François Lutzoni's Lab -- Primer sequences

Primer sequences for PCR amplification and sequencing from nuclear ribosomal DNA, RPB2, and beta-tubulin

Nuclear ribosomal DNA
RNA polymerase II subunit,RPB2
Beta-tubulin

Nuclear ribosomal DNA

We used the primer list from Vilgalys’ lab at Duke University as the basis to which we added primers developed in our lab. We update this list as new primers are published. Please let us know if new primers that we are not aware of should be included here. Contrary to Vilgalys’ lab who are designing primers mainly for phylogenetic studies of basidiomycota, the target organisms for our primers are lichenized and non-lichenized Ascomycota. Primers that work best for us are highlighted in blue. Many of these primers are likely to work on Zygomycetes or Chytridiomycetes. Several of these primers work well on plants, as well as on algae found in lichens.
Link to: Vilgalys’ lab at Duke University

Small subunit rDNA primer sequences		ssu ref.	nb pb
BMB-'A' (Lan)	GWA TTA CCG CGG CKG CTG	583-566	18
BMB-'B' (Lan)	CCG TCA ATT CMT TTR AGT TT	1147-1128	20
BMB-'C' (Lan)	ACG GGC GGT GTG TRC	1641-1637	15
BMB-BR (Lan)	CTT AAA GGA ATT GAC GGA A	1131-1149	19
BMB-CR (Lan)	GTA CAC ACC GCC CGT CG	1637-1643	17
nssu97a (Kau)	TAT ACG GTG AAA CTG CGA ATG GC	75-97	23
nssu97b (Kau)	CGG TGA AAC TGC GAA TGG C	79-97	19
nssu634 (Kau)	CCC CAG AAG GAA AGI CCC GIC C	705-684	22
nssu897R (Kau)	AGA GGT GAA ATT CTT GGA	897-914	18
nssu1088 (Kau)	TGA TTT CTC GTA AGG TGC CG	1088-1069	20
nssu1088R (Kau)	CGG CAC CTT ACG AGA AAT CA	1069-1088	20
nssu131 (Kau)	CAG TTA TCG TTT ATT TGA TAG TAC C	107-131	25
NS1 (Whi)	GTA GTC ATA TGC TTG TCT C	20-38	19
NS2 (Whi)	GGC TGC TGG CAC CAG ACT TGC	573-553	21
NS3 (Whi)	GCA AGT CTG GTG CCA GCA GCC	553-573	21
NS4 (Whi)	CTT CCG TCA ATT CCT TTA AG (sim. to BMB-b)	1150-1131	20
NS5 (Whi)	AAC TTA AAG GAA TTG ACG GAA G (sim. to BMB-BR)	1129-1150	22
NS6 (Whi)	GCA TCA CAG ACC TGT TAT TGC CTC	1436-1413	24
NS7 (Whi)	GAG GCA ATA ACA GGT CTG TGA TGC	1413-1436	24
NS8 (Whi)	TCC GCA GGT TCA CCT ACG GA	1788-1769	20
NS17 (Gar)	CAT GTC TAA GTT TAA GCA A	54-72	19
NS18 (Gar)	CTC ATT CCA ATT ACA AGA CC	516-497	20
NS19 (Gar)	CCG GAG AAG GAG CCT GAG AAA C	381-402	22
NS20 (Gar)	CGT CCC TAT TAA TCA TTA CG	871-852	20
NS21 (Gar)	GAA TAA TAG AAT AGG ACG	802-819	18
NS22 (Gar)	AAT TAA GCA GAC AAA TCA CT	1312-1297	20
NS23 (Gar)	GAC TCA ACA CGG GGA AAC TC	1184-1203	20
NS24 (Gar)	AAA CCT TGT TAC GAC TTT TA	1769-1750	20
SR1 (VilU)	ATT ACC GCG GCT GCT (similar to BMB-'A')	581-567	15
SR1R (Vil0)	TAC CTG GTT GAT TCT GC	21-1	21
SR2 (Elw)	CGG CCA TGC ACC ACC	1277-1263	15
SR3 (VilU)	GAA AGT TGA TAG GGC T	321-306	16
SR4 (VilU)	AAA CCA ACA AAA TAG AA	841-825	17
SR5 (VilU)	GTG CCC TTC CGT CAA TT	1155-1139	17
SR6 (VilU)	TGT TAC GAC TTT TAC TT	1763-1747	17
SR7 (VilU)	GTT CAA CTA CGA GCT TTT TAA	637-617	21
SR7R (VilU)	TTA AAA AGC TCG TAG TTG AAC	617-637	21
SR8R (VilU)	GAA CCA GGA CTT TTA CCT T	732-750	19
SR9R (Elw)	YAG AGG TGA AAT TCT	896-910	15
SR10R (VilU)	TTT GAC TCA ACA CGG G	1181-1196	16
SR11R (Spa)	GGA GCC TGA GAA ACG GCT AC	389-408	20
1 (DeP)	GCT CTT TCT TGA TTT TGT	1241-1258	18
3 (DeP)	TTT GAG GCA ATA ACA GGT	1410-1427	18
4 (DeP)	CAT CTA AGG GCA TCA CAG	1445-1428	18
5 (DeP)	TTG TCT CTG TCA GTG TAG	1482-1465	18
6 (DeP)	CAA CGA GGA ATT CCT AGT	1566-1583	18
9 (DeP)	AAT GAT CCT TCC GCA GGT	1797-1780	18

Large subunit rDN primer sequences		lsu ref.	nb pb
GUADET P1 (Gua)	GAG TCG AGT TGT TTG GGA ATG CAG C	275-300	25
GUADET P2 (Gua)	GAA AAG AAC TTT GAA AAG AGA GTG AA	379-403	26
GUADET P3 (Gua)	CCC GTC TTG AAA CAC GGA CCA AGG	661-685	24
P2R (Reh)	CTC TCT TTT CAA AGT TCT TTT CAT CT	1412-1396	17
LR0R (VilU)	ACC CGC TGA ACT TAA GC	26-42	17
LR0R (Reh)	GTA CCC GCT GAA CTT AAG C	24-42	19
LR1R (Mon)	AGG AAA AGA AAC CAA CC	57-73	17
LR2	TTT TCA AAG TTC TTT TC	395-379	17
LR2R	AAG AAC TTT GAA AAG AG	382-398	17
LR3 (Vil0)	GGT CCG TGT TTC AAG AC	677-661	17
LR3R	GTC TTG AAA CAC GGA CC	664-680	17
LR4	ACC AGA GTT TCC TCT GG	887-871	17
LR5 (Vil0)	ATC CTG AGG GAA ACT TC	997-981	17
LR5R (VilU)	GAA GTT TCC CTC AGG AT	981-997	17
LR6 (Vil0)	CGC CAG TTC TGC TTA CC	1173-1157	17
LR7 (Vil0)	TAC TAC CAC CAA GAT CT	1483-1467	17
LR7R (Vil0)	AGA TCT TGG TGG TAG TA	1465-1481	17
LR8	CAC CTT GGA GAC CTG CT	1917-1901	17
LR8R	AGC AGG TCT CCA AGG TG	1901-1917	17
LR9	AGA GCA CTG GGC AGA AA	2292-2276	17
LR10	AGT CAA GCT CAA CAG GG	2508-2492	17
LR10R	GAC CCT GTT GAG CTT GA	2490-2506	17
LR11	GCC AGT TAT CCC TGT GGT AA	2919-2900	20
LR12 (Vil0)	GAC TTA GAG GCG TTC AG	3221-3204	17
LR12R (VilU)	CTG AAC GCC TCT AAG TCA GAA	3205-3225	21
LR13	CAT CGG AAC AAC AAT GC	3464-3448	17
LR14	AGC CAA ACT CCC CAC CTG	2701-2694	18
LR15	TAA ATT ACA ACT CGG AC	161-145	17
LR16 (Mon)	TTC CAC CCA AAC ACT CG	716-700	17
LR17R	TAA CCT ATT CTC AAA CTT	1066-1083	18
LR20R	GTG AGA CAG GTT AGT TTT ACC CT	3058-3080	23
LR21	ACT TCA AGC GTT TCC CTT T	442-424	19
LR22	CCT CAC GGT ACT TGT TCG CT	371-352	20
F377	AGA TGA AAA GAA CTT TGA AAA GAG AA	375-400	26
R377	CTC TCT TTT CAA AGT TCT TTT CAT CT	400-375	26
LIC15R (Mia2)	GGA GGA AAA GAA ACC AAC AG	55-74	20
* LIC24R (Mia1)	GAA ACC AAC AGG GAT TG	64-80	17
LIC2044 (Kau)	ACG CCT GCC TAC TCG CC	26-42	17

IGS and 5S rDNA p rimer sequences
5S RNA (VilU)	ATC AGA CGG GAT GCG GT		17
5S RNAR (VilU)	ACQ GCA TCC CGT CTG AT		17
NS1R	GAG ACA AGC ATA TGA CTA C	38-20 (SSU)	19
LR13R	GCA TTG TTG TTC CGA TG	3448-3464(lsu)	17

ITS and 5.8S rDNA primer sequences
ITS1 (Whi)	TCC GTA GGT GAA CCT GCG G	1769-1787(ssu)	19
ITS1F (Gad)	CTT GGT CAT TTA GAG GAA GTA A		22
ITS2 (Whi)	GCT GCG TTC TTC ATCG ATG C (sim. to 5.8S)		20
ITS3 (Whi)	GCA TCG ATG AAG AAC GCA GC (sim. to 5.8SR)		20
ITS4 (Whi)	TCC TCC GCT TAT TGA TAT GC	60-41(lsu)	20
ITS4B (Gad)	CAG GAG ACT TGT ACA CGG TCC AG		23
ITS4S (Kre)	CCT CCG CTT ATT GAT ATG CTT AAG	59-36 (lsu)	24
ITS5 (Whi)	GGA AGT AAA AGT CGT AAC AAG G (sim. to SR6R)	1745-1766(ssu)	22
ITS5R	CCT TGT TAC GAC TTT TAC TTC C	1766-1745 (ssu)	22
5.8S (Vil0)	CGC TGC GTT CTT CAT CG		17
5.8SR (Vil0)	TCG ATG AAG AAC GCA GC		18
SR6R (VilU)	AAG TAP AAG TCG TAA CAA GG	1747-1766(ssu)	20
LR1 (Vil0)	GGT TGG TTT CTT TTC CT	73-57(lsu)	17
SLG-1 (Gro)	TTG CGC AAC CTG CGG AAG GAT	1773-1793(ssu)	21
NS24R	TAA AAG TCG TAA CAA GGT TT	1750-1769(ssu)	20

ssu ref. Mankin et al., 1986. Gene 44:143-145
lsu ref. Lapeyre et al., 1993. Nucleic Acids Res. 21:3322-3322

* Primers designed in Lutzoni's lab
(DeP) DePriest,1993. Gene 134:67-74
(Elw) Elwood et al., 1985. Mol. Biol. Evol. 2:399-410
(Gad) Gardes and Bruns, 1993. Mol. Ecol. 2:113-118
(Gar) Gargas and Taylor, 1992. Mycologia 84(4): 589-592
(Gro) Groner and LaGreca, 1997. Lichenologist 29(5):441-454
(Gua) Guadet et al., 1989. Mol. Biol. Evol. 6:227-242
(Kau) Kauff and Lutzoni, 2002. Mol. Phylogen. Evol. 25:138-156
(Kre) Kretzer et al., 1996. Mycologia 88(5): 776-785
(Lan) Lane et al., 1985. PNAS USA 82:6955-6959
(Mia1) Miadlikowska and Lutzoni, 2000. Int. J. Plant Sci. 161(6)
(Mia2) Miadlikoska, McCune, Lutzoni,2001. Bryologist 105:1-10
(Mon) Moncalvo et al., 1993. Mycologia 85(5):788-794
(Reh) Rehner and Samuels, 1994. Mycol. Res. 98(6):625-634
(Spa) Spatafora et al, 1995. J. Clinical Microbiol. 33:1322-1326
(Vil0) Vilgalys and Hester, 1990. Journal of bacteriology 172:4238-4246
(VilU) Vilgalys unpublished [http://www.botany.duke.edu/fungi/mycolab]
(Whi) White et al., 1990. pp 315-322 in PCR protocols: A guide to methods and applications

RNA Polymerase II Subunit RPB2

Liu et al. (1999) were the first to design primers from this molecule to resolve phylogenetic relationships within the Ascomycota. Their study was restricted to non-lichenized ascomycetes. Primers that work best for us are highlighted in blue.

We have developed additional primers that work on both lichenized and non-lichenized Ascomycota. The following image shows all of the
RPB2 primers that work best for us. Please let us know if new primers that we are not aware of should be included here.

Beta-tubulin

This compilation of primers is from Glass & Donaldson (1995) and O’Donnell & Cigelnik (1997). Please let us know if new primers that we are not aware of should be included here. We are currently designing new primers for lichenized and non-lichenized Ascomycota.

Department of Biology, Duke University, Box 90338, Durham, NC 27708

Control panel

Last update April 2003