Oligonucleotides(Manfred Binder and David Hibbett 9/18/2003)

Resuspending freeze-dried oligonucleotides
Oligonucleotides are usually shipped in dry form. The dried DNA pellet becomes dislodged
from the bottom of the tube during shipping and it can easily fly out of the tube when firstopened, particularly as electrostatic attraction is present. For this reason:
Always briefly centrifuge oligos before opening for the first time.
We dissolve the stock oligo in sterile dH2O which must be freshly autoclaved. Alternatively,TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) can be used.
For convenience, make a freezer stock at 100 µM concentration (which should be thawed
infrequently). Adding a volume of dH2O (µL) equal to ten times the number of nanomoles of
DNA present in the tube (as noted on the spec sheet provided with the oligo) will produce astock solution at this concentration. [1 µM = 1 µmole/L or 1 pmole/µL].
For example, dissolve 50 nmoles (= quantity) of oligo in 500 µL dH2O to make a 100 µM
stock solution (= concentration). Dilute from this stock 1:10 in dH2O (1 part 100 µM oligo
solution, 9 parts dH2O) to make a working solution at 10 µM for use in setting up PCR
reactions.
Most PCR reactions use 0.1 - 0.5 µM primer. Addition of 1 µL of the 10 µM primer to a 20 µl
PCR reaction (total volume) will result in a final primer concentration of 0.5 µM, or a 10picomoles quantity of the oligo in a 20 µl volume.
Oligos used in sequencing reactions have lower concentrations at 2 pmoles/µl. For example,
use a 10 µM stock and prepare a 1:5 dilution. We use up to 3 picomoles of primer in 12 µlsequencing reactions.

Primer sequences:
The conserved rDNA primers that we use for PCR and sequencing have been mostly
developed in the Bruns lab and the Vilgalys lab. Visit their web pages for additional
information and for a greater choice of primer sequences. The DeepHypha web page
provides several primer links and is also summing-up primer sequences for protein coding
genes like atp6 (Bruns lab), RPB1 and RPB2 (Hall lab), EF-1α (Steve Rehner), and newprimers developed for the AFTOL project, partly including appendant protocolls.
DeepHyphaThe Bruns lab
The Hall lab
The Vilgalys lab
AFTOL

http://ocid.nacse.org/research/aftol/primers.php
http://plantbio.berkeley.edu/~bruns/primers.html
http://protist.biology.washington.edu/bio2/people/bio.html?parecID=142
http://www.biology.duke.edu/fungi/mycolab/primers.htm
https://aftol.biology.duke.edu/pub/primers/viewPrimers

The following is a list of primers currently in use in the Hibbett lab. Check the protocol page for
updates on primers of protein-coding regions. Primer sequences (5”—3’), hybridization regions
and their relative position are given where applicable. It is also indicated which primers areused for PCR and which primers are used for sequencing (SEQ) purpose.

NameLRORLR15LR22LR3LR3RLR5LR7

Nuclear large subunit rDNA(nuc-lsu, 25S, 28S)
Sequence 5’-3’
Position
Notes
Vilgalys lab
ACC CGC TGA ACT TAA GC
26-42
TAA ATT ACA ACT CGG AC
154-138
CCT CAC GGT ACT TGT TCG CT
364-344
CCG TGT TTC AAG ACG GG
651-635
GTC TTG AAA CAC GGA CC638-654
TCC TGA GGG AAA CTT CG
964-948
TAC TAC CAC CAA GAT CT
1418-1432

nuc-lsu primer map

PCR: LR0R—LR5 (LR7)
SEQ: LR0R, LR22, LR3, LR3R, LR5, (LR7)ITS1
ITS1-F
ITS2
ITS3
ITS4
ITS4-B
ITS5
5.8S
5.8SR

Internal transcribed spacer region (ITS region, including the 5.8S gene)
TCC GTA GGT GAA CCT GCG G
1773-1791 (18S) White et al. 1990
CTT GGT CAT TTA GAG GAA GTA A
1735-1756 (18S) Gardes & Bruns 1993
White et al. 1990
GCT GCG TTC TTC ATC GAT GC
53-34
White et al. 1990
GCA TCG ATG AAG AAC GCA GC
34-53
White et al. 1990
TCC TCC GCT TAT TGA TAT GC
57-38 (25S)
Gardes & Bruns 1993
CAG GAG ACT TGT ACA CGG TCC AG 194-172 (25S)
GGA AGT AAA AGT CGT AAC AAG G
1749-1770 (18S) White et al. 1990
Vilgalys lab
CGC TGC GTT CTT CAT CG
54-38
Vilgalys lab
TCG ATG AAG AAC GCA GCG
37-54

ITS primer map

PCR: ITS1 (ITS1F, ITS5)—ITS4 (ITS4, LR15, ITS4-B) alternatives in parentheses.
SEQ: see above. It is recommended using 5.8SR (ITS3) and 5.8S (ITS2) to sequence largerproducts (> 800 bp).

PNS1NS19bc
NS19bNS41
NS51
NS6NS7NS8SR1SR1cSR4SR6Nuclear small subunit rDNA (nuc-ssu, 18S)
CCA AGC TTG AAT TCG TAG TCA TAT
1-31
GCT TGT C
GTT TCT CAG GCT CCC TCT CCG G399-378
CCG GAG AGG GAG CCT GAG AAA C
378-399
CCC GTG TTG AGT CAA ATT A
1200-1182
GGG GGA GTA TGG TCG CAA GGC
1108-1128
GCA TCA CAG ACC TGT TAT TGC CTC
1439-1416
GAG GCA ATA ACA GGT CT GTG ATG C 1416-1439
TCC GCA GGT TCA CCT ACG GA
1792-1773ATT ACC GCG GCT GCT
578-564
AGC AGC CGC GGT ATT564-578
AAA CCA ACA AAA TAGA A
838-820
TGT TAC GAC TTT TACT T
1760-1744

K. O’Donnell(s. Hibbett 1996)
Bruns lab

White et al. 1990

Vilgalys lab

nuc-ssu primer map

PCR: 1) PNS1—NS41 and 2) NS19b—NS8; alternatively use PNS1—NS8.
SEQ: for product 1) PNS1, NS19bc, NS41 and 2) NS19b, NS51, NS7, NS8. SR primers andNS6 are alternatives for sequencing.

ML5
ML6
MLIN3
CML7.5

Mitochondrial large subunit rDNA (mt-lsu)
CTC GGC AAA TTA TCC TCA TAA GCAG TAG AAG CTG CAT AGG GTCCGA CAC AGG TTC GTA GGT AG
CCG CCC CAG TCA AAC TGC C

mt-lsu primer map

PCR and SEQ: ML5—ML6. MLIN3 and CML7.5 are alternatives

White et al. 1990
Bruns lab (see webpage for intron sites)

MS1
MS2
U1
CU6

Mitochondrial small subunit rDNA (mt-ssu)
White et al. 1990
CAG CAG TCA AGA ATA TTA GTC AAT G
GCG GAT TAT CGA ATT AAA TAA C
Bruns lab
TAA TTT TGG TGC CGA TTG AAC GTGT GGC ACG TCT ATA GCC CAmt-ssu primer map

PCR and SEQ: MS1—MS2. U1 and CU6 are alternatives.

ATP6-1
ATP6-3
ATP6-2
ATP6-4
ATP6-5f
ATP6-6r

Mitochondrial ATPase subunit 6, atp6
ATT AAT TSW CCW TTA GAW CAA TT
TCT CCT TTA GAA CAA TTT GA
TAA TTC TAN WGC ATC TTT AAT RTA
AAG TAC GAA WAC WTG WGM TTG
In experimental stage
WAT RGT WAG AGA WCA AWT AGG
AAC TAA TAR AGG AAC TAA AGC TA

Kretzer & Bruns 1999

Binder unpublished,
Hibbett lab

atp6 primer map

PCR and SEQ: ATP6-1 (ATP6-3)—ATP6-2 (ATP6-4) in any combination.
Note: ATP6-5f and ATP6-6r are not yet extensively tested but they work fine as SEQ primers.

COX3-1
COX3-2

Cytochrome oxidase subunit 3, cox3
CAT TTA GTA TCG CCT TCA CCA TGG CC
AAC AAC CAA ACA ACA TCT ACA AAG TG

cox3 primer map

Kretzer & Bruns 1999

EF1-526F
EF1-983F
EF1-1577F
EF1-1567R
EF1-1953R
EF1-2218R
Efcf
Efdf
Efgr
Efir
Efjr

Translation elongation factor 1α, EF-1α
GTC GTY GTY ATY GGH CAY GT
1-20
GCY CCY GGH CAY CGT GAY TTY AT
336-358
CAR GAY GTB TAC AAG ATY GGT GG
908-930
ACH GTR CCR ATA CCA CCR ATC TT
942-920
CCR GCR ACR GTR TGT CTC AT
1519-1490
ATG ACA CCR ACR GCR ACR GTY TG
1553-1530
ATY GCY GCN GGT ACY GGY GAR TTC GA 408-433
AAG GAY GGN CAR ACY CGN GAR CAY GC 447-472
GCA ATG TGG GCR GTR TGR CAR TC
1311-1289
GCR TGY TCN CGR GTY TGN CCR TC
472-450TGY TCN CGR GTY TGN CCR TCY TT
469-447

Rehner,(DeepHypha webpage)

EF-1α primer map

PCR: 1) 526F—1567R, 2) EF-df—2218R, 3) 983F—1953R
SEQ: 1) 526F, EF-ir, 1567R; 2) EF-df, 1577F,EF-gr, 2218R; 3) 983F, 1953R
Note: the suggestions made above are the most reliable combinations in our experience tocreate overlapping sequences. The whole gene, however, can be amplified in one or twopieces, while additional PCR products occur more frequently.

Lac 1FLac 3R
Lac 4R

LaccaseAGC AYT GGC AYG GCT TYT TYC
AGA CCR TCA CAR TAY TGR GTG GATA TCG AAG RAT GRC RGA ATT GATOmon Isikhuemhen,Vilgalys lab, (Pleurotus,Lentinus, Ganoderma)

Note: PCR conditions and primer combinations are currently being improved, suggestions
will follow later.

E2FB
E8R

Manganese dependant peroxidases and Lignin peroxidases (MnP, LiP)GAC CTS CAG AAG AAC CTG TTC SA
Omon Isikhuemhen,(Pleurotus)
CGG AGY TGS GTC TCG ATG AAG A

Note: PCR conditions and primer combinations are currently being improved, suggestions
will follow later.

RPB1-Af
RPB1-Cf
RPB1-Df
RPB1-Dr
RPB1-Ff
RPB1-Fr
RPB1-G1f
RPB1-G2raRPB1-Br

DNA-directed RNA polymerase II subunit 1, RPB1Hall lab
GAR TGY CCD GGD CAY TTY GG
CCN GCD ATN TCR TTR TCC ATR TA
TAC AAT GCY GAY TTY GAY GG
TTC ATY TCR TCD CCR TCR AAR TC
CAY GCD ATG GGD GGD MGD GAR GG
CCY TCN CKW CCW CCC ATD GCR TG
TGR AAD GTR TTD AGD GTC ATY TG
GTC ATY TGD GTD GCD GGY TCD CC
TCC GCR CCY TCT TCY TTG G

Matheny et al. 2002

RPB1 primer map

Note: PCR conditions and primer combinations are currently being improved, suggestions
will follow later.

RPB2-3bF
RPB2-6F
RPB2-6R
RPB2-7F
RPB2-7R
RPB2-11aR
RPB2-11bR
fRPB2-5f
fRPB2-5R
fRPB2-7cF
fRPB2-7cR
fRPB2-11aR
bRPB2-3.1F
bRPB2-6F
bRPB2-6.3F
bRPB2-7R
bRPB2-7.1R
bRPB2-10.9R

DNA-directed RNA polymerase II subunit 2, RPB2
Hall lab, general
GGW GGW TAY TTY ATY ATY AAT GG
TGG GGK WTG GTY TGY CCT GC
GCA GGR CAR ACC AWM CCC CAATG GGK AAG CAR GCW ATG GG
CCC ATW GCY TGC TTM CCC ATGTG WAT YTT RTC RTC MAC CCAA TCW CGY TCC ATY TCW CCHall lab, fungal
GAY GAY MGW GAT CAY TTY GGspecific
CCR AAR TGA TCW CKR TCR TC
ATG GGY AAR CAA GCY ATG GG
CCC ATR GCT TGY TTR CCC AT
GCR TGG ATC TTR TCR TCS ACCHall lab,ATY GCY CAA GAR MGN ATG GC
basidiomycetes specific
TGG GGY ATG GTN TGY CCY GC
GTY ATY GGT GTN TGG ATG GGGAY TGR TTR TGR TCR GGG AAV GG
CCC ATR GCY TGY TTM CCC ATD GC
GTR AAS GGY GTG GCR TCY CCRPB2-3F1RPB2-6F1
RPB2-7F1
RPB2-6R1
RPB2-7R1
RPB2-10R1

In experimental stage
AAR GTY YTK ATY GCM CAR GAG CG
CAC AAY CAN CAY TGG GGW ATG GT
ATG GAT ACN ATG GCS AAY AT
ACC ATW CCC CAR TGN TGR TTG TG
ATR TTG GCC ATN GTR TCC AT
ACC CTT YTG MCC RTG ACR AGA

Y.W. Lim unpublished,
polypore specific,
Hibbett lab

RPB2 primer map

Note: PCR conditions and primer combinations are currently being improved, suggestions
will follow later. See P. Brandon Matheny’s recently posted updates on RPB2.

LITERATURE CITED:••••••••••

Gardes M, Bruns TD. 1993. ITS primers with enhanced specifity for Basidiomycetes: application
to identification of mycorrhizae and rusts. Mol Ecol 2:113-118.
Hibbett DS. 1996. Phylogenetic evidence for horizontal transmission of Group I introns in thenuclear ribosomal DNA of mushroom-forming fungi. Mol Biol Evol 13:903-917.
Kretzer AM, Bruns TD. 1999. Use of atp6 in fungal phylogenetics: An example from the Boletales.
Mol Phyl Evol 13:483-492.
Matheny PB, Liu YJ, Ammirati JF, Hall BD. 2002. Using RPB1 sequences to improve phylogenetic
inference among mushrooms (Inocybe, Agaricales). Am J Bot 89:688-698.
Liu YJ, Wheelen S, Hall BD. 1999. Phylogenetic relationships among ascomycetes: evidence froman RNA polymerase II subunit. Mol Biol Evol 16:1799-1808.
O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC. 1998b. Multiple evolutionary origins of thefungus causing Panama disease of banana: Concordant evidence from nuclear andmitochondrial gene genealogies. Proc Natl Acad Sci 95:2044-2049.
O’Donnell K, Lutzoni FM, Ward TJ, Benny GL. 2001. Evolutionary relationships amongmucoralean fungi (Zygomycota): Evidence for family polyploidy on a large scale. Mycologia
93:286-296.
Rehner S. 2001. Primers for Elongation Factor 1-α (EF1-α).
http://ocid.NACSE.ORG/research/deephyphae/EF1primer.pdf
Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified
ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238-4246.
White TJ, Bruns TD, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ eds.
PCR protocols, a guide to methods and applications. San Diego, California: Academic Press.
p315-322.