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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
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  • 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
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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  • 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
• • 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 teachings generally relate to methods of directly amplifying nucleic acids from crude samples.
• the present teachings provide a method of performing a polymerase chain reaction (PCR) comprising; providing a crude sample comprising: deoxyribonucleic acid; optionally incubating the crude sample with NaOH at 5 mM to 25mM; mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and performing a PCR on the deoxyribonucleic acid, wherein the direct buffer comprises at least 3%-8% glycerol, 0.2%-0.9% non-ionic surfactants, and 1000-3000 ug/ml BSA.
• Suitable non- ionic surfactants include, but not limited to, polysorbate (Tween), Tween 20, Triton-X 100 and the like.
• the present teachings provide methods of performing a polymerase chain reaction (PCR) comprising; providing a crude sample comprising: deoxyribonucleic acid; optionally incubating the crude sample with NaOH at 5 mM to 25 mM; mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and performing PCR on the solution, wherein the direct buffer comprises at least 5 PCR primer pairs, 10- 50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1000-3000 ug/ml BSA, and 0.10-0.35 U/ul of DNA polymerase.
• PCR polymerase chain reaction
• the present teachings provide methods of determining the identity of a human comprising; providing a crude sample comprising deoxyribonucleic acid from the human; optionally incubating the crude sample with NaOH at 5 mM to 25 mM; mixing the crude sample with the direct buffer forming a nucleic acid containing solution; wherein the direct buffer comprises a plurality of primer pairs; wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); performing PCR on the solution to form a plurality of PCR amplicons; wherein each PCR amplicon has an ascertainable size; and identifying the human by reference to size of the PCR amplicons; wherein the direct buffer further comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100- 350 uM of each dNTPs,
• nucleic acids for a downstream enzymatic manipulation comprising; providing a crude sample comprising: deoxyribonucleic acid; optionally incubating the crude sample with NaOH at 5 mM to 25 mM; mixing the crude sample with a direct buffer to form a nucleic acid containing solution; and performing a downstream enzymatic manipulation on the solution, wherein the direct buffer comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1000-3000 ug/ml BSA, and 0.10-0.35U/ul of DNA polymerase.
• the downstream enzymatic manipulation is PCR.
• kits for genetic analysis comprising; a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); and a direct buffer, wherein the direct buffer comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1000-3000 ug/ml BSA, and 0.10 -0.35U/ul of DNA polymerase.
• the kits as set forth above can optionally include NaOH or other strong alkaline compounds, or other agents for cell lysis.
• kits comprising; a direct buffer and a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR), and wherein the direct buffer comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1200-3000 ug/ml BSA, and 0.10 - 0.35 U/ul of DNA polymerase.
• the kits as set forth above can optionally include NaOH or other strong alkaline compounds, or other agents for cell lysis.
• Figure 1 is an electropherogram of an STR profile of genomic DNA from a blood sample.
• Figures 2A-2D are electropherograms of failed STR profiles of four blood samples spotted onto FTA paper.
• Figures 3A through 12B are comparative electropherograms of STR profiles from 10 individuals whose blood was spotted onto FTA paper, a punch of which was placed into the direct buffer of the present teachings herein and designated by "A". While the electropherograms designated by "B” represent STR profiles of the same 10 individuals' blood prepared using the PowerPlex 16 HS purification system sold by Promega. Description of Exemplary Embodiments
• the term "crude sample” refers to a specimen of biological origin suspected of containing nucleic acids, which has not undergone procedures for the isolation or purification of those nucleic acids.
• a sample of blood is a crude sample.
• the cells in crude sample can optionally be lysed.
• a sample of blood spotted on paper containing a lysis reagent, such as FTA paper (commercially available from Whatman) is a crude sample.
• a buccal swab of cheek cells is another example of a crude sample.
• Crude samples include, but are not limited to, blood, diluted blood, blood on paper, buccal swabs, and buccal swabs on a substrate for samples storage, such as FTA paper.
• FTA paper commercially available from Whatman
• the term "direct buffer” refers to a buffer into which a crude sample can be placed.
• the direct buffer contains primers and enzyme for performing a downstream enzymatic manipulation, such as a polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• the direct buffer allows for the liberation of the nucleic acids and for their amplification directly in the direct buffer, without the need for any nucleic acid isolation or purification. Illustrative cycling times and temperatures for PCR can be found in Sambrook et al., Molecular Cloning, 3 rd Edition 1993.
• the direct buffer of the present teachings focus on the use of the direct buffer for PCR, it will be appreciated that one of skill in the art can easily employ the direct buffer of the present teachings as a front-end procedure for other types of downstream enzymatic manipulations, for example reverse transcription using a reverse transcriptase, or an oligonucleotide ligation assay using a ligase.
• downstream when used in reference to methods and manipulations performed on a target nucleic acids refers to methods and manipulations performed on a target nucleic acid sample subsequent to a method for liberating a nucleic acid from a biological sample, including, but not limited to, lysis of a cell to release the nucleic acid from the cell.
• downstream enzymatic manipulation refers to procedures performed on a nucleic acid sample, including, but not limited to PCR using a polymerase, reverse transcription using a reverse transcriptase, or an oligonucleotide ligation assay using a ligase.
• strong alkaline compound as used herein includes, but is not limited to, NaOH. Strong alkaline compounds can be used to lyse cells prior to the addition of some embodiments of direct buffer.
• genomic DNA refers to the chromosomal DNA sequence of a gene or segment of a gene, including the DNA sequences of non-coding as well as coding regions. Genomic DNA also refers to DNA isolated directly from cells or chromosomes or the cloned copies of all or part of such DNA.
• allele refers to a genetic variation associated with a gene or a segment of DNA, i.e., one of two or more alternate forms of a DNA sequence occupying the same locus.
• locus refers to a specific position on a chromosome or a nucleic acid molecule. Alleles of a locus are located at identical sites on homologous chromosomes.
• STR locus refers to any region of the human genome which contains short, repetitive sequence elements of 3 to 7 base pairs in length.
• genomic locus containing a short tandem repeat refers to an STR locus in which the number of repetitive sequence elements (and net length of sequence) in a particular region of genomic DNA varies from allele to allele, and from individual to individual.
• amplification primer and “primer” refers to an oligonucleotide, which is capable of site-specifically annealing to an RNA or DNA region adjacent a target sequence, and serving as an initiation primer for DNA synthesis under suitable conditions in which synthesis of a primer extension product is induced, e.g., in the presence of nucleotides and a polymerization-inducing agent such as a DNA-dependent DNA polymerase and at suitable temperature, pH, metal concentration, and salt concentration.
• a polymerization-inducing agent such as a DNA-dependent DNA polymerase and at suitable temperature, pH, metal concentration, and salt concentration.
• a PCR reaction employs a pair of amplification primers also known as “primer pairs” including an "upstream” or “forward” primer and a “downstream” or “reverse” primer, which delimit a region of the RNA or DNA to be amplified.
• primer pairs also known as “primer pairs” including an "upstream” or “forward” primer and a “downstream” or “reverse” primer, which delimit a region of the RNA or DNA to be amplified.
• amplifying refers to a process whereby a portion of a nucleic acid is replicated using, for example, any of a broad range of primer extension reactions.
• primer extension reactions include, but are not limited to, PCR.
• amplifying refers to a single replication or to an arithmetic, logarithmic, or exponential amplification.
• amplicon and "PCR amplicon” as used herein refers to a broad range of techniques for increasing polynucleotide sequences, either linearly or exponentially and can be the product of an amplification reaction.
• An amplicon can be double-stranded or single-stranded, and can include the separated component strands obtained by denaturing a double-stranded amplification product.
• the amplicon of one amplification cycle can serve as a template in a subsequent amplification cycle.
• Exemplary amplification techniques include, but are not limited to, PCR or any other method employing a primer extension step.
• amplification examples include, but are not limited to, ligase detection reaction (LDR) and ligase chain reaction (LCR).
• LDR ligase detection reaction
• LCR ligase chain reaction
• Amplification methods can comprise thermal-cycling or can be performed isothermally.
• the term "amplification product" includes products from any number of cycles of amplification reactions.
• nucleic acid sequence can refer to the nucleic acid material itself and is not restricted to the sequence information (i.e. the succession of letters chosen among the five base letters A, C, G, T, or U) that biochemically characterizes a specific nucleic acid, for example, a DNA or RNA molecule.
• polynucleotide refers to a linear polymer of natural or modified monomers or linkages, including deoxyribonucleic acid, deoxyribonucleosides, ribonucleosides, polyamide nucleic acids, and the like, joined by inter-nucleosidic linkages and have the capability of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, and capable of being ligated to another oligonucleotide in a template-driven reaction.
• oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several hundreds of monomeric units.
• a polynucleotide such as an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5' ⁇ 3' order from left to right and that "A” denotes deoxyadenosine, "C” denotes deoxycytidine, “G” denotes deoxyguanosine, and "T” denotes deoxythymidine, unless otherwise noted.
• A, C, G, and T can be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.
• the inter-nucleoside linkage is typically a phosphodiester bond, and the subunits are referred to as "nucleotides.”
• sequence determination includes determination of partial as well as full sequence information. That is, the term includes sequence comparisons, fingerprinting, and like levels of information about a target polynucleotide, as well as the express identification and ordering of each nucleoside of the target polynucleotide within a region of interest.
• sequence determination comprises identifying a single nucleotide, while in other embodiments more than one nucleotide is identified. Identification of nucleosides, nucleotides, and/or bases are considered equivalent herein. It is noted that performing sequence determination on a polynucleotide typically yields equivalent information regarding the sequence of a perfectly complementary polynucleotide and thus is equivalent to sequence determination performed directly on a perfectly complementary polynucleotide.
• oligonucleotide probes includes sets of two or more oligonucleotide probes where there may be a single "common” oligonucleotide probe that is usually specific for a non-variable region of a target polynucleotide and one or more "wild-type” and/or “mutant” oligonucleotide probes that are usually specific for a region of a target polynucleotide that contains allelic or mutational variants in sequence.
• Nucleic acid polymerase or “polymerase” refers to any polypeptide that catalyzes the synthesis of a polynucleotide using an existing polynucleotide as a template.
• DNA polymerase refers to a nucleic acid polymerase that catalyzes the synthesis of DNA using an existing polynucleotide as a template.
• the present teachings include direct buffers comprising 3%-8% glycerol, 0.2%-0.9% non-ionic surfactants, and 1200-3000 ug/ml BSA.
• the present teachings include a direct buffers comprising 10-50 mM Tris- HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% non-ionic surfactants, 1200-3000 ug/ml BSA, and 0.10l-0.35U/ul of DNA polymerase.
• a direct buffers comprising 10-50 mM Tris- HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% non-ionic surfactants, 1200-3000 ug/ml BSA, and 0.10l-0.35U/ul of DNA polymerase.
• the non-ionic surfactants can be polysorbate (Tween, Tween 20), Triton-X 100 and the like.
• DNA polymerase is commercially available from Applied Biosystems. Suitable DNA polymerases include Pol A, Pol B, Pol C, Pol D, Pol X, and Pol Y DNA polymerases and a Type I, Type Il and Type III DNA polymerase. Generally, thermostable DNA polymerases such as Taq, Pfu, VentTMDeepVentTM, 9 Q NorthTM, are used in PCR. The DNA polymerases may be modified so as to be inactive prior to subsequent treatment, e.g.
• DNA polymerases are known to those skilled in the art. DNA polymerases include DNA-dependent polymerases, which use DNA as a template, or RNA-dependent polymerases, such as reverse transcriptase, which use RNA as a template.
• DNA-dependent DNA polymerases fall into one of six families (A, B, C, D, X, and Y), with most falling into one of three families (A, B, and C). See, e.g., lto et al. (1991 ) Nucleic Acids Res. 19:4045- 4057; Braithwaite et al. (1993) Nucleic Acids Res. 21 :787-802; Filee et al. (2002) J. MoI. Evol. 54:763-773; and Alba (2001 ) Genome Biol.
• Certain DNA polymerases may be single-chain polypeptides (e.g., certain family A and B polymerases) or multi-subunit enzymes (e.g., certain family C polymerases) with one of the subunits having polymerase activity.
• a fusion protein may comprise a DNA polymerase selected from a family A, B, C, D, X, or Y polymerase.
• Pol A Family A polymerases
• Replicative members from this family include T7 DNA polymerase and the eukaryotic mitochondrial DNA Polymerase y.
• repair polymerases are E. coll DNA Pol I, Thermus aquaticus Pol I (Taq DNA polymerase), and Bacillus stearothermophilus Pol I. Excision repair and processing of Okazaki fragments generated during lagging strand synthesis are performed by the repair polymerases Because most thermostable Pol A enzymes do not possess the 3' to 5' exonuclease activity, they are incapable of proofreading the newly synthesized nucleic acid strand and consequently have high error rates.
• Pol B Family B polymerases
• Pol B polymerases are substantially replicative polymerases including the major eukaryotic DNA polymerases ⁇ , ⁇ , ⁇ , and also DNA polymerase ⁇ .
• Pol B polymerases also include DNA polymerases encoded by some bacteria and bacteriophages, of which the best characterized are from T4, Phi29 and RB69 bacteriophages.
• Pol B enzymes are involved in both leading and lagging strand synthesis and are noteworthy for their remarkable accuracy during replication as many have strong 3'-5' exonuclease activity the exceptions being DNA polymerase ⁇ and ⁇ which lack proofreading activity.
• BSA is commercially available from a variety of sources, for example catalog number 1071 1454001 from Roche.
• FTA paper is commercially available from Whatman. In some embodiments, FTA paper is used herein with bloodstains. For example, Bloodstain Card from Whatman (Cat# WB 10 0014).
• the FTA paper containing blood or buccal cells is cut out to remove a small region containing biological material, e.g., a circular punch of 0.5mm to 1.2mm in diameter, or1.0-1.5mm in diameter, or 1.5-2.0mm in diameter.
• the paper is placed directly into the PCR mix comprising the direct buffer for PCR reaction. In some embodiments, a wash of the FTA paper is not required before placing the FTA paper in the direct buffer.
• a NaOH solution at 5-25 mM is incubated with non-FTA before mixing with the direct buffer and performing a PCR reaction. In other embodiments, the cells may be lysed by exposure to reagents other than NaOH.
• the direct buffer further comprises a plurality of PCR primer pairs.
• the direct buffer comprises 5 primer pairs.
• the direct buffer comprises 10 primer pairs.
• the direct buffer comprises greater than 10 primer pairs.
• the direct buffer does not comprise PCR primer pairs, but rather the PCR primer pairs are added at a separate time.
• Some embodiments of the subject methods eliminate the need for a sample purification procedure prior to performing PCR.
• the same protocol can be applied to both blood and buccal cell samples on FTA cards.
• the physical sample handling is significantly reduced, therefore this can prevent possible cross-contamination between samples as well as eliminate the possibility of human errors.
• the amount of time required from FTA card to STR profiles can be reduced by at least 1 1/2 hours.
• the method is compatible with the current automation instruments available in the forensic market.
• the costs associated with the purification of DNA from FTA can be eliminated.
• the success rate of getting a full STR profile is comparable when compared to the traditional standard sample preparation method.
• a sample of genomic DNA used directly with the Applied Biosystems Identifier HID v.1 Kit provides a full STR profile.
• the STR profile is either partial or undetectable when blood from genomic DNA is taken from blood spotted on an FTA disc punch (1.2 mm punch) without washing the disc to remove materials which interfere with subsequent sample testing, e.g., STR profiling via direct PCR, see Figures 2A-2D.
• buccal swab samples were collected and placed in 50OuI TE buffer. The resulting suspension was heated at 97 0 C for 5 minutes. 10 ul of the resulting suspension was used to set up a PCR in the direct buffer.
• kits designed to expedite performing certain methods.
• kits serve to expedite the performance of the methods of interest by assembling two or more components used in carrying out the methods.
• kits may contain components in pre-measured unit amounts to minimize the need for measurements by end-users.
• kits may include instructions for performing one or more methods of the present teachings.
• the kit components are optimized to operate in conjunction with one another.
• the present teachings provide a kit comprising; a plurality of primer pairs, wherein each primer pair flanks a genomic locus containing a short tandem repeat (STR); and a direct buffer, wherein the direct buffer comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% non-ionic surfactants, 1200-3000 ug/ml BSA, and 0.10 -0.35 U/ul of AmpliTaq Gold DNA polymerase.
• the direct buffer comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.
• kits, methods and reaction mixtures provided by the present teachings can be used with procedures for multiplexed PCR of degraded samples, as found for example in WO05054515 to Dimsoski and Woo.
• the direct buffer in the kit comprises 0.2%-0.9% polysorbate, 3%-8% glycerol, 1200-3000 ug/ml BSA.
• the direct buffer in the kit further comprises 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI, 1.4-2.4 mM MgCI 2 , 0.01 %-0.04% Sodium azide, 3%-8% glycerol, 100-350 uM of each dNTPs, 0.2%-0.9% polysorbate, 1200-3000 ug/ml BSA, and 0.10 -0.35U/ul of AmpliTaq Gold DNA polymerase.
• the direct buffer in the kit feature comprises NaOH at 5 mM-25 mM.
• the NaOH is supplied in a vial separate from the direct buffer.

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