Degraded, inhibited bone extract: re-extract or amplify now?
A femur fragment recovered from a shallow grave is submitted for identification. The laboratory grinds 2 g of bone, demineralizes it, and extracts with a silica-based method, eluting in 50 µL. Real-time PCR quantitation targeting a short (approximately 100 bp) segment gives 0.006 ng/µL. The internal positive control shows a 2.4-cycle delay relative to the calibrator, and the ratio of short-amplicon to long-amplicon signal is 9. The extract is dark brown. The amplification protocol uses 15 µL of extract in a 25 µL reaction at 28 cycles. A reference sample from the presumptive mother quantifies at 0.9 ng/µL with an IPC delay of 0.1 cycles. The disaster-victim identification deadline is two weeks away.
1. Interpret the quantitation results. What do the concentration, IPC delay, and amplicon ratio indicate about this sample?
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The concentration is 0.006 ng/µL, which over a 50 µL elution is about 0.3 ng total. That is low but not zero.
The 2.4-cycle IPC delay means the internal control amplified later than the calibrator, which is the signature of PCR inhibition. The brown color of the extract supports co-extraction of humic or other inhibitory material.
The short-to-long amplicon ratio of 9 means the long product is roughly nine-fold reduced relative to the short product, which is consistent with degradation: the DNA is fragmented, so long amplicons are lost first.
So the sample is both inhibited and degraded. The reported concentration may be an underestimate, because inhibition suppresses the very signal used to quantify it. Treating 0.006 ng/µL as a precise value would be misleading. The mother's reference is clean (0.9 ng/µL, negligible IPC delay), so the problem is sample-specific, not a failed run.
2. Should the laboratory amplify this extract now, re-extract, or purify and concentrate? Justify your choice with numbers.
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Do not amplify this extract as it stands.
At 0.006 ng/µL, a 15 µL input delivers about 0.09 ng (90 pg), well below the roughly 0.5–1 ng target for a robust autosomal profile, and the same 15 µL carries a concentrated dose of inhibitor. Raising the input volume is not possible beyond the reaction's validated limit, and concentrating the current extract would concentrate the inhibitor along with the DNA.
Prefer re-extraction from a larger mass of bone (for example 5 g) with thorough demineralization and an inhibitor-removal or silica clean-up step, eluting in a smaller volume such as 20–30 µL. That addresses both problems at once rather than trading one for the other.
If the bone is exhausted, purify the existing extract through an inhibitor-removal column and re-quantify before deciding on input. Verify improvement by a reduced IPC delay and a lower amplicon ratio, not by concentration alone.
3. If amplification must proceed, what modifications and controls would you require?
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Proceed only under a validated low-template protocol.
- Use the maximum validated input volume, with the remaining volume made up with water or dilution buffer, rather than exceeding the validated fraction of the reaction.
- Increase cycle number (for example to 29–30) only if the laboratory has validated that cycle number for sensitivity, stutter, and drop-in behavior. Unvalidated extra cycles generate artifacts that mimic alleles.
- Consider a mini-STR or SNP-based assay designed for fragmented DNA, since the amplicon ratio predicts poor performance at longer loci.
- Run an extraction negative, an amplification negative, a reagent blank, and a low-input positive control (for example 0.1 ng) in the same batch to monitor sensitivity and contamination simultaneously.
- Expect and document allelic drop-out; a locus with no result should not be reported as a homozygous genotype.
If the resulting profile is partial, comparison to the maternal reference should be framed as a kinship assessment rather than a direct match.