Analysis of seung yeo moon primers (Moon et al lo 2000)




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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

Number of sequences

Forward primer

Reverse primer

0

593

654

1, not in 3’- nucleotide

188

106

2, not in 3’- nucleotide

27

39

3, not in 3’- nucleotide

47

23

4, not in 3’- nucleotide

5

10

>4 or mismatch in 3’- nucleotide

6

34

Total

866

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.




  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 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.

  2. 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

Number of sequences

Forward primer

Reverse primer

0

421

162

1, not in 3’- nucleotide

278

437

2, not in 3’- nucleotide

134

96

3, not in 3’- nucleotide

69

34

4, not in 3’- nucleotide

61

18

>4 or mismatch in 3’- nucleotide

70

48

Total

1033

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 :

  1. species with >4 mismatches or a terminal mismatch with the forward primer

  2. 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 C G U gap other

A | 145 101 18 255 0 94

A | 501 19 3 10 0 80

C | 10 432 0 123 1 47

C | 1 551 3 28 0 30

U | 39 17 2 549 0 6

- | 0 0 1 1 610 1

G | 1 0 599 4 1 8

G | 22 1 586 3 0 1

U | 1 1 0 609 1 1

U | 0 9 4 590 4 6

- | 1 0 0 0 611 1

- | 0 0 0 1 611 1

G | 2 0 606 1 1 3

A | 609 0 1 1 1 1

U | 3 1 0 606 1 2

C | 2 520 0 89 0 2

C | 0 575 0 37 0 1

U | 0 1 0 609 0 3

G | 5 2 602 1 0 3

- | 0 1 0 0 611 1

C | 0 600 2 10 0 1

C | 1 610 0 1 0 1

A | 559 0 11 42 0 1

G | 8 1 601 0 0 3

- | 0 0 1 19 592 1

U | 50 16 9 517 0 21

A | 509 39 52 8 0 5


reverse primer: 5'- GATCCTTCTGCAGGTTCACCTAC

complement: GUAGGUGAACCUGCAGAAGGAUC

Total number of sequences examined: 795

species with > 0 mismatches: total = 633

pr | A C G U gap other

G | 4 2 621 5 0 2

U | 1 1 2 629 0 1

- | 1 0 0 1 631 1

A | 603 3 0 26 0 2

G | 0 2 624 0 2 6

G | 16 0 578 3 36 1

U | 28 2 4 599 0 1

G | 2 0 630 1 0 1

A | 630 0 0 2 0 2

A | 631 1 1 0 0 1

- | 1 4 0 0 628 1

C | 1 595 3 16 18 1

C | 0 626 2 2 2 2

U | 33 0 0 600 0 1

- | 0 0 2 0 631 1

G | 2 1 608 19 1 3

C | 3 599 6 20 2 4

A | 80 21 505 3 1 24

- | 0 0 2 0 631 1

G | 18 4 605 3 2 2

A | 563 56 8 4 1 2

A | 509 7 61 52 2 3

G | 4 2 624 1 1 2

G | 7 3 613 2 8 1

A | 617 1 4 10 1 1

U | 4 9 5 615 0 1

C | 10 611 3 9 0 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;D85503;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


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