Sequences of degenerate primers used for cloning of short gastrulation (sog), single-minded (sim), prospero (pros), engrailed (en) and optomotor blind (omb) genes
Primer name . | Nucleotide sequence(5′-3′)* . | Amino acid sequence . |
---|---|---|
sog1 | tggcayccnttygtnccncc | WHPFVPP |
sog2 | ggrcaytcyttrcarcankc | (A/D)CCKQCP |
sog3 | gayytnggnccnccnttyg | DLGPPFG |
sog4 | acncknckccanacnccrca | CGVWRRV |
sog5 | tgymrnaayathaaraaygantg | C(K/R)NIKN(D/E)C |
sog6 | ccnggrcangtyttrcarca | CCKTCPG |
sim1 | gnatgaartgygtnytngc | RMKCVLAK |
sim2 | tanswytgnacccanaccca | WVWVQSY |
sim3 | ccnswrcartgdatnacytt | KVIHCSG |
pros1 | aargcnaarytnatgttyttyt | KAKLMFF(W/Y) |
pros2 | gcytgnckngcrwayttytc | EK(Y/F)ARQA |
en1 | tggccngcntgggtntwytg | WPAWV(F/Y)C |
en2 | ttrtanarnccytgngccat | MAQGLYN |
en3 | aakgakaatmgktatttkackga | (N/Q)(D/E)NRYLTE |
en4 | ttrttytgraaccadatytt | KIWFQNK |
omb1 | ayggnmgnmgnatgttycc | NGRRMFP |
omb2 | aangcyttngcraanggrtt | NPFAKAF |
Primer name . | Nucleotide sequence(5′-3′)* . | Amino acid sequence . |
---|---|---|
sog1 | tggcayccnttygtnccncc | WHPFVPP |
sog2 | ggrcaytcyttrcarcankc | (A/D)CCKQCP |
sog3 | gayytnggnccnccnttyg | DLGPPFG |
sog4 | acncknckccanacnccrca | CGVWRRV |
sog5 | tgymrnaayathaaraaygantg | C(K/R)NIKN(D/E)C |
sog6 | ccnggrcangtyttrcarca | CCKTCPG |
sim1 | gnatgaartgygtnytngc | RMKCVLAK |
sim2 | tanswytgnacccanaccca | WVWVQSY |
sim3 | ccnswrcartgdatnacytt | KVIHCSG |
pros1 | aargcnaarytnatgttyttyt | KAKLMFF(W/Y) |
pros2 | gcytgnckngcrwayttytc | EK(Y/F)ARQA |
en1 | tggccngcntgggtntwytg | WPAWV(F/Y)C |
en2 | ttrtanarnccytgngccat | MAQGLYN |
en3 | aakgakaatmgktatttkackga | (N/Q)(D/E)NRYLTE |
en4 | ttrttytgraaccadatytt | KIWFQNK |
omb1 | ayggnmgnmgnatgttycc | NGRRMFP |
omb2 | aangcyttngcraanggrtt | NPFAKAF |
Achaearanea sog (At-sog) was amplified using the sog1 and sog2 primers, and Pholcus sog (Pp-sog) and Artemia sog (Af-sog) were amplified using the sog3 and sog4 primers for first PCR and the sog5 and sog6 primers for nested PCR. Achaearanea sim (At-sim) was amplified using the sim1 and sim2 primers for first PCR and the sim1 and sim3 primers for nested PCR. Achaearanea pros (At-pros) was amplified using the pros1 and pros2 primers. Achaearanea en (At-en) was amplified using the en1 and en2 primers for first PCR and the en3 and en4 primers for nested PCR. Achaearanea omb (At-omb) was amplified using the omb1 and omb2 primers. The PCR conditions were as follows: 1 cycle of 95°C for 5 minutes, 50°C for 2 minutes 30 seconds, 72°C for 40 seconds; 35 cycles of 95°C for 40 seconds, 50°C for 40 seconds, 72°C for 40 seconds;1 cycle of 72°C for 10 minutes; and then a 4°C soak. For amplification of At-en and At-omb, the annealing temperatures were 45°C and 40°C, respectively, instead of 50°C. To obtain full-length cDNAs for At-sog At-en, At-omb and Artemia sog,cDNA libraries (Akiyama-Oda and Oda,2003; Oda et al.,2005) were screened with DIG-labeled DNA probes for the PCR-amplified fragments. To obtain full-length cDNAs for At-sim,At-pros and Pp-sog, 5′ and 3′ RACE were performed using a SMART RACE cDNA amplification kit (Clontech) and ExTaq polymerase(Takara)
d is a, g or t; h is a, c or t; k is g or t; m is a or c; n is a, c, g or t; r is a or g; s is c or g; w is a or t; y is t or c