American Society for Clinical Pathology (ASCP) Technologist in Cytogenetics certification (ASCP CG) Practice Test

How can the stringency for each probe be adjusted if the same hybridization incubator is used for several different probe strategies?

1. Changing the incubation time

2. Adjusting the formamide and salt concentrations

3. Altering the temperature of the incubator

4. Changing the type of probes used

The correct answer is: Adjusting the formamide and salt concentrations

The stringency of hybridization is crucial for ensuring specific binding between a probe and its target sequence. When using different probes in the same hybridization incubator, adjusting the formamide and salt concentrations is the most effective way to manage stringency. Formamide acts to lower the melting temperature of DNA duplexes, which can reduce the stringency of the hybridization. Higher concentrations of formamide will allow for more mismatches between the probe and the target, leading to less specific binding. Conversely, lower concentrations of formamide increase stringency by enhancing specificity. Salt concentration also plays a key role; higher salt concentrations promote binding by stabilizing duplex formation, whereas lower salt levels can increase stringency and reduce nonspecific binding. While changing the incubation time or temperature can influence the overall conditions of the hybridization, these adjustments do not directly target the molecular interactions that define stringency. Furthermore, simply changing the type of probes does not inherently address stringency issues across different hybridization scenarios, as the conditions may still be inappropriate for optimal binding for the specific probes used. Thus, adjusting formamide and salt concentrations directly impacts the specificity and overall success of hybridization for various probes.