ANALYSIS OF SEUNG YEO MOON PRIMERS (Moon et al LO 2000)
By Jan Wuyts and Rupert De Wachter, Universiteit Antwerpen, Belgium
We tried to determine how large a fraction of the known SSU rRNA gene sequences would be amplified by the primer set used
in the work by Moon et al.. This proved to be more complicated than it looks at first sight, as explained below.
1. Our database presently contains 5260 eukaryotic SSU rRNA sequences. We only collect and align sequences that are at
least 70% complete. However, most incomplete sequences are incomplete at the termini, probably because they were
amplified by means of primers further inward than those used in the work by Moon et al.. As a result, only 866 sequences
contain both PCR primer binding sites used in the manuscript by Moon et al. So only for these sequences can it be
examined which fraction of them would be amplified by the chosen primer set.
2. Although the primers are situated in conserved areas, a considerable fraction of the 866 sequences shows a difference of
one or more nucleotides with the primer sequences. Therefore one cannot predict exactly how many sequences would be
amplified by the primers, because a difference of a limited number of bases with the primer sequence does not necessarily
impair amplification. However one cannot predict exactly how many differences are tolerable. Sometimes a primer does
not work although its sequence exactly matches that of the gene, sometimes it works although several nucleotides are
different. We assumed that a mismatch of the sequence with the 3’-terminal nucleotide is prohibitive for amplification.
Our calculations were performed as follows:
 For each primer, we counted:
 the number of sequences with zero mismatches
 the number of sequences with 1, 2, 3, 4 mismatches, but not in the 3'-terminal nucleotide of the primer.
 For each primer a list of the distribution of bases at each primer position in the 866 avaiable complete sequences.
Number of mismatches with primer
0
1, not in 3’- nucleotide
2, not in 3’- nucleotide
3, not in 3’- nucleotide
4, not in 3’- nucleotide
>4 or mismatch in 3’- nucleotide
Total
Forward primer
593
188
27
47
5
6
866
Number of sequences
Reverse primer
654
106
39
23
10
34
866
What you wanted to have, I think, is an estimate of the fraction of eukaryotic species whose SSU rRNA genes would not be
amplified by your primers. There is no exact reply to this question, but we have made lists of the species that have the most
mismatches with the primers and therefore are least likely to be detectable. Document Primers3.doc contains 4 lists of species
with their taxonmy :
1. species with >4 mismatches or a terminal mismatch with the forward primer
2. species with >3 mismatches or a terminal mismatch with the forward primer
3. species with >4 mismatches or a terminal mismatch with the reverse primer
4. species with >3 mismatches or a terminal mismatch with the reverse primer
Note that list 2 includes the species of list 1 and list 4 includes those of list 3. You should print these lists in landscape
otherwise the lists look too scrambled.
Document Primers2.doc contains 2 Tables where you can see for each position of each primer how many times each of the 4
bases occurs. These tables could be useful if you want to develop other primers or primers with more ambiguities.
Total number of sequences examined: 866
forward primer: 5'-ACCTGGTTGATCCTGCCAG
reverse primer: 5'-TGATCCTTCYGCAGGTTCAC
complement: GUGAACCUGCRGAAGGAUCA
forward primer, species with > 0 mismatches: total = 273
pr
A
C
G
U
gap
other
A
223
16
4
26
0
4
C
9
161
0
100
0
3
C
1
250
3
18
0
1
U
33
11
3
224
0
2
0
0
1
1
271
0
G
1
0
267
4
1
0
G
9
2
260
2
0
0
U
0
0
0
273
0
0
U
0
5
2
261
4
1
0
1
0
0
272
0
G
2
0
270
1
0
0
A
270
0
1
1
1
0
U
2
2
0
268
1
0
C
1
192
0
79
0
1
C
0
244
0
29
0
0
U
0
0
0
273
0
0
G
5
1
266
0
0
1
C
2
261
1
9
0
0
C
0
272
0
1
0
0
A
227
1
9
36
0
0
G
3
1
266
1
0
2
reverse primer, species with > 0 mismatches: total = 211
pr
A
C
G
U
gap
other
G
13
1
194
2
1
0
U
32
2
4
172
0
1
G
1
0
208
2
0
0
A
208
0
0
2
0
1
A
210
1
0
0
0
0
1
3
0
0
207
0
C
1
178
4
13
15
0
C
1
204
3
2
1
0
U
35
0
0
175
0
1
0
0
2
0
209
0
G
2
1
185
21
1
1
C
1
178
5
24
2
1
G
138
19
46
3
3
2
G
13
3
186
3
5
1
A
139
58
7
5
2
0
A
125
4
23
58
1
0
G
5
1
202
1
1
1
G
7
1
194
1
8
0
A
199
2
4
3
2
1
U
17
8
2
182
0
2
C
7
190
1
12
0
1
A
157
41
6
7
0
0
List of species with most mismatches
Forward primer:
Species with more than 4 mismatches or mismatch in last nucleotide:
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium falciparum (malaria parasite P. falciparum) : X57167
Eukaryota Fungi Ascomycota {Ascomycota incertae sedis} Pleomassariaceae {anamorphic Pleomassariaceae} Dendryphiopsis : Dendryphiopsis atra
:AF053731
Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Archosauria Aves Palaeognathae Tinamiformes Tinamidae Nothoprocta : Nothoprocta ornata :
AJ002921
Eukaryota Metazoa Nematoda Chromadorea Spirurida Gnathostomatoidea Gnathostomatidae Gnathostoma : Gnathostoma binucleatum : Z96946
Eukaryota Metazoa Nematoda Chromadorea Spirurida Gnathostomatoidea Gnathostomatidae Gnathostoma : Gnathostoma neoprocyonis : Z96947
Eukaryota Metazoa Nematoda Chromadorea Spirurida Gnathostomatoidea Gnathostomatidae Gnathostoma : Gnathostoma turgidum : Z96948
Species with more than 3 mismatches or mismatch in last nucleotide:
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium falciparum (malaria parasite P. falciparum) : X57167
Eukaryota Diplomonadida Hexamitidae Giardiinae Giardia : Giardia intestinalis : M54878
Eukaryota Diplomonadida Hexamitidae Giardiinae Giardia : Giardia intestinalis : U09491
Eukaryota Diplomonadida Hexamitidae Giardiinae Giardia : Giardia intestinalis : U09492
Eukaryota Diplomonadida Hexamitidae Giardiinae Giardia : Giardia intestinalis : X52949
Eukaryota Fungi Ascomycota {Ascomycota incertae sedis} Pleomassariaceae {anamorphic Pleomassariaceae} Dendryphiopsis : Dendryphiopsis atra
:AF053731
Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Archosauria Aves Palaeognathae Tinamiformes Tinamidae Nothoprocta : Nothoprocta ornata :
AJ002921
Eukaryota Metazoa Nematoda Chromadorea Spirurida Gnathostomatoidea Gnathostomatidae Gnathostoma : Gnathostoma binucleatum : Z96946
Eukaryota Metazoa Nematoda Chromadorea Spirurida Gnathostomatoidea Gnathostomatidae Gnathostoma : Gnathostoma neoprocyonis : Z96947
Eukaryota Metazoa Nematoda Chromadorea Spirurida Gnathostomatoidea Gnathostomatidae Gnathostoma : Gnathostoma turgidum : Z96948
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon cuniculi : AJ005581
Reverse primer:
Species with more than 4 mismatches or mismatch in last nucleotide:
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium falciparum (malaria parasite P. falciparum) : X57167
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium vivax (malaria parasite P. vivax) : U93095
Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Archosauria Aves Palaeognathae Tinamiformes Tinamidae Nothoprocta : Nothoprocta ornata :
AJ002921
Eukaryota Microsporidia Burenellidae Vairimorpha : Vairimorpha lymantriae : AF033315
Eukaryota Microsporidia Burenellidae Vairimorpha : Vairimorpha necatrix : Y00266
Eukaryota Microsporidia Burenellidae Vairimorpha : Vairimorpha sp. : D85502
Eukaryota Microsporidia : Microsporidium 57864 : U90885
Eukaryota Microsporidia Nosematidae Nosema : Nosema apis : U26534
Eukaryota Microsporidia Nosematidae Nosema : Nosema apis : U26534
Eukaryota Microsporidia Nosematidae Nosema : Nosema apis : U97150
Eukaryota Microsporidia Nosematidae Nosema : Nosema bombycis : D85503
Eukaryota Microsporidia Nosematidae Nosema : Nosema ceranae : U26533
Eukaryota Microsporidia Nosematidae Nosema : Nosema furnacalis : U26532
Eukaryota Microsporidia Nosematidae Nosema : Nosema necatrix : U11051
Eukaryota Microsporidia Nosematidae Nosema : Nosema oulemae : U27359
Eukaryota Microsporidia Nosematidae Nosema : Nosema portugal : AF033316
Eukaryota Microsporidia Nosematidae Nosema : Nosema sp. : D85501
Eukaryota Microsporidia Nosematidae Nosema : Nosema sp. : D85501
Eukaryota Microsporidia Nosematidae Nosema : Nosema trichoplusiae : U09282
Eukaryota Microsporidia : Pleistophoridae : D85500
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon cuniculi : X98467
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon cuniculi : X98469
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon cuniculi : X98470
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon hellem : AF118142
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon hellem : AF118143
Eukaryota Microsporidia Visvesvaria : Visvesvaria acridophagus : AF024658
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Acetabularia : Acetabularia acetabulum : Z33461
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Acetabularia : Acetabularia major : Z33462
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Acicularia : Acicularia schenkii : Z33470
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Cymopolia : Cymopolia van bosseae : Z33467
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Neomeris : Neomeris dumentosa : Z33469
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Polyphysa : Polyphysa peniculus : Z33472
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Polyphysa : Polyphysa peniculus : Z33473
Species with more than 3 mismatches or mismatch in last nucleotide:
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium falciparum (malaria parasite P. falciparum) : X57167
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium vivax (malaria parasite P. vivax) : U93095
Eukaryota Fungi Basidiomycota Hymenomycetes Ceratobasidiales Ceratobasidiaceae {anamorphic Ceratobasidiaceae} Rhizoctonia : Rhizoctonia praticola :
M92990
Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Archosauria Aves Palaeognathae Tinamiformes Tinamidae Nothoprocta : Nothoprocta ornata :
AJ002921
Eukaryota Metazoa Cnidaria Epiactis : Epiactis japonica : Z92904
Eukaryota Metazoa Cnidaria Hydrozoa Hydroida Sertulariidae Selaginopsis: Selaginopsis cornigera : Z92899
Eukaryota Microsporidia Burenellidae Vairimorpha : Vairimorpha lymantriae : AF033315
Eukaryota Microsporidia Burenellidae Vairimorpha : Vairimorpha necatrix: Y00266
Eukaryota Microsporidia Burenellidae Vairimorpha : Vairimorpha sp. : D85502
Eukaryota Microsporidia : Microsporidium 57864 : U90885
Eukaryota Microsporidia Nosematidae Nosema : Nosema apis : U26534
Eukaryota Microsporidia Nosematidae Nosema : Nosema apis : U26534
Eukaryota Microsporidia Nosematidae Nosema : Nosema apis : U97150
Eukaryota Microsporidia Nosematidae Nosema : Nosema bombycis : D85503
Eukaryota Microsporidia Nosematidae Nosema : Nosema ceranae : U26533
Eukaryota Microsporidia Nosematidae Nosema : Nosema furnacalis : U26532
Eukaryota Microsporidia Nosematidae Nosema : Nosema necatrix : U11051
Eukaryota Microsporidia Nosematidae Nosema : Nosema oulemae : U27359
Eukaryota Microsporidia Nosematidae Nosema : Nosema portugal : AF033316
Eukaryota Microsporidia Nosematidae Nosema : Nosema sp. : D85501
Eukaryota Microsporidia Nosematidae Nosema : Nosema sp. : D85501
Eukaryota Microsporidia Nosematidae Nosema : Nosema trichoplusiae :U09282
Eukaryota Microsporidia : Pleistophoridae : D85500
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon cuniculi : X98467
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon cuniculi : X98469
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon cuniculi : X98470
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon hellem : AF118142
Eukaryota Microsporidia Unikaryonidae Encephalitozoon : Encephalitozoon hellem : AF118143
Eukaryota Microsporidia Visvesvaria : Visvesvaria acridophagus :AF024658
Eukaryota stramenopiles Chrysophyceae Synurales Synura : Synura uvella : U73222
Eukaryota stramenopiles Labyrinthulida Thraustochytriidae Labyrinthuloides : Labyrinthuloides haliotidis : U21338
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Acetabularia : Acetabularia acetabulum : Z33461
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Acetabularia : Acetabularia major : Z33462
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Acicularia : Acicularia schenkii : Z33470
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Batophora : Batophora occidentalis : Z33465
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Batophora : Batophora oerstedii : Z33463
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Bornetella : Bornetella nitida : Z33464
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Cymopolia : Cymopolia van bosseae : Z33467
Eukaryota Viridiplantae Chlorophyta Ulvophyceae DasycladalesDasycladaceae Neomeris : Neomeris dumentosa : Z33469
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Polyphysa : Polyphysa parvula : Z33471
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Polyphysa : Polyphysa peniculus : Z33472
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae Polyphysa : Polyphysa peniculus : Z33473
ANALYSIS OF MEDLIN PRIMERS
Dear Daniel,
My collaborator Jan Wuyts has done the calculations on the Medlin primers
and I am attaching his results. We have simply made the required changes in
the 3 files and they are now called Medlin1.doc to Medlin3.doc. As you will
see, the addition of extra nucleotides results in more species possessing
differences, especially at the 3'-terminus of the forward primer. So if you
want to amplify the 18S genes of the maximum possible diversity of species
I think Mooon's primers are better.
Best wishes,
Rupert
We tried to determine how large a fraction of the known SSU rRNA gene sequences would be amplified by the primer set used
by Medlin. This proved to be more complicated than it looks at first sight, as explained below.
3. Our database presently contains 5260 eukaryotic SSU rRNA sequences. We only collect and align sequences that are at
least 70% complete. However, most incomplete sequences are incomplete at the termini, probably because they were
amplified by means of primers further inward than those used by Medlin. As a result, only 1033 sequences contain the PCR
primer binding sites at the 5' terminus of the molecule and only 795 sequences contain the PCR primer binding sites at the
3' terminus. So only for these sequences can it be examined which fraction of them would be amplified by the chosen
primer set.
4. Although the primers are situated in conserved areas, a considerable fraction of the sequences shows a difference of one or
more nucleotides with the primer sequences. Therefore one cannot predict exactly how many sequences would be amplified
by the primers, because a difference of a limited number of bases with the primer sequence does not necessarily impair
amplification. However one cannot predict exactly how many differences are tolerable. Sometimes a primer does not work
although its sequence exactly matches that of the gene, sometimes it works although several nucleotides are different. We
assumed that a mismatch of the sequence with the 3’-terminal nucleotide is prohibitive for amplification.
Our calculations were performed as follows:
 For each primer, we counted:
 the number of sequences with zero mismatches
 the number of sequences with 1, 2, 3, 4 mismatches, but not in the 3'-terminal nucleotide of the primer.
 For each primer a list of the distribution of bases at each primer position in the 1033 and 795 avaiable sequences with
complete termini.
Number of mismatches with primer
0
1, not in 3’- nucleotide
2, not in 3’- nucleotide
3, not in 3’- nucleotide
4, not in 3’- nucleotide
>4 or mismatch in 3’- nucleotide
Total
Forward primer
421
278
134
69
61
70
1033
Number of sequences
Reverse primer
162
437
96
34
18
48
795
What you wanted to have, I think, is an estimate of the fraction of eukaryotic species whose SSU rRNA genes would not be
amplified by your primers. There is no exact reply to this question, but we have made lists of the species that have the most
mismatches with the primers and therefore are least likely to be detectable.
Document Medlin2.doc contains 2 Tables where you can see for each position of each primer how many times each of the 4
bases occurs. These tables could be useful if you want to develop other primers or primers with more ambiguities.
Document Medlin3.doc contains 2 lists of species with their taxonmy :
5. species with >4 mismatches or a terminal mismatch with the forward primer
6. species with >4 mismatches or a terminal mismatch with the reverse primer
forward primer: 5'- AACCTGGTTGATCCTGCCAGTA
Total number of sequences examined: 1033
species with > 0 mismatches: total = 612
pr
A
A
C
C
U
G
G
U
U
G
A
U
C
C
U
G
C
C
A
G
U
A
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
A
145
501
10
1
39
0
1
22
1
0
1
0
2
609
3
2
0
0
5
0
0
1
559
8
0
50
509
C
101
19
432
551
17
0
0
1
1
9
0
0
0
0
1
520
575
1
2
1
600
610
0
1
0
16
39
G
18
3
0
3
2
1
599
586
0
4
0
0
606
1
0
0
0
0
602
0
2
0
11
601
1
9
52
U
255
10
123
28
549
1
4
3
609
590
0
1
1
1
606
89
37
609
1
0
10
1
42
0
19
517
8
gap
0
0
1
0
0
610
1
0
1
4
611
611
1
1
1
0
0
0
0
611
0
0
0
0
592
0
0
other
94
80
47
30
6
1
8
1
1
6
1
1
3
1
2
2
1
3
3
1
1
1
1
3
1
21
5
reverse primer: 5'- GATCCTTCTGCAGGTTCACCTAC
complement: GUAGGUGAACCUGCAGAAGGAUC
Total number of sequences examined: 795
species with > 0 mismatches: total = 633
pr
G
U
A
G
G
U
G
A
A
C
C
U
G
C
A
G
A
A
G
G
A
U
C
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
A
4
1
1
603
0
16
28
2
630
631
1
1
0
33
0
2
3
80
0
18
563
509
4
7
617
4
10
C
2
1
0
3
2
0
2
0
0
1
4
595
626
0
0
1
599
21
0
4
56
7
2
3
1
9
611
G
621
2
0
0
624
578
4
630
0
1
0
3
2
0
2
608
6
505
2
605
8
61
624
613
4
5
3
U
5
629
1
26
0
3
599
1
2
0
0
16
2
600
0
19
20
3
0
3
4
52
1
2
10
615
9
gap
0
0
631
0
2
36
0
0
0
0
628
18
2
0
631
1
2
1
631
2
1
2
1
8
1
0
0
other
2
1
1
2
6
1
1
1
2
1
1
1
2
1
1
3
4
24
1
2
2
3
2
1
1
1
1
List of species with most mismatches
Forward primer:
Species with more than 4 mismatches or mismatch in last nucleotide:
1
Eukaryota Metazoa Arthropoda Crustacea Malacostraca Eumalacostraca Eucarida Decapoda Dendrobranchiata Penaeoidea Penaeidae Penaeus
Farfantepenaeus : Penaeus (Farfantepenaeus) aztecus (brown shrimp) : M34362
1
Eukaryota Metazoa Arthropoda Crustacea Malacostraca Eumalacostraca Eucarida Decapoda Pleocyemata Stenopodidea Stenopodidae Stenopus :
Stenopus hispidus (banded coral shrimp) : M34361
1
Eukaryota Metazoa Chordata Craniata Hyperotreti Myxiniformes Myxinidae Eptatretinae Eptatretus : Eptatretus stouti (Pacific hagfish) : M97572
1
Eukaryota Metazoa Chordata Craniata Hyperotreti Myxiniformes Myxinidae Myxininae Myxine : Myxine glutinosa (Atlantic hagfish) : M97574
1
Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Amphibia Gymnophiona Caeciliidae Caeciliinae Grandisonia : Grandisonia
alternans : M59391
1
Eukaryota Metazoa Gastrotricha Chaetonotida Paucitubulatina Chaetonotidae Chaetonotus : Chaetonotus sp. : AJ001735
8
Eukaryota Metazoa Nematoda Chromadorea : X03680;Z96946;Z96947;Z96948;L04153;L04154;L04152;U01230
1
Eukaryota Metazoa Arthropoda Pentastomida Porocephalida Porocephaloidea Porocephalidae Porocephalus : Porocephalus crotali : M29931
1
Eukaryota Fungi Ascomycota Saccharomycetes Saccharomycetales {anamorphic Saccharomycetales} Candida : Candida tropicalis : M60308
1
Eukaryota Fungi Basidiomycota Hymenomycetes Auriculariales Auriculariaceae Auricularia : Auricularia polytricha : L22255
4
Eukaryota Viridiplantae Embryophyta Tracheophyta Spermatophyta Magnoliophyta eudicotyledons : X16604;X16603;L24408;L24415
1
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium falciparum (malaria parasite P. falciparum) : X57167
1
Eukaryota Cryptophyta Cryptomonadaceae Geminigera : Geminigera cryophila : U53123
33
Eukaryota Microsporidia :
U68473;U68474;AF027685;AF024655;AF027683;AF027684;Z19563;X98467;X98470;X98469;AJ005581;L07123;AF023245;AF024657;U26534;U97150;D
85503;D85504;U26533;U26532;U11051;U27359;D85501;U09282;AF027682;U10342;D85500;AF033197;AJ002605;Y00266;D85502;AF024658;AF024656
;
1
Eukaryota Cercozoa Cercomonadidae Cercomonas : Cercomonas ATCC50319 : U42451
6
Eukaryota Diplomonadida Hexamitidae Giardiinae Giardia : M54878;X52949;U09491;U09492;AF006676;AF006677
Reverse primer:
Species with more than 4 mismatches or mismatch in last nucleotide:
1
Eukaryota Metazoa Arthropoda Crustacea Maxillopoda Cirripedia Thoracica Sessilia Balanomorpha Chthamaloidea Chthamalidae Chthamalus :
Chthamalus fragilis : L26515
1
Eukaryota Metazoa Cnidaria Hydrozoa Hydroida Sertulariidae Selaginopsis : Selaginopsis cornigera : Z92899
1
Eukaryota Fungi Ascomycota Taphrinales Taphrinaceae Taphrina : Taphrina deformans (peach leaf curl fungus) : X69852
1
Eukaryota Fungi Ascomycota Pezizales Helvellaceae Helvella : Helvella lacunosa : U53378
1
Eukaryota Fungi Basidiomycota Hymenomycetes Ceratobasidiales Ceratobasidiaceae {anamorphic Ceratobasidiaceae} Rhizoctonia : Rhizoctonia
praticola : M92990
1
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium falciparum (malaria parasite P. falciparum) : X57167
1
Eukaryota Alveolata Apicomplexa Haemosporida Plasmodium : Plasmodium vivax (malaria parasite P. vivax) : U93095
13
Eukaryota Viridiplantae Chlorophyta Ulvophyceae Dasycladales Dasycladaceae :
Z33461;Z33460;Z33468;Z33462;Z33470;Z33465;Z33463;Z33464;Z33467;Z33469;Z33471;Z33472;Z33473;
1
Eukaryota stramenopiles Chrysophyceae Synurales Synura : Synura uvella : U73222
1
Eukaryota stramenopiles Labyrinthulida Thraustochytriidae Labyrinthuloides : Labyrinthuloides haliotidis : U21338
17
Eukaryota Microsporidia :
L15741;X98467;X98470;X98469;U26534;U97150;D85503;U26533;U26532;U11051;U27359;D85501;U09282;D85500;Y00266;D85502;AF024658