Question

Adman degradation process followed by oligonucleotide probe construction testing your probe including the critical factors of Tm Gc vs AT richness and degenerate probe，write as much as detail you can, plz type the answer, i can't read the hand writing, Thank you.

Answer 1

Edman degradation is the process of purifying protein by sequentially removing one residue at a time from the amino end of a peptide. To solve the problem of damaging the protein by hydrolyzing conditions, Pehr Edman created a new way of labeling and cleaving the peptide. Edman thought of a way of removing only one residue at a time, which did not damage the overall sequencing. This was done by adding Phenyl isothiocyanate, which creates a phenylthiocarbamoyl derivative with the N-terminal. The N-terminal is then cleaved under less harsh acidic conditions, creating a cyclic compound of phenylthiohydantoin PTH-amino acid. This does not damage the protein and leaves two constituents of the peptide. This method can be repeated for the rest of the residues, separating one residue at a time.Edman degradation is very useful because it does not damage the protein. This allows sequencing of the protein to be done in less time. Edman sequencing is done best if the composition of the amino acid is known.

Probe construction

Probes

You have a choice of using single-quenched or double-quenched probes. IDT recommends use of double-quenched probes because they provide consistently lower background, resulting in higher signal compared to single-quenched probes. Double-quenched probes that include the IDT ZEN™ molecule as a secondary, internal quencher allow for longer probe lengths to be used in addition to providing strong quenching and increased signal. (Read more about the ZEN quencher in the article, Two Quenchers are Better Than One!,) If designing single-quenched probes, ensure that they are 20−30 bases in length. This will help you to achieve an ideal Tm without increasing the distance between the dye and quencher such that the quencher will no longer optimally absorb the fluorescence of the dye. Design your PCR probes to conform to the following guidelines:

• Location: Ideally, the probe should be in close proximity to the forward or reverse primer, but should not overlap with a primer-binding site on the same strand. Probes can be designed to bind to either strand of the target.
• Melting temperature (Tm): Preferably, probes should have a Tm 6–8°C higher than the primers. If the melting temperature is too low, the percentage of probe bound to target will be low. In this case, the primers may amplify a product, but sensitivity may be compromised as all target sites are not saturated with probe resulting in reduced fluorescence signal that does not truly represent the true amount of target present in the sample.
• Annealing temperature (Ta): The annealing temperature should be set no more than 5°C below the lower primer Tm. Use this as a general guideline, but note that optimization may still be necessary.
• GC content: As with primer sequences, aim for a GC content of 35−65% and avoid a G at the 5’ end to prevent quenching of the 5’ fluorophore.

A popular algorithm could easily be applied to calculate Tm of a degenerate oligonucleotide: Tm (°C) = 2 ´ AT + 4 ´ GC where AT and GC are the numbers of AT and GC base pairs, respectively .