Sample Preparation & Nucleic Acid Extraction Flashcards

Which blood component is a well-known PCR inhibitor that can carry over into DNA extracts?

Answer: Heme

Heme and its derivatives inhibit Taq polymerase and must be removed during blood DNA extraction.

Which anticoagulant is generally preferred for blood samples intended for PCR testing?

Answer: EDTA

EDTA is preferred because heparin strongly inhibits PCR and is difficult to remove during extraction.

Humic and fulvic acids are common PCR inhibitors in which type of sample?

Answer: Soil and sediment

Humic substances from decaying organic matter co-purify with DNA from environmental samples and inhibit polymerase.

How can a lab best determine whether a negative PCR result is due to inhibition rather than absence of target?

Answer: Include an internal amplification control in each reaction

An internal amplification control that fails to amplify flags inhibition, distinguishing a true negative from a false negative.

A stool extract shows inhibition, but target concentration is expected to be high. What is a simple first step to overcome inhibition?

Answer: Dilute the extract (e.g., 1:10) and retest

Diluting the extract reduces inhibitor concentration below inhibitory levels while leaving enough target to detect.

Which PCR additive is commonly used to relieve inhibition by binding inhibitors such as phenolics and humic acids?

Answer: Bovine serum albumin (BSA)

BSA binds and sequesters many inhibitors, protecting the polymerase.

Melanin, which can inhibit PCR, is most likely to be carried over from which specimen?

Answer: Hair and pigmented tissue

Melanin from hair shafts and pigmented tissues binds polymerase and can co-purify with DNA.