| Repeat motifs |
Description |
Effect |
| Simple repeats |
A repeated sequence of four or more nucleotides, which is repeated: (…AATCGA…AATCGA…) |
Simple repeats can generate secondary binding sites for primers. Stable hybridization to secondary binding sites results in
nonspecific amplification. Repeats >3-4.
|
| Inverse repeats |
A self-complementary sequence motif of four or more nucleotides (stem-loop or hairpin motifs): (…AATGGC…GCCATT…) |
Inverse repeats can cause inefficient priming because they lead to formation of stable hairpins in the binding region, or
within the amplicon.
|
| Homopolymeric runs |
A sequence of four or more identical nucleotides: (…AAAAA…) |
Homopolymeric runs can be considered a special case of direct repeats. These can cause ambiguous binding of primers to their
target site (“slippage effect”). Poly (A) and poly (T) stretches should also be avoided because these will “breathe” and open
up stretches of the primer-template complex. Additionally, runs of three or more G residues can cause problems due to intermolecular
stacking.
|
