Degenerate primers

Primers

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 GA Y AC N GC N GG N CA R 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 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. 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 GCGCG GAATTC ( EcoRI ) and (5' end) of reverse primer can have GCGCGC AAGCTT ( 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

9. 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

10. 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

11. 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