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. 2012 Jul 25;7(7):e42149. doi: 10.1371/journal.pone.0042149

Table 3. Properties of group-specific primers and their coverage for phylogenetic and environmental groupings in the nifH database.

nifH a (%) Specific groupingsb (%) Environ.c (%)
Primerd Name Tg.e Pos.f Deg.g Tm (°C) 0 1 2 Pr Cy III IA Fr Pb Ep IV Soil Mat Sea Ref.h
CGCIWTYTACGGIAARGGIGG ChenBR1 BR 18–38 512 66.6–69.8 42 81 94 63 6 10 60 85 60 27 9 0 0 0 [52]
GCSTTCTACGGMAAGGGTGG nifH-f1-forA Fr 19–38 4 63.9–66.7 3 7 24 0 0 0 0 85 0 0 0 0 0 0 [21]
GCRTTYTACGGYAARGGSGG nifH-a1-forA AP 19–38 32 60.6–69.1 14 38 70 26 20 0 4 0 18 0 0 0 100 11 [21]
TACGGNAARGGSGGNATCGGCAA nifHF R 25–47 64 66.7–73.9 20 47 78 32 4 11 12 0 17 7 10 17 100 11 [53]
GGTATYGGYAARTGYACYAC primer-3 RA 37–56 32 52.6–64.8 0 19 56 0 0 0 0 0 0 0 0 0 0 0 [54]
GGCAAGTCCACCACCCAGC nifHf1 Fr 43–61 1 67.0 1 2 4 0 0 0 0 30 0 0 0 0 0 0 [55]
ATYGTCGGYTGYGAYCCSAARGC Olsen1 AM 106–128 64 65.0–73.6 37 66 81 53 2 3 58 57 0 14 0 38 100 0 [56]
CGTAGGTTGCGACCCTAAGGCTGA cyanoF Cy 108–131 1 68.8 0 0 1 0 1 0 0 0 0 0 0 0 0 0 [56]
GGCTGCGATCCCAAGGCTGA nifH-b1-forB AB 112–131 1 68.3 1 10 32 2 0 0 1 0 0 0 0 2 0 0 [21]
GGTTGTGACCCGAAAGCTGA nifH-g1-forB GP 112–131 1 64.1 0 3 10 1 1 0 0 0 0 0 0 1 0 0 [21]
GGWTGTGATCCWAARGCVGA nifH-c1-forB AN 112–131 24 58.7–64.3 1 8 25 1 1 1 0 0 0 4 0 1 0 4 [21]
GGCTGCGATCCGAAGGCCGA nifH-a2-forB AP 112–131 1 70.3 1 10 33 2 0 0 0 0 23 0 0 1 0 0 [21]
GGMTGCGAYCCSAARGCSGA nifH-a1-forB AP 112–131 32 66.2–72.7 27 58 73 29 1 31 41 22 33 5 8 15 20 0 [21]
GGBTGYGACCCSAASGCYGA nifH-f1-forB Fr 112–131 48 65.9–72.9 22 48 74 15 1 9 17 91 2 3 3 22 20 0 [21]
ACCCGCCTGATCCTGCACGCCAAGG nifHFor MS 136–160 1 74.7 11 20 32 21 0 0 5 0 0 0 0 16 0 9 [57]
TAARGCTCAAACTACCGTAT cylnif-F Cs 156–175 2 56.2–57.9 1 1 3 0 3 0 0 0 0 0 0 0 0 1 [58]
GAAGGTCGGCTACCAGAACA NIFH2F TB 231–250 1 63.1 0 2 6 1 0 0 0 0 0 0 0 2 0 0 [59]
AAGTTGATCGAGGTGATGACG NIFH5R TB 306–326 1 61.6 10 22 36 21 0 0 0 0 0 0 0 18 0 7 [59]
CCGGCCTCCTCCAGGTA nifH-269 Fr 325–341 1 64.2 3 3 3 0 0 0 0 85 0 0 0 4 0 0 [60]
ATTTAGACTTCGTTTCCTAC cylnif-R Cs 356–375 1 54.6 1 1 4 0 3 0 0 0 0 0 0 0 0 1 [58]
ACGATGTAGATTTCCTGGGCCTTGTT NifHRev MS 427–452 1 67.5 13 29 43 23 0 0 6 0 3 0 0 15 1 3 [57]
GACGATGTAGATYTCCTG primer 4 = AQE RA 436–453 2 53.8–55.1 24 54 81 33 8 21 12 69 7 0 1 19 24 13 [54]
GCATACATCGCCATCATTTCACC cyanoR Cy 460–482 1 63.6 4 8 23 2 25 0 1 0 0 0 0 1 0 8 [56]
GCGTACATSGCCATCATCTC nifH-f1-rev Fr 463–482 2 62.2–62.3 23 44 60 14 0 3 35 94 0 7 0 20 0 0 [21]
GCGTACATGGCCATCATCTC nifH-b1-rev AB 463–482 1 62.3 8 32 53 9 0 3 33 6 0 7 0 18 0 0 [21]
GCGTACATGGCCATCATCTC nifH-g1-rev GP 463–482 1 62.3 8 32 53 9 0 3 33 6 0 7 0 18 0 0 [21]
GCATAYASKSCCATCATYTC nifH-c1-rev AN 463–482 8 55.4–62.3 1 13 58 1 0 3 1 0 0 4 0 2 0 0 [21]
GCGTAGAGCGCCATCATCTC nifH-a2-rev AP 463–482 1 64.0 2 17 43 4 0 0 1 0 0 1 0 3 0 0 [21]
GCATAGAGCGCCATCATCTC nifH-a1-rev AP 463–482 1 62.0 9 16 33 17 0 1 0 0 0 0 0 2 0 0 [21]
ATGGTGTTGGCGGCRTAVAKSGCCATCAT Olsen2 AM 466–494 24 71.5–75.3 0 32 54 0 0 0 0 0 0 0 0 0 0 0 [56]
CTCGATGACGGTCATCCGGC nifHr Fr 671–690 1 65.9 0 3 6 0 0 0 0 0 0 24 0 0 0 0 [55]
GGIKCRTAYTSGATIACIGTCAT ChenBR2 BR 676–698 1024 63.6–69.1 31 67 87 40 0 0 7 71 0 0 0 39 0 0 [52]
GAAGACGATCCCGACCCCGA FGPH750 Fr 759–778 1 66.8 0 1 1 0 0 0 0 25 0 0 0 0 0 0 [48]
AGCATGTCYTCSAGYTCNTCCA nifHI R 785–806 32 63.3–68.8 24 41 51 44 0 0 0 0 0 0 0 100 0 0 [53]
GGTCGGGACCTCATCCTCGA FGPD913′ Fr NAi 1 66.3 10 10 10 0 0 NA NA 100 NA NA 0 NA NA NA [48]
a

Data indicate primer binding to all nifH sequences in the database with 0, 1, and 2 mismatches allowed. In some cases highly degenerate primers bind to multiple positions in the sequence generating coverage values that exceed 100%.

b

Data indicate primer binding to specific groupings in the nifH phylogeny. Abbreviations are as follows: Alpha-, Beta-, and Gammaproteobacteria (Pr); Cyanobacteria (Cy); Cluster III (III); Cluster IA (IA); Paenibacillus (Pb); Frankia (Fr); Epsilonproteobacteria Containing Cluster (Ep); paralogous sequences in Cluster IV (IV).

c

Primer coverage queried against sequences recovered from specific environments (Environ.) as described in methods. Environments include: soils (Soil), microbial mats (Mat), and pelagic marine samples (Sea).

d

Sequences are given in the 5′ to 3′ direction, IUPAC characters are used, and I =  Inosine.

e

Abbreviations indicate the Target Group (Tg.) which the primer was intended to amplify as follows: β-Rhizobia (BR); Frankia (Fr); Alphaproteobacteria (AP); Symbiotic rhizobia (R); reamplification of Cluster I (RA); aerobic and microaerophilic diazotrophs (AM); Cyanobacteria (Cy); Alpha- and Betaproteobacteria (AB); Gammaproteobacteria (GP); alternative nitrogenase cluster (AN); designed to match multiple species of Azospirillum, Burkholderia, Gluconoacetobacter, Azotobacter, Herbaspirillum and Azoarcus (MS); species of the cyanobacterial genus Cylindrospermopsis (Cs); Bradyrhizobium sp. prevalent in truffles (TB).

f

Position is relative to A. vinelandii nifH (Genbank ACCN# M20568).

g

Degeneracy is given as the number of oligonucleotides that comprise the primer.

h

References in which the primers are described.

i

This binding position for this primer sequence lies beyond the stop codon of Frankia sp. (Genbank ACCN# M21132) and cannot be represented using the A. vinelandii numbering system.

NA: Data not available as described in Methods.