forensic dna lab quiz 2

What is the, main advantage of doing a multiplex PCR instead of running three separate PCRs for cow, goat, and sheep DNA?

Multiplex PCR allows simultaneous amplification of multiple species-specific targets in one reaction, saving time and reagents and enabling direct comparison under identical conditions.

Explanation

It increases efficiency and consistency, as all samples are amplified under the same thermocycling regime.

If a cheese sample produces two distinct

PCR bands (e.g. 172 bp and 256 bp), what might this indicate?

The cheese contains milk from two different species — likely a mixture (e.g. sheep and cow milk).

Explanation

Each band corresponds to a unique amplicon size linked to one species' primers. A single-origin product should yield only one band.

What is the purpose of including both a

'positive control' (known goat DNA) and a

'negative control' (no DNA) in your PCR setup?

To confirm that the PCR system works correctly (positive) and that results are not due to contamination (negative).

Explanation

The positive verifies reagents and cycling conditions; the negative ensures no amplification occurs without DNA input.

Why is it important to avoid transferring the creamy fat layer during the lysate clarification step (Step 6 of extraction)?

Because fats and proteins can inhibit PCR by interfering with DNA polymerase or binding DNA.

Explanation

Lipids and proteins reduce amplification efficiency; careful phase separation ensures a clean DNA extract.

If you observe a low A260/A280 ratio (e.g.

1.3) after DNA quantification, what does this suggest and how might it affect PCR?

It suggests protein contamination, which may inhibit Taq polymerase activity and reduce amplification yield.

Explanation

Pure DNA typically has an A260/A280 ratio around 1.7 or greater. Protein contamination absorbs more at 280 nm, lowering the ratio.

What would happen if MgCl2 concentration was too low or too high in the PCR mix?

Too low → insufficient enzyme activity and weak/no amplification; Too high → nonspecific binding and multiple unwanted products.

Explanation

Mg?+ stabilises DNA-primer interactions and is essential for Taq function; its concentration must be optimised.

If your gel shows a single bright band at ~326 bp, which species' DNA is most likely present in your cheese?

Capra hircus (goat).

Explanation

326 bp corresponds to the goat-specific 12S rRNA amplicon described in the assay.

What is the role of the 100 bp DNA ladder in your gel electrophoresis?

It provides reference band sizes to estimate the length (in base pairs) of the PCR products.

Explanation

Comparing sample bands to known ladder bands enables species identification based on fragment size.

Why might a sample yield no visible PCR band, even though the cheese was goat-derived? (Name two possible explanations.)

PCR inhibitors remained in the DNA extract. DNA was too degraded or below detectable concentration.

Explanation

Cheese DNA can be fragmented or contaminated with inhibitors (like fats or salts), preventing amplification.

Suppose a student's 'negative control' lane shows a faint band at 256 bp. What conclusion should they draw, and what should they do next?

It indicates contamination with bovine DNA; results are unreliable and the PCR should be repeated with stricter aseptic technique.

Explanation

A band in the negative control means foreign

DNA entered the reaction, invalidating species identification.

Why is it important to document chain-of-custody information during this practical, even in a teaching setting?

To maintain traceability and integrity of samples, reflecting real forensic standards where sample mishandling could invalidate evidence.

Explanation

Practising proper documentation reinforces habits required in professional forensic workflows.

After amplification, the ovine band (172 bp) appears much fainter than expected. What are two possible experimental reasons for this?

Suboptimal primer efficiency or low primer concentration. Partial inhibition or insufficient template DNA.

Explanation

Uneven amplification between primer sets can occur if one pair is less efficient or if DNA concentration is limiting.