Degenerate primers are designed from conserved amino acid sequences aligned from multiple species. They contain nucleotide degeneracies that allow binding to related gene sequences. Key steps are: 1) Identifying conserved regions over 5+ amino acids long and within 200-600 bp for primers. 2) Calculating primer degeneracy based on contributing amino acids to minimize values over 64. 3) Optimizing primers by avoiding amino acids with 6-fold degeneracy and adding 5' tails.

1. Degenerate Primers
• useful for searching out a part of similar genes
from a variety of species
• or only when the protein sequence of a gene
is known
• very powerful tool to find "new" genes or
gene families
The more distant those related organisms, the
more difficult it can be to design primers

2. • designed by gathering sequences from a large range of
organisms
• aligning the translated amino acid sequence
Trp Asp Thr Ala Gly Gln Glu
5' TGG GAY ACN GCN GGN CAR GA 3‘
This gives a mix of 256 different oligonucleotides.
• Based on these alignments, it is possible to identify regions
of the sequence which are highly conserved at the amino
acid level
• These conserved regions can be then exploited for
designing degenerate primers

3. Two important things
• product length
• the degeneracy
1. needs two conserved regions for locating the
forward and the reverse primers - each should be
atleast 5 AA long (preferably 6 or 7 AA long) and fairly
close together (reasonable target is 400 bp, 200-600
bp also work). If primers are less degenerate, then
target can be further apart also.
2. second thing is to determine which of the possible
primers shows least degeneracy. It is evident from the
genetic code, some amino acids are coded for by
more triplet codon possibilities than others

4. amino acids and their corresponding
number of codons
Fold sites Amino acids
1 M W
2 F Y H Q N K D E C
3 I
4 V P T A G
6 L S R

5. Degeneracy
• While designing degenerate primers, avoid 6 fold
sites (L, S and R) and maximize the number of 1
or 2 fold sites in the region. This can be done by
comparing the degeneracies of the possible
primers
• To compute degeneracy, multiply the
degeneracies of each of the contributing amino
acids. For example, if a primer which matches the
amino acid sequence F H M T G, this would
correspond to a degeneracy of 2 * 2 * 1 * 4 * 4 =
64. just need to weigh the factors of degeneracy
and distance separating the forward and reverse
primers to select a pair to try

6. • Inosine residues can pair with any nucleotide,
and hence it can be used at sites where there
is complete degeneracy (substitute I for N).
• This can reduces the number of oligos that
have to be synthesised.
• Try and avoid degeneracy at the 3’ end of the
oligo (note that it is not necessary to have
whole codons), and especially avoid ending in
inosine

7. • adding tails to the degenerate primers on the 5'
ends increases the PCR efficiencies. These
primers increase primer length and hence
annealing temperature. Although the tails do not
help in the first few rounds of PCR when only the
genomic template is being amplified, the tails do
match in subsequent PCR cycles when amplifying
the short PCR products containing the primers at
each end. For example, the tails (5' end) of
forward primer can have GCGCGGAATTC (EcoRI)
and (5' end) of reverse primer can have
GCGCGCAAGCTT (HindIII) - including restriction
sites can be used for directional cloning.
Alternatively, they end with terminal G's which
encourages Taq to add overhanging A's for use in
TA cloning

8. D-Primer degenerate primer designing tool

11. Degenerate nucleotide codes
R = A/G Y = C/T
M = A/C K = G/T
S = G/C W = A/T
H = A/T/C D = G/A/T
B = G/T/C V = G/A/C
N = A/T/G/C

12. • These would in fact be a set of primers which
have a number of options at several positions in
the sequence so as to allow annealing to and
amplification of a variety of related sequences.
• For example, in the primer GG{C,G}A{C,G,T}A, the
third position is C or G, and the fifth is C, G, or T.
• That is, if you do not know exactly the sequence
of the gene you are going to amplify, you insert
"wobbles" in the PCR primers where there is
more than one possibility

13. • Degeneracy obviously reduce the specificity
of the primer(s), meaning mismatch
opportunities are greater, and background
noise increases
• Also, increased degeneracy means
concentration of the individual primers
decreases; thus, greater than 512-fold
degeneracy should be avoided