Guidelines for Sequence Design of PNA Oligomers
Specific Design Rules:
- Length: We will not synthesize a sequence with more than 18 bases,
excluding linkers, amino acids and labels.
- Amino Acid Content: We are able to attach one Lysine or one Cysteine
to either the N-terminus or the C-terminus.
- Purine Content: Purine-rich PNA oligomers tend to aggregate and
have low solubility in aqueous solutions. To avoid aggregation, please
follow the following specific guidelines:
Avoid self-complementary: sequences with inverse repeats, hairpins
and palindromes. These types of probes are prone to aggregate,
as PNA/PNA interactions are even stronger than PNA/DNA interactions.
- Limit the purine content to 60%.
GAT TAG CAG TCT ACG (Acceptable - Purine content < 60%)
- The maximum purine-stretch is 4 in a row. However, the maximum stretch
of guanines is 3-in-a-row
ATT AGG GGC
ATC TAC (Not acceptable - 4 G's in a row)
CTA GAT AGA AGG TTC (Not Acceptable
- 6 purines in a row)
- Consider probing the complementary strand if you violate the above
GTA GAT GCC CCT AAT (Acceptable -Complement of the above sequence
does not violate any of the guidelines)
GAA CCT TCT ATC TAG(Acceptable -Complement of the above sequence)
- Four base complements are acceptable, unless they contain only C and
G. However CG complements are acceptable when interrupted by one or
GAT AATT GCA (Acceptable - four bases complement)
GAT CCGG TAC (Not acceptable -
TAT CCT GGT A (Acceptable CC, GG interrupted by a base)
- Complements that are 6 bases or more in length are unacceptable.
CTA TTA ATG CA (Not acceptable -
six bases complement)
- Complements of 6 bases are acceptable when interrupted by one or more
bases, unless only C and G are used.
AGT GCT ACT (Acceptable - interrupted six bases complement
GCG GCT CGC
(Not acceptable - G and C only complement
- Complements of 8 bases are unacceptable even when interrupted by one
or more bases.
ACTG T CAGT
(Not acceptable - eight bases complement)
For Research Use Only.
- We strongly recommend that you design anti-parallel custom
PNA probes. PNAs can form duplexes in either orientation, but an anti-parallel
orientation is strongly preferred and forms the most regular duplex. Anti-parallel
is the preferred configuration for antisense and DNA probe-type applications.
When the orientation of the PNA is anti-parallel, the N-terminal of the
PNA probe is equivalent to the 5'-end of the DNA.
- PNA melting temperatures differ from those of DNA oligonucleotides.
Because the PNA strand is uncharged, a PNADNA-duplex will have a higher
Tm than the corresponding DNA-DNA-duplex. Typically there will
be an increase in Tm of about 1°C per base pair at 100 mM NaCl.
At lower salt concentrations the Tm differences will be even
more dramatic. A 10-mer PNA will typically have a Tm of about
50°C, and a 15-mer PNA will typically have a Tm of 70° C.
- A PNA oligomer length of 12 to 17 units is optimal. Sequence
length is primarily determined by the required specificity of the application.
DNA applications that require more than 25 bases can be routinely performed
with much shorter PNA probes. Long PNA oligomers, depending on the sequence,
tend to aggregate and are difficult to purify and characterize. However,
the shorter a sequence is, the more specific it is. Consequently, the
impact of mismatch is greater for a short sequence.
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