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Testing Ambiguous Primers With Degenerate Target Sequences

You can check ambiguous primers against degenerate sequences

It may be desirable to use ambiguous primers for amplifying targets against mutiple sequences where you do not know the exact sequence, or if you want the same set of primers to amplify many sequences in different organisms. Many suppliers of oligonucleotides will generate ambiguous primers by accepting sequences where ambiguities are specified using the IUPAC set of characters (other than ATCGU).

Select IUPAC Codes on the Window menu to display the IUPAC ambiguity codes for nucleic acid and amino acids.

For Nucleic Acids

A Adenine
C Cytosine
G Guanine
T Thymine
U Uracil
R puRine (A or G)
Y pYrimidine (C or T/U)
K Keto (G or T/U)
M aMino (A or C)
S Strong (C or G)
W Weak (A or T)
B not A (C or G or T/U)
D not C (A or G or T/U)
H not G (A or C or T/U)
V not T/U (A or C or G)
N aNy (A or C or G or T/U)

For Amino Acids

A Ala Alanine
B Asx Asparagine or aspartic acid
C Cys Cysteine
D Asp Aspartic acid
E Glu Glutamic acid
F Phe Phenylalanine
G Gly Glycine
H His Histidine
I Ile Isoleucine
K Lys Lysine
L Leu Leucine
M Met Methionine
N Asn Asparagine
P Pro Proline
Q Gln Glutamine
R Arg Arginine
S Ser Serine
T Thr Threonine
U Sec Selenocysteine
V Val Valine
W Trp Tryptophan
X Xxx Any amino acid; unknown
Y Tyr Tyrosine
Z Glx Glutamine or glutamic acid

You can design and test ambiguous primers using a combination of Test Primers and Primer Pairs.

Use PCR Primer Pairs (or Sequencing Primers/Probes) to generate useful primers to amplify the desired region, then manually add in the required ambiguities using the IUPAC codes.

After obtaining the ambiguous primer sequences, use Test PCR Primer Pairs (or Test Sequencing Primer/Probe) to test them. Select the Parameters button and ensure the Allow ambiguous residues option is selected and click OK. Finally, enter the ambiguous primer sequences in the two boxes, and select OK.

The results will show matches between bases and ambiguous residues characters other than A,C,G,T/U. These are as defined by the IUPAC specification. In the following example, we can see that GC matches with GY. Y stands for any pYrimidine, C or T/U, and so GY would bind to CG or CT.

5'-GCNTGYGAYGAR- 3' (primer 1)
|| || || ||
1 5'-GCTTGCGATGAA..<- 226bp ->..
3'-CGAACGCTACTT.. ..

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