Thus, many sequences (up to 40) can be amplified and quantified using just a single primer pair.

Specific products obtained with each primer pair from various organs and in several independent experiments were verified by sequencing.

We could design primer pairs for 56 out of 83 unique fragments (see Additional File 1).

NetPrimer can be used to determine the best primer pairs for a given set of experimental conditions.

This requires at least one primer (for a given primer pair) to have the specified number of mismatches to unintended targets.

Increasing this number can increase the chance of finding a specific primer pair but the process will take longer.

The annealing temperatures for each primer pair are listed in Table 1.

From 56 primer pairs, 19 could detect a polymorphism among 24 peanut genotypes (see Table 1).

All positive clones were confirmed by PCR with appropriate primer pairs.

However, such multiplex amplifications have the inherent problem that each primer pair has its own specific conditions of hybridization and thus control is not perfect.