Stringency is a term that many molecular technologists are all very familiar with. It is a term that describes the combination of conditions under which a target is exposed to the probe. Typically, conditions that exhibit high stringency are more demanding of probe to target complementarity and length. Low stringency conditions are much more forgiving.
- If conditions of stringency are too HIGH → Probe doesn’t bind to the target
- If conditions of stringency are too LOW → Probe binds to unrelated targets
Important Factors That Affect Stringency and Hybridization
- Temperature of hybridization and salt concentration
- Increasing the hybridization temperature or decreasing the amount of salt in the buffer increases probe specificity and decreases hybridization of the probe to sequences that are not 100% the same.
- Concentration of the denaturant in the buffer
- For example: Deionized Formamide and SDS can be used to reduce non-specific binding of the probe
- Length and nature of the probe sequence
|STRINGENCY AND BINDING|
|– Long Probe
– Probe has increased number of G and C bases
|Binding occurs under more stringent conditions|
|– Short Probe
– Probe has increased number of A and T bases
|Binding occurs under less stringent conditions|
Melting Temperature (Tm) Long Probes
- The ideal hybridization conditions are estimated from the calculation of the Tm.
- The Tm of the probe sequence is a way to express the amount of energy required to separate the hybridized strands of a given sequence.
- At the Tm: Half of the sequence is double stranded and half of the sequence is single stranded.
- Tm = 81.5°C + 16.6logM + 0.41(%G+C) – 0.61(%formamide) – (600/n)
Where M = Sodium concentration in mol/L
n = number of base pairs in smallest duplex
- If we keep in mind that RNA is single stranded (ss) and DNA is double stranded (ds), then the following must be true:
RNA : DNA Hybrids More stable
DNA : RNA Hybrids ↓
DNA : DNA Hybrids Less stable
- Tm of RNA probes is higher, therefore RNA : DNA hybrids increase the Tm by 20 – 25°C
Calculating the Tm for Short Probes (14 – 20 base pairs)
- Tm = 4°C x number of G/C pairs + 2°C x number of A/T pairs
- The hybridization temperature (annealing temp) of oligonucleotide probes is approximately 5°C below the melting temperature.
Sequence Complexity (Cot)
- Sequence complexity refers to the length of unique, non-repetitive nucleotide sequences.
- Cot = Initial DNA Concentration (Co) x time required to reanneal it (t)
- Cot1/2 = Time required for half of the double-stranded sequence to anneal under a given set of conditions.
- Short probes can hybridize in 1 – 2 hours, where long probes require more time.
Test Your Knowledge
- Calculate the melting temperature of the DNA sequence below:
If the number of G/C pairs = 11, and the number of A/T pairs = 9. The calculation is as follows:
4(11) + 2(9) = X
X = 62°C
-LeAnne Noll, BS, MB(ASCP)CM is a molecular technologist in Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.