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| Product Name | Ancient DNA (aDNA) Extraction Kit, Silica-Based, Bone and Teeth |
| Catalog No. | DREK-0034 |
| Description | Specialized silica-based extraction kit optimized for the recovery of highly fragmented, chemically modified ancient DNA (aDNA) from archaeological and paleontological samples including bone, teeth, dental calculus, and sediments. The protocol incorporates decalcification with EDTA, proteinase K digestion, and a modified silica binding method tailored for short DNA fragments (<100 bp) that are characteristic of ancient specimens. The kit includes specialized guanidinium-based binding buffer optimized for short fragment recovery, a nucleic acid carrier to enhance precipitation efficiency of trace quantities of DNA, and pre-treatment solutions for removing environmental contaminants including humic acids and other PCR inhibitors prevalent in ancient samples. All reagents are prepared in a dedicated clean facility and pre-screened for the absence of contemporary human DNA contamination. |
| Intended Use | Extraction of degraded, fragmented ancient DNA (<50-500 bp) from archaeological bone, teeth, dental calculus, paleofeces, and sediments for downstream applications including targeted PCR, NGS library preparation (with uracil-DNA glycosylase treatment for damage pattern analysis), whole genome shotgun sequencing, mitochondrial genome capture, and metagenomic analysis of ancient microbiomes. |
| Principle / Technology | Decalcification of bone/tooth powder in 0.5 M EDTA (pH 8.0) releases DNA from hydroxyapatite matrix over 24-48 hours at controlled temperature; proteinase K digestion removes collagen and other proteins; modified guanidinium-silica binding protocol with optimized ethanol concentration for retention of short DNA fragments (>=35 bp); nucleic acid carrier (linear polyacrylamide or glycogen) co-precipitates ultra-low quantities of DNA; multiple wash steps remove humic acids, fulvic acids, and other co-extracted inhibitors; elution in low-EDTA TE buffer. |
| Detection Method | Grind bone/tooth to fine powder (approximately 50-100 mg) using dedicated clean equipment in a controlled environment; incubate powder in 1 mL 0.5 M EDTA pH 8.0 for 24-48 h at 37 C or 56 C with agitation; add proteinase K (0.5 mg/mL final), digest 12-24 h at 37-56 C; centrifuge, retain supernatant; add binding buffer and carrier to supernatant; bind to silica column; wash with inhibitor removal buffer; wash 2x with ethanol-based wash buffer; dry column; elute in 30-50 uL pre-warmed TE (10 mM Tris, 0.1 mM EDTA, pH 8.0). |
| Sample Type | Archaeological bone (50-200 mg powder, cortical bone preferred — petrous portion of temporal bone optimal), teeth (dental pulp or dentine powder 50-100 mg), dental calculus (10-50 mg), sediment (0.5-5 g after pre-treatment), paleofeces (50-200 mg). Samples should be handled under strict ancient DNA protocols (dedicated clean room, full PPE, UV decontamination, negative controls at every step). |
| Performance Range / Specifications | DNA yield: variable (0-500 ng per gram of bone powder depending on preservation); fragment size: 35-500 bp (median typically 50-100 bp for Pleistocene samples, longer for well-preserved Holocene specimens); A260/A280 ratio 1.6-1.9; PCR inhibitor removal efficiency >99% of humic acids. |
| Sensitivity / LOD | Recovery of DNA fragments as short as 35 bp; capable of extracting DNA from samples with <0.1% endogenous DNA content; Qubit fluorometric detection of dsDNA as low as 0.01 ng/uL. |
| Specificity | Silica binding in high chaotropic salt and optimized ethanol concentration is selective for >35 bp double-stranded and single-stranded DNA; EDTA decalcification specific for calcium phosphate (hydroxyapatite) matrix dissolution; proteinase K specific for protein digestion; carrier precipitation non-specific — also precipitates contaminant DNA if present. |
| Reaction Conditions / Protocol | EDTA decalcification: 24-48 h at 37-56 C; proteinase K digestion: 12-24 h; binding, washing, elution: 1-2 h; total protocol: 2-4 days (most time is incubation). |
| Components / Formulation | EDTA Solution (0.5 M, pH 8.0, 50 mL), Proteinase K (lyophilized, 100 mg), Digestion Buffer (10x, 15 mL), Binding Buffer with Carrier (25 mL), Inhibitor Removal Buffer (15 mL), Wash Buffer (concentrate, 10 mL — add ethanol before use), Elution Buffer (TE, pH 8.0, 5 mL), Silica Membrane Spin Columns with Collection Tubes (50 columns), Certificate of Human DNA-Free (QC lot-specific). |
| Storage Conditions | EDTA solution at RT; Proteinase K at -20 C; all other components at RT (15-25 C); after addition of ethanol to Wash Buffer, store at RT. |
| Shelf Life | 18 months from date of manufacture; reconstituted Proteinase K stable at 2-8 C for 12 months. |
| Package Specifications | 20 preparations, 50 preparations. |
| Product Form | Liquid buffers; lyophilized proteinase K; silica membrane spin columns. |
| Quality Control | Each lot tested for: DNA recovery from pre-extracted aDNA standard (40-150 bp fragments): >=70% recovery; inhibitor removal: >99% humic acid removal; absence of amplifiable human DNA by qPCR (Alu repeat assay, 35 cycles Cq >35 or undetermined); absence of DNase and RNase contamination; pH and conductivity verification of all buffers. |
| Key Features | Optimized for short ancient DNA fragments; decalcification protocol included; inhibitor removal for humic acids; carrier for ultra-low DNA recovery; pre-screened for contemporary human DNA absence; dedicated clean manufacturing facility. |
| Purity | Human DNA-free by qPCR; DNase/RNase-free; inhibitor-free (humic acid removal >99%); DNA fragment size enriched 35-500 bp. |
| Concentration | Variable yield dependent on sample preservation; elution volume 30-50 uL; typical [DNA] 0.01-5 ng/uL. |
| Activity / Unit Definition | Proteinase K: >=30 mAnson units/mg; activity verified after reconstitution. |
| Molecular Weight | Proteinase K: approximately 28.9 kDa. |
| Source / Origin | All reagents manufactured in DNA-free facility; proteinase K from recombinant source (Tritirachium album); silica membrane synthetic; EDTA and buffer salts: molecular biology grade. |
| pH Range / Optimal pH | EDTA decalcification pH 8.0; proteinase K digestion pH 8.0-8.5; binding buffer pH 5.0-6.0; elution buffer pH 8.0. |
| Shipping Conditions | Ambient temperature; Proteinase K on blue ice. |
| Expiration Date / Stability | 18 months for buffers and columns; proteinase K stable at -20 C for 24 months; reconstituted proteinase K stable at 2-8 C for 12 months. |
| Regulatory / Compliance | For research use only; not for diagnostic, forensic, or clinical use. Users must comply with all applicable laws and regulations regarding the handling and export of archaeological and cultural heritage samples. Ethical approvals and sample export permits are the user's responsibility. |
| Compatibility | Eluted aDNA compatible with: NGS library preparation (NEBNext, AccuPrime, KAPA — use uracil-DNA glycosylase treatment for authentic aDNA damage pattern verification), targeted enrichment (myBaits, SureSelect for mtDNA or nuclear capture), PCR (AmpliTaq Gold, Platinum Taq HiFi, Phusion with appropriate primers for short amplicons), qPCR with short amplicon design (<100 bp). Avoid using high-fidelity polymerases with proofreading activity for direct PCR of non-UDG-treated aDNA as they stall at deaminated cytosines (uracils). |
| Recommended Buffer System | EDTA: 0.5 M EDTA disodium, adjusted to pH 8.0 with NaOH; digestion: 50 mM Tris-HCl pH 8.0, 50 mM EDTA, 1% SDS, 0.5 mg/mL proteinase K; binding: guanidine HCl, sodium acetate, isopropanol, carrier; inhibitor removal: Tris-HCl, guanidine HCl, isopropanol; elution: 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA. |
| Application Notes / Precautions | Ancient DNA research requires specialized laboratory infrastructure: physically isolated pre-PCR clean room with positive air pressure, HEPA filtration, UV decontamination, full PPE (full-body suit, double gloves, face mask, hair net, shoe covers), and dedicated equipment. Include multiple extraction blank controls (no sample powder) and PCR negative controls in every batch. For highly degraded samples, use the entire DNA extract for library preparation without quantification to minimize losses. The petrous portion of the temporal bone and dental cementum typically yield the highest proportion of endogenous human DNA. Fragment length distribution should be assessed by Bioanalyzer or TapeStation prior to library preparation. Store extracted aDNA at -20 C (short term) or -80 C (long term). |
| Batch-to-Batch Consistency | DNA recovery of spiked control oligonucleotides (40 bp) >70%; inhibitor removal >99% humic acids; human DNA-free by qPCR. |
For research use only, not for clinical use.
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