Low template dna

What is low-template DNA (ltDNA)?

DNA samples containing a very small quantity of DNA available for analysis.

What are the two main reasons a DNA sample may be low-template?

The original sample contains very little DNA, or DNA has been lost or degraded during collection, storage or processing.

Why is low-template DNA challenging to analyse?

There is a greater chance of stochastic effects during PCR, making DNA profiles incomplete or difficult to interpret.

What is the most important consideration when analysing low-template DNA?

The method of interpretation is particularly important because results may be affected by stochastic effects.

What is locus drop-out?

When an entire genetic locus fails to amplify and produces no detectable result.

What is allele drop-out?

When one allele at a heterozygous locus fails to amplify to a detectable level.

What is allele drop-in?

When additional alleles appear in a DNA profile that are not genuinely part of the contributor's profile.

What is a stutter peak?

A PCR artefact that produces a peak usually one repeat unit shorter than the true allele.

What happens to stutter peaks in low-template DNA samples?

Stutter peaks may increase in size relative to the true allele, making interpretation more difficult.

Why does allele drop-out occur?

Insufficient DNA molecules may be available for amplification, meaning an allele is not detected.

Why does allele drop-in occur?

Additional DNA or sporadic amplification products may produce unexpected alleles.

What is stochastic variation?

Random variation in amplification caused by low quantities of starting DNA.

What happens to the risk of stochastic effects as DNA quantity decreases?

The risk increases.

What is the approximate DNA quantity associated with the traditional LCN threshold discussed in the lecture?

Approximately 100 pg, although definitions and thresholds vary.

Why is there no universal definition of low-template DNA?

Definitions vary depending on DNA quantity, laboratory methods and interpretation approaches.

What are additional anti-contamination measures used for?

To reduce the risk of introducing foreign DNA into low-template samples.

Why are contamination controls especially important for ltDNA?

Even a very small amount of contaminating DNA may affect the resulting profile.

What are the main stages of forensic DNA analysis?

DNA sampling, extraction, quantification, amplification, visualisation and interpretation.

How can PCR be enhanced for low-template DNA?

Increase the number of PCR cycles, reduce reaction volume and use PCR enhancers.

How many PCR cycles are discussed for enhanced amplification of low-template DNA?

Approximately 30–34 cycles.

Why are additional PCR cycles used?

To increase the amplification of small quantities of DNA.

What is a disadvantage of increasing PCR cycles?

It can increase stochastic effects and PCR artefacts.

Why might the PCR reaction volume be reduced?

To increase the ratio of DNA to PCR reaction components.

What is an example of a reduced PCR reaction volume discussed in the lecture?

As little as 5 microlitres.

What are PCR enhancers?

Additional substances that improve the efficiency of DNA amplification.

When might PCR enhancers be particularly useful?

When samples contain high GC content, homopolymeric repeats or PCR inhibitors.

What is post-PCR clean-up?

A process that removes unwanted components from a PCR product before capillary electrophoresis.

Why is post-PCR clean-up useful for low-template DNA?

It reduces competition during electrokinetic injection, allowing more DNA to enter the capillary.

What is one advantage of post-PCR clean-up?

It can increase peak heights by allowing more DNA to enter the electrophoresis capillary.

What is one disadvantage of post-PCR clean-up?

There is a risk of losing some DNA during the cleaning process.

Does post-PCR clean-up introduce a high risk of contamination?

No, it has a relatively low contamination risk.

How can capillary electrophoresis (CE) be enhanced for low-template DNA?

Use highly deionised formamide and increase injection time or voltage.

Why is highly deionised formamide used?

It helps prepare DNA for electrophoresis by reducing unwanted ionic interference.

How can increasing injection voltage improve DNA detection?

It can increase the amount of DNA entering the capillary.

What enhanced CE conditions are described by Cowen et al. (2011)?

4 kV for 30 seconds compared with standard conditions of 3 kV for 10 seconds.

What improvement was reported using enhanced CE conditions?

Approximately 50% of previously dropped-out alleles were detected.

What does RFU stand for?

Relative Fluorescence Units.

What does an RFU threshold represent?

The minimum fluorescence signal required for a peak to be considered detectable.

What RFU threshold is mentioned in the lecture?

25 RFU is often used as a standard threshold in the discussed context.

Why might standard RFU thresholds be reduced for low-template DNA?

To detect weaker peaks that may represent genuine alleles.

Why is reducing RFU thresholds risky?

It may increase the likelihood of interpreting artefacts or contamination as genuine alleles.

Why is designating a low-template DNA profile as homozygous risky?

One allele may have dropped out, making a heterozygous individual appear homozygous.

What is consensus profiling?

A method involving multiple independent amplifications of the same DNA extract to identify reliable alleles.

How many amplifications are required in the consensus method described?

At least two independent amplifications.

How are results assessed during consensus profiling?

DNA profiles are analysed independently and then compared.

Which alleles are recorded during consensus profiling?

Only alleles observed in at least two amplifications.

Why is consensus profiling useful?

It helps distinguish genuine alleles from sporadic amplification artefacts.

Which artefact is consensus profiling particularly useful for reducing?

Allele drop-in.

By approximately how much can consensus profiling reduce allele drop-in?

At least 95%, according to the study discussed in the lecture.

What is direct PCR?

A technique where a sample is added directly into the PCR reaction without a separate DNA extraction step.

What are the main advantages of direct PCR?

Minimises sample loss, reduces contamination opportunities, saves time and reduces reagent costs.

Why does direct PCR reduce sample loss?

It removes the need for separate extraction and transfer steps.

What is a disadvantage of direct PCR?

Any PCR inhibitors present in the sample enter the PCR reaction.

Why is the lack of a quantification step a disadvantage of direct PCR?

The amount of DNA entering the reaction is not measured, so input must be standardised another way.

Why is direct PCR becoming increasingly popular in forensic science?

It can be automated and is suitable for high-throughput analysis.

What is high-throughput analysis?

Processing large numbers of samples efficiently, often using automated systems.

What is Low Copy Number (LCN) DNA analysis?

An approach designed to obtain DNA profiles from very small quantities of DNA.

Why has LCN DNA analysis been controversial?

Low DNA quantities can produce stochastic effects, incomplete profiles and increased risks of contamination or misinterpretation.

What was the R v Hoey case?

A 2007 trial presided over by Judge Weir, discussed in the lecture in relation to the controversy surrounding LCN DNA evidence.

Why is the interpretation of LCN DNA important in court?

Because low-template results may be incomplete or affected by artefacts, so their evidential reliability must be carefully evaluated.

What should forensic scientists consider when interpreting low-template DNA?

Drop-out, drop-in, stutter, contamination, RFU thresholds and reproducibility.

What is the main limitation of low-template DNA analysis?

The increased possibility of unreliable or ambiguous DNA profiles due to stochastic effects.