Data Analysis & Troubleshooting Flashcards

What does a clear single band on a gel indicate after PCR?

Answer: Successful target amplification

A clear, single band on an agarose gel at the expected size after PCR indicates that the reaction successfully amplified the specific target DNA sequence. This result confirms that the primers were specific, the reaction conditions were optimal, and the desired product was generated efficiently. It also suggests the absence of significant non-specific amplification or primer dimers.

What might cause no bands to appear in a PCR gel?

Answer: Missing enzyme or wrong temperature

The absence of any bands on a PCR gel strongly suggests that the amplification reaction failed. This can be caused by critical missing components, such as the DNA polymerase enzyme which is essential for DNA synthesis, or by incorrect temperature settings. Improper temperatures can prevent denaturation, annealing, or extension from occurring effectively, thus halting the amplification process.

What does a smear on the gel indicate?

Answer: Degraded DNA or over-cycling

A smear on an agarose gel after PCR typically indicates a range of DNA fragment sizes rather than a distinct, specific band. This can result from degraded template DNA, which leads to fragmented amplification products. Alternatively, over-cycling can cause the accumulation of non-specific products, primer dimers, and even degradation of the desired product, all contributing to a smeared appearance.

Which factor helps confirm primer specificity?

Answer: Melt curve analysis

Melt curve analysis, commonly used in real-time PCR, involves slowly increasing the temperature and monitoring the dissociation of double-stranded DNA. Each specific PCR product has a unique melting temperature (Tm) at which its strands separate. The presence of a single, sharp peak at the expected Tm confirms the specificity of the primers and the absence of non-specific products or primer dimers.

Why is a negative control used in PCR?

Answer: To check for contamination

A negative control in PCR contains all reaction components except the template DNA, with water replacing the template. If amplification occurs in this control, it indicates contamination of reagents, primers, or equipment with exogenous DNA. This contamination could lead to false positive results in experimental samples, making the negative control essential for validating results.

What is a common cause of non-specific bands?

Answer: Low primer specificity or annealing temp

Non-specific bands arise when primers bind to unintended sites on the template DNA or to each other, forming primer dimers. This is often caused by primers that are not highly specific to the target sequence, meaning they have partial complementarity to other regions. Additionally, an annealing temperature that is too low can allow primers to bind to these partially complementary sequences, leading to unwanted amplification.

What does high background noise on a gel suggest?

Answer: Primer dimers or contamination

High background noise on a PCR gel, appearing as a faint smear or multiple indistinct bands, often suggests the presence of primer dimers or contamination. Primer dimers are short, non-specific products formed when primers anneal to each other and are amplified. Contamination introduces extraneous DNA that can be amplified non-specifically, both contributing to a noisy background.

How can yield be increased if amplification is weak?

Answer: Adjust template or cycling conditions

If PCR amplification is weak, increasing the amount of template DNA can provide more starting material for the reaction, potentially boosting yield. Alternatively, optimizing cycling conditions, such as extending annealing or extension times, increasing the total cycle number, or adjusting the annealing temperature, can improve reaction efficiency. These adjustments help ensure more efficient amplification and higher product yield.

What is an internal control in PCR?

Answer: A known DNA sequence to check reaction success

An internal control in PCR is a known DNA sequence, often from a housekeeping gene, that is co-amplified in the same reaction as the target DNA. Its successful amplification confirms that the PCR reaction itself is working correctly, ruling out issues with reagents or thermocycler function. This is particularly useful when the target DNA is absent or at very low levels, ensuring that a negative result is not due to reaction failure.