Designing Degenerate Primers

Objective To learn by practice how to design degenerate primers.

Bindu Sree & Rex Arunraj

Principles Degenerate primers may be used to amplify DNA in situations where only the protein sequence of a gene is known, or where the aim is to isolate similar genes from a variety of species. A six or seven residue peptide sequence should be selected, corresponding to an oligo of about 20 nucleotides. If the oligo is designed to amplify several similar protein sequences, then the most conserved regions of the proteins needs to be selected. If some of the residues are not completely conserved, then the oligo sequence will need to accommodate all possible codons of all amino acid residues a that site (e.g. if one protein has a E at a particular site, while all the others have a D, then the corresponding oligo sequence will be GAN see table overleaf). The peptide sequence should avoid amino acids that have a lot of codons, such as leucine(L), arginine(R) and serine(S). Instead, aim for regions that are rich in amino acids that have only one or two possible codons (i.e M, W, C, D, E, F, H, K, N, Q, Y see table overleaf).

Principles contd

Inosine residues can pair with any nucleotide, and so can be used at sites where there is complete degeneracy (i.e. substitute I for N in the table below0. This reduces the number of oligos that have to be synthesized. Note that only some manufacturers use inosine, however. 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. The table overleaf may be used to help with primer design: Primer should have minimum of six amino acids Good to have Methionine or Tryptophan at 3 end Distance between forward and reverse primer should be 200-600 base pairs Last three or four amino acid degeneracy should be low (<8) For higher efficiency, use tailGCGCGC or CCCCC

Exercises 1.Design degenerate primers for the given protein sequence. 2.Give reasons why it is the best primer set?

Practice Sequence MEVISTNTNGSTIFKSGAIPMNGHHNGTSKHQNGHKNGTSEQ QNGTISLDNGNELLGNSNCIKPGWFSEFSALWPGEAFSLKVEK LLFQGKSDYQDVMLFESATYGKVLTLDGAIQHTENGGFPYTE MIVHLPLGSIPNPKKVLIIGGGIGFTLFEMLRYPTIEKIDIVEIDD VVVDVSRKFFPYLAANFNDPRVTLVLGDGAAFVKAAQAEYY DAIIVDSSDPIGPAKDLFERPFFEAVAKALRPGGVVCTQAESIW LHMHIIKQIIANCRQVFKGSVNYAWTTVPTYPTGVIGYMLCST EGPEIDFKNPVNPIDKETAQVKSKLAPLKFYNSDIHKAAFILPS FARSMIES

MEVISTNTNGSTIFKSGAIPMNGHHNGTSKHQNGHKNGTSEQQNGTISLDNGNELLGNSNCIKPGWFSEFSALWPGE 12436424246432264434124222446222242224462222443662242266426223244126226461442
1X2X4X2X2X2=64

AFSLKVEKLLFQGKSDYQDVMLFESATYGKVLTLDGAIQHTENGGFPYTEMIVHLPLGSIPNPKKVLIIGGGIGFTL 42662422662242622224162264424246462443224224424242134264646342422463344434246
2*2*2*2*4*1=64

FEMLRYPTIEKIDIVEIDDVVVDVSRKFFPYLAANFNDPRVTLVLGDGAAFVKAAQAEYYDAIIVDSSDPIGPAKDL 22166244322323423224442466222426442222464464642444242442422224334266243444226
2*4*2*2*2*2=128

FERPFFEAVAKALRPGGVVCTQAESIWLHMHIIKQIIANCRQVFKGSVNYAWTTVPTYPTGVIGYMLCSTEGPEIDF 22642224442466444442424263162123342334226242246424414444424444342162642442322
2*3*2*2*2*2=96

KNPVNPIDKETAQVKSKLAPLKFYNSDIHKAAFILPSFARSMIES 224424322244242626446222262322442364624661326

Forward primer

1.MNGHHN
Degeneracy 1*2*4*2*2*2 = 64 Sequence 5ATGAAT/CGGT/C/A/GCAT/CCAT/CAAT/C3 5ATGAAYGGNCAYCAYAAY3

2.DYQDVM
Degeneracy 2*2*2*2*4*1=64 Sequence 5GAT/CTAT/CCAG/AGAT/CGTT/C/A/GATG3 5GAYTAYCARGAYGTNATG3

Degeneracy Code R K S Y M W B H N D V A or G G or T G or C C or T A or C A or T G, C or T A, C or T A, G, C or T A, G, or T A, C, or G Y M S R K W V D N H B

Complement

Reverse primer EIDFKN Degeneracy 2*3*2*2*2*2=96 QAEYYD Degeneracy 2*4*2*2*2*2=128

Sequence 5GAA/GATT/C/AGAT/CTTT/CAAA/GAAT/C3 5GARATHGAYTTYAARAAY3 Reverse primer 5G/ATTC/TTTG/AAAG/ATCT/G/AATC/TTC3 5RTTYTTRAARTCDATYTC3

Inference Did you have hands on practice on primer design? What are the important characteristics of primer? What was the outcome of the excercises?