amplification of unwashed HeLa cell spotted iFTAe (replicate no. 1).
• Figure 2 presents the STR profile from the PCR amplification of unwashed HeLa cell spotted iFTAe (replicate no. 2).
• Figure 3 presents the STR profile from the PCR amplification of unwashed HeLa cell spotted iFTAe (replicate no. 3).
• Figure 4 presents the STR profile from the PCR amplification of control DNA sample.
• Figure 5 presents the DNA yield from the qPCR amplification of washed blood spotted iFTAe amplified directly.
• Figure 6 presents the DNA yield from the qPCR amplification of unwashed HeLa cell spotted iFTAe either amplified directly or after being eluted.
• Figure 7 presents the amplification of DNA targets from homogenised museum samples applied to ambient storage FTA cards using optimised FTA Express
• Potential use of the proposed methods of the invention includes but are not limited to those discussed infra.
• FTA Express enables an efficient and loss-less/reduced-loss process in typical automated HID workflows.
• FTA Express enables an efficient and loss-less/reduced-loss process in typical automated HID workflows.
• the industry uses 1.2 mm FTA punches in either an Identifiler Direct or Powerplex 18D workflow where special amplification reaction mixtures and enzymes have been formulated to overcome the detergent chemistry contained in the non- washed FTA punch. The polymerase at this level of detergent would otherwise be denatured and render the STR reaction less efficient.
• the typical method is based on direct addition of one 1.2 mm punch into one tube or well containing a special STR formulation and having a total volume between 10- 25 ⁇ .
• the invention proposes adding one 1.2 mm punch directly to a high pH elution buffer where a substantial amount of the DNA is rendered single stranded and diffuses rapidly into the general volume of the solution.
• a volume of neutralizing agent can be added to provide a combined total volume of 100-200 ⁇ and containing approximately 0.05 to 0.2ng/ ⁇ genomic DNA.
• This stock solution of DNA can then be used for multiple standard STR reactions (example disclose using Powerplex 16 HS) where multiples of 5-10 ⁇ can be removed and added to parallel reactions. It is also possible to add a portion of the solution to qPCR reactions or general PCR reactions for amplification and adaption of amplicons for any molecular biology procedure.
• nucleic acid types that can be found in freshly spotted whole blood including small molecular weight nucleic acid species including microRNA, circulating nucleic acid (CNA), small viral genomes of various kinds and fragmented DNAs.
• small molecular weight nucleic acid species including microRNA, circulating nucleic acid (CNA), small viral genomes of various kinds and fragmented DNAs.
• CNA circulating nucleic acid
• conventional methods that teach or rely upon active washing and removal of denaturing chemistry are unsuitable to sustain such low molecular weight species on cellulose filters and other membranes.
• washing steps may simply wash off such nucleic acid types thereby resulting in their loss before any detection strategies can be applied.
• a feature of the present invention aims to eliminate such loss.
• CNAs are a pool of low molecular weight nucleosome-derived DNAs that are typically less than 200bp's and are described as being cell-free DNA. Circulating fetal- specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma and can emanate from the unborn fetus. Because of this they are viewed by the scientific community as a "window on fetal genetics" and is different to amniocentesis that has some associated risk to the fetus.
• Cell free nucleic acids have been proposed as biomarkers for various diseases exemplified by cancer, diabetes, Sickel Cell Disease, auto-immune diseases, myocardial infarction, Multiple Sclerosis etc. as and as well for particular physiological conditions such as intense physical exercise, hemodialysis, pregnancy. More, their presence or absence has also been linked to some clinical conditions such as trauma, sun bum, sepsis. Indeed, CNAs have been detected in many biological fluids, such as blood, urine, feces, milk, bronchial lavage and ascite. Refer to EP 2426217 Al.
• Fetal aneuploidy e.g., Down syndrome, Edward syndrome, and Patau syndrome
• other chromosomal aberrations affect 9 of 1 ,000 live births (Cunningham et al. in Williams Obstetrics, McGraw-Hill, New York, p. 942, 2002).
• abnormalities are generally diagnosed by karyotyping of fetal cells obtained by invasive procedures such as chorionic villus sampling or amniocentesis. Those procedures are associated with potentially significant risks to both the fetus and the mother. Noninvasive screening using maternal serum markers or ultrasound are available but have limited reliability (Fan et al., PNAS, 105 (42): 16266- 16271, 2008). In the coming years, the detection of cell free nucleic acid, such as circulating nucleic acids e.g.
• cirDNA DNA
• cirRNA cirRNA
• nucleosomes levels correlate with tumor stage and the presence of metastases is in gastrointestinal cancers.
• circulating nucleosomes like CNAs are likely to find use in monitoring cytotoxic therapy wherein strongly decreasing levels are mainly found in patients with disease remission while constantly high or increasing values can be associated with progressive disease during chemo- and radiotherapy.
• Holdenrieder supra reports that therapy outcome can be indicated by nucleosomal levels during the first week of chemo- and radiotherapy in patients with lung, pancreatic, and colorectal cancer as well as hematological malignancies.
• RNA-mediated gene silencing A large part of gene regulation in eukaryotes takes place at the level of RNA- molecules.
• miRNAs endogenous regulatory RNA-molecules
• sRNA small RNA molecules
• microRNA' s are a group of small regulatory RNA species that are typically 20 - 30nt in length and have been implicated in health and disease including controlled cellular differentiation or uncontrolled cellular proliferation. They are generated in cells from miRNA precursors as a result of a series of RNA processing steps. microRNAs (miRNAs) have been shown to regulate gene expression through both translational attenuation and messenger RNA (mRNA) degradation. These small noncoding RNAs typically target multiple genes simultaneously, inducing subtle but reproducible shifts in target gene expression. Although several basic questions regarding their biological principles still remain to be answered, many specific characteristics of microRNAs in combination with compelling therapeutic efficacy data and a clear involvement in human disease have triggered the biotechnology community to start exploring the possibilities of viewing microRNAs as therapeutic entities.
• miRNAs miRNAs
• RNAs-RNA molecules on the order of 100 nucleotides or fewer-from various tissues in many organisms, as well as cultured cells, is an area of extreme interest now, and promises to remain one for the future.
• Disease-associated changes of miRNA expression patterns may provide new prognostic and diagnostic opportunities as well as potential clinical markers.
• WO 2009/126650 discloses the use of mi- 126 and inhibitors of mi-126 for modulating angiogenesis.
• WO 2008/137867 ('867) describes compositions comprising miR-34 therapeutic agents for treating cancer.
• Alzheimers disease 14, p 27-41, 2008, describes the identification of miRNA changes in Alzheimer's disease brain and CSF yields putative biomarkers and insights into disease pathways.
• WO 2008/137867 discloses that miRNA expression is altered in Alzheimer's CSF and that that changes for miRNA in CSF of normal and Alzheimer's disease can be detected.
• WO 2008/154333 details methods and compositions for identifying genes or genetic pathways modulated by miR-34, using miR-34 to modulate a gene or gene pathway, as well as of using this profile in assessing the condition of a patient and/or treating the patient with an appropriate miRNA.
• MicroRNA's have also been implicated in a range of cancers. Lodes MJ, Caraballo M, Suciu D, et al. Detection of cancer with serum microRNAs on an oligonucleotide microarray. PLoS ONE. 2009;14:e622.
• RNA extraction is to remove protein and DNA, as these provide the greatest interference in the use of RNA.
• Lipid and carbohydrate moieties can usually be dissolved away with the aid of a detergent. Protein can be stripped off RNA (and DNA) with the aid of detergents and denaturants, but still must be removed from the common solution. See US Patent Application, US 2005/0059024 ( ⁇ 24).
• the second method proposes selectively immobilizing the RNA on a solid surface and rinsing the protein away, followed by releasing the RNA in an aqueous solution. While, both procedures can reduce the amount of DNA contamination or carryover, its efficiency varies with the precise conditions employed.
• phenol and phenol-chloroform extractions provide an extremely protein- and lipid-free solution of nucleic acid, much of the carbohydrate is also lost in this procedure.
• acid phenol-chloroform is known to extract some of the DNA out of the aqueous solution.
• the solution is high in denaturing agents such as guanidinium hydrochloride, guanidinium thiocyanate, or urea, all of which are incompatible with downstream enzymatic analysis.
• RNA is usually separated from these mixtures by selective precipitation, usually with ethanol or isopropanol. Note, however, that use of this procedure is very ineffective for small nucleic acid molecules, such as, for example, small RNAs.
• Solid-phase extraction on the other hand relies on high salt or salt and alcohol to decrease the affinity of RNA for water and increase it for the solid support used.
• glass sica
• the use of glass (silica) as a solid support has been demonstrated to work for large RNAs in the presence of high concentrations of denaturing salts - (U.S. Pat. Nos. 5,155,018;
• miRNAs Quantitative and qualitative isolation of miRNAs from various biological samples has been hampered for several reasons, including, but not limited to, labor-intensive and time consuming protocols; the nature of small size of miRNA invariably leads to easy loss of the targets during extraction; miRNAs, like other RNAs, are not stable and can be degraded easily during processing and storage; the miRNA detection rate from real patient samples is usually low or undetectable, i.e., not quantitative; and further the extracted miRNA targets normally are poorly correlated between related study objects.
• RNAs are present in a sample at low concentrations which are prone to be depleted during the purification steps or even getting lost.
• the art also makes clear that while it is possible to purify short RNAs from body fluids such as whole blood and also cell-free blood fractions (plasma or serum) by methods known in the prior art, this is done only with comparatively low yield.
• body fluids such as whole blood and also cell-free blood fractions (plasma or serum)
• plasma or serum cell-free blood fractions
• isolation reagents or kits available from, for example, QIAGEN-PAXGENE blood RNA kit, AMBION-LEUKOLOCK etc. US 20120/208189 argues that such reagents/kits suffer from one or more of the following drawbacks: low sensitivity and detectability; low yield; not reproducible; large sample volume requirement; microRNA extraction does not scale well; low throughput.
• Aged blood samples It is known that aged blood samples applied to FTA are often of poor quality with respect to the molecular integrity of the total RNA and DNA in the sample. However modern methods have been developed like next generation sequencing that can exploit fragmented nucleic acid. The method(s) of the invention would further support these methodologies by helping preserve the molecular integrity of aged nucleic acids for use in next generation sequencing methodologies by limiting the loss of appreciable amounts of informative nucleic acid that would undoubtedly occur using current FTA procedures that call for aggressive washing steps. What becomes clear from the above discussion is that current methods of isolating and preserving small nucleic acids such as CNA's, microRNAs, aged blood samples etc. were very unreliable and fraught with numerous drawbacks.
• Genomic DNA (Promega product code G152A);
• Harris Uni-core punch, 1.2mm (Sigma, Catalogue number Z708860-25ea, lot 3110);
• TaqMan RNase P Detection Reagents (Applied Biosystems part number 4316831) - contains RNase P primer; Quantifiler qPCR kit, Applied Biosystems Part: 4343895
• PowerPlex 18D (Promega code DC 1802) - contains primers
• PowerPlex 16 HS (Promega Code: DC2101 - contains primers;
• HeLa Huma cervical epithelial cells (ATCC code CCL-2) and
• Figure 1 shows STR profile of unwashed HeLa cell spotted indicating FTA elute card combined with STR PCR reagents (replicate 1). The average peak height was 2313 RFU.
• Figure 2 shows STR profile of unwashed HeLa cell spotted indicating FTA elute card combined with STR PCR reagents (replicate 2).
• the average peak height was 2260 RFU.
• Figure 3 shows STR profile of unwashed HeLa cell spotted indicating FTA elute card combined with STR PCR reagents (replicate 3). The average peak height was 5386 RFU.
• Figure 4 shows STR profile of purified genomic DNA with STR PCR reagents. The average peak height was 1904 RFU. Quantification of DNA from HeLa cell spotted and blood spotted FTA elute cards using qPCR (Table 5).
• 3mm punch was added into a 96 well PCR plate, 200 ⁇ 1 of sterile water was added to each well, the plate was sealed and pulse vortexed three times (5 seconds each). The plate was centrifuged at 1200rpm for 2min. The water was aspirated and discarded and 60 ⁇ 1 of sterile water was added to each well and the plate was sealed again. The plate was centrifuged at 1200rpm for 2 min and placed on a thermal cycler at 98°C for 30min. The plate was then pulse vortexed 60 times (one pulse/sec) using a vortex mixer set on maximum speed. The plate was centrifuged at 1200rpm for 2mins and the eluate was removed from the wells using a pipette and transferred to another plate/well for quantification.
• Table 5 shows the qPCR results of washed and unwashed blood spotted or cell spotted iFTAe card.
• the table shows the average yield of DNA from three qPCR reactions in ng/ ⁇ .
• the first 3 samples are replicates of DNA eluted from two 3mm punches of iFTAe cards and the 4 th sample is of a 1.2mm punch of blood spotted iFTAe card that was washed with 1ml of elution buffer and then amplified using real - time PCR (the data is the average of 3 separate samples).
• Samples 5 to 7 are replicates of DNA eluted from two 3mm punch of an iFTAe card spotted with HeLa cells and the 8 th sample is of a 3mm punch HeLa spotted iFTAe card that was washed with 1ml of elution buffer and then used in the real - time PCR machine.
• the last sample was of a 1.2mm punch of HeLa spotted iFTAe card that was not washed and used directly in the real - time PCR machine (the data is the average of 3 separate samples).
• An unspotted negative punch did not yield any detectable DNA.
• Table 5 qPCR results from the PCR amplification of samples of unwashed blood spotted and HeLa spotted iFTAe and pure blood samples.
• iFTAe card Cultured HeLa cells at a concentration of 7.54x10 6 cell/ml or whole blood was spotted onto an iFTAe card.
• a 1.2mm (HeLa and blood samples) punch was taken from the iFTAe card and washed with 1ml of sterile water, vortexed and water was removed or the sample was left unwashed.
• a 3mm (HeLa and blood samples) punch was taken from the iFTAe card and eluted using the iFTAe high throughput elution protocol. Either the sample spotted iFTAe card or 2 or 5 ⁇ 1 of the eluate was added to the qPCR reaction containing TaqMan Rnase P detection reagents and TaqMan Universal PCR master mix. The PCR sample mix was added to individual wells of a 96 well PCR plate prior to amplification.
• PCR reaction was set up as described above using Applied Biosystems 7900 Real- Time PCR System.
• Figure 5 shows DNA yield of washed blood spotted iFTAe cards used directly in a qPCR reaction. Three different batches of iFTAe cards were used in the experiment (A, B and C).
• Figure 6 shows DNA yield of unwashed HeLa cell spotted iFTAe cards either used directly in a qPCR reaction or eluated first and then used in a qPCR reaction. Three different batches of iFTAe cards were used in the experiment (A, B and C).
• STR PCR was carried out using 5 ⁇ and 10 ⁇ aliquots from the duplicate samples from both scales of total eluted volume (100 or 200 ⁇ ). The samples were then placed on a Geneamp 9700 Thermal cycler using the standard conditions in the instruction manual. All STR products were separated using a 3130x1 Genetic analyzer and the results analyzed using Genemapper ID software.
• Table 6 Presents the STR profiles from the PCR amplification of DNA eluted from blood spotted FTA cards using the FTA Express method, Shown are the average peak height (relative fluorescence units) for each loci using different volumes of eluate following high pH buffer wash. 100 & 200 represent the total volume of buffer used during the wash stage.
• Pass criteria - STR allelic peaks are expected to be higher than 75 RFU's heterozygote peaks and higher than 150 RFU's for homozygote peaks.
• Negative controls are expected to produce no profiles, i.e. no peaks on ladder above 50 RFU's.
• Positive controls are expected to be higher than 75 RFU' s for heterozygote peaks and higher than 150 RFU's for homozygote peaks.
• Table 7 The total number of alleles present after STR profiling using different volumes of eluate following high pH elution and neutralisation. The total number of alleles that were possible to obtain was noted only if a partial profile (PP) was obtained. A & B are represented as replicate, 100 & 200 represent the volume of the total buffer used.
• Table 8 Average yields returned for each elution volume used to extract DNA from blood spotted FTA cards. Both elution volumes returned an average yield close to 0.12 ng/ ⁇ .
• Solution 1 0.1N NaOH, 0.3mM EDTA, pH13.0
• Figure 7 shows results of amplification of DNA targets from homogenised museum samples applied to ambient storage FTA cards using optimised FTA Express methodology.
• the agarose gel of amplified samples shows that the approach of eluting DNA from FTA punches in the absence of any removal of impregnated chemistry by exhaustive washing provides DNA solutions suitable for successful PCR (lanes 1-3, 15- 17 and 29-31: standard wash; lanes 4-6, 18-20 and 32-34: single wash; lane 7: No washes + 2 % Neutralizer, Sscoxl-1/1-2 All; lane 21: No washes + 2 % Neutralizer, Sscoxl-1/1-3 All; lane 35: No washes + 2 % Neutralizer, Sscoxl-1/1-4 All; lane 8: No washes + 2 % Neutralizer products 138 bp; lane 22: No washes + 2 % Neutralizer products 265 bp; lane 36: No washes + 2 % Neutralizer products 451
• RNA species like miRNA are naturally small (-22 nucleotides) and are therefore easily lost during conventional extraction and purification steps.
• alkaline pH solutions catalyze 2'-hydroxyl trans- esterification reactions in RNA, but not DNA, and may exacerbate RNA degradation and loss.
• FTA cards were spotted with synthetic miRNAs and analyzed by RT-PCR using the FTA Express method as follows:
• RNA solution 0.1M NaOH, 0.3mM EDTA, pH 13.0
• Solution 1 0.1M NaOH, 0.3mM EDTA, pH 13.0
• Solution 2 0.1M Tris-HCl, pH 7.0
• Samples were further incubated for 5 minutes at room temperature, flash vortexed to mix, and the punch was removed from solution and pressed to recover a maximum volume of RNA solution ( ⁇ 100 ⁇ 1).
• RT reaction mixtures were prepared using the miScript RT kit (Qiagen, cat#218161). Briefly, 4 ⁇ 1 of 5x miScript HiSpec Buffer, 2 ⁇ 1 of lOx miScript Nucleics Mix, 2 ⁇ 1 of miScript RT Mix, 4 ⁇ 1 of 8% ot-cyclodextrin (w/v), 6 ⁇ 1 nuclease-free water, and 2 ⁇ 1 of FTA Express eluate (-2% of total volume extracted above) were combined in a 0.2ml tube. Control reactions were prepared in parallel using synthetic miRNAs at equivalent input. Reactions were incubated at 37°C for 1 hour, followed by 5 minutes at 95°C to inactivate enzymes.
• cDNA template preparation 4 ⁇ 1 of 5x miScript HiSpec Buffer, 2 ⁇ 1 of lOx miScript Nucleics Mix, 2 ⁇ 1 of miScript RT Mix, 4 ⁇ 1 of 8% ot-cyclodextrin (w/v), 6 ⁇ 1 nuclease-free
• a 1 :10 dilution of cDNA was prepared by adding 180 ⁇ 1 of nuclease-free water to each of the 20 ⁇ 1 RT reactions described above. This cDNA stock was serially diluted to 1 :100 and 1 :400 in nuclease-free water.
• qPCR reactions were prepared using the miScript SYBR Green PCR kit (Qiagen, cat#218073). Briefly, 12.5 ⁇ 1 of 2x SYBR Green PCR Master mix, 2.5 ⁇ 1 of lOx miScript Universal Primer, 5.5 ⁇ 1 of nuclease-free water, and 2.5 ⁇ 1 of lOx miScript Primer (Hs_miR21_2, cat#MS00009079 or Hs_Let7i_l , cat#MS00003157) were combined as a master mix. Diluted cDNA template (1:100 and 1:400 stocks) was added in 2 ⁇ 1 aliquots into a 96-well PCR plate and subsequently mixed with 23 ⁇ 1 of master mix per well.
• FTA cards spotted with MCF-7 total RNA were analyzed using the FTA Express method as follows:
• RNA solution 0.1M NaOH, 0.3mM EDTA, pH 13.0
• Solution 1 0.1M NaOH, 0.3mM EDTA, pH 13.0
• Solution 2 0.1M Tris-HCl, pH 7.0
• Samples were further incubated for 5 minutes at room temperature, flash vortexed to mix, and the punch was removed from solution and pressed to recover a maximum volume of RNA solution ( ⁇ 100 ⁇ 1).
• RT reaction mixtures were prepared using the miScript RT kit (Qiagen, cat#218161). Briefly, 4 ⁇ 1 of 5x miScript HiSpec Buffer, 2 ⁇ 1 of lOx miScript Nucleics Mix, 2 ⁇ 1 of miScript RT Mix, 4 ⁇ 1 of 8% ot-cyclodextrin (w/v), 6 ⁇ 1 nuclease-free water, and 2 ⁇ 1 of FTA Express eluate (-2% of total volume extracted above) were combined in a 0.2ml tube. Reactions were incubated at 37°C for 1 hour, followed by 5 minutes at 95°C to inactivate enzymes.
• cDNA template preparation 4 ⁇ 1 of 5x miScript HiSpec Buffer, 2 ⁇ 1 of lOx miScript Nucleics Mix, 2 ⁇ 1 of miScript RT Mix, 4 ⁇ 1 of 8% ot-cyclodextrin (w/v), 6 ⁇ 1 nuclease-free water, and 2 ⁇ 1 of FTA Express eluate (-
• a 1 :10 dilution of cDNA was prepared by adding 180 ⁇ 1 of nuclease-free water to each of the 20 ⁇ 1 RT reactions described above. This cDNA stock was serially diluted to 1 :100 and 1 :400 in nuclease-free water.
• Quantitative PCR detection qPCR:
• qPCR reactions were prepared using the miScript SYBR Green PCR kit (Qiagen, cat#218073). Briefly, 12.5 ⁇ 1 of 2x SYBR Green PCR Master mix, 2.5 ⁇ 1 of lOx miScript Universal Primer, 5.5 ⁇ 1 of nuclease-free water, and 2.5 ⁇ 1 of lOx miScript Primer
• Hs_miR21_2, cat#MS00009079 or Hs_Let7i_l, cat#MS00003157 were combined as a master mix.
• Diluted cDNA template (1:10, 1:100, and 1:400 stocks) was added in 2 ⁇ 1 aliquots into a 96-well PCR plate and subsequently mixed with 23 ⁇ 1 of master mix per well. Each sample and dilution was prepared in this manner in triplicate, and qPCR reactions were incubated in a 7500 Fast Real-Time PCR instrument according to the following parameters: 95°C/15min, (95°C/15sec, 55°C/30sec, 70°C/30sec) x 40 cycles.

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