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PolyA Polymerase Modification and Reverse Transcriptase
PCR Amplification of Environmental RNA

Lina M. Botero1 , Seth D’Imperio1 , Timothy R. McDermott1*, Mark Burr1 , Mark Young1 & Daniel J. Hassett2

1 Thermal Biology Institute
Montana State University
Bozeman, MT 59717

2 Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524

Figure 1 Caption. Cartoon describing the steps involved in polyA modification and reverse transcriptase amplification of environmental (m)(r)RNA. Step 1: PolyA tail modification of 3' termini. Step 2: First strand cDNA synthesis with MMLV-RT; requires the addition of polyT-oligo primer, Primer #1. Step 3: MMLV-RT terminal transferase activity adds Cs to the 3' end of the first strand cDNA. Step 4: MMLV-RT template switching; requires primer #2 that base-pairs with the dC added by the terminal transferase. Step 5: Amplification of double stranded cDNA using Primer #3. Steps 1-4 are carried out in a single step. Figure significantly modified from Fig. 1 in Clontech protocol #PT3656-1.

 

Table 1. Detailed list of major cDNA clone groups obtained from polyA-modified RNA and amplified using RT-PCR and PCR as depicted in Figure 1. Clone groups are categorized based on type of rRNA and by (sub-)phylum. For each (sub-)phylum designated clone group, descriptive information is shown as the total number of clones contained in that (sub-)phylum category. For each individual clone group within the (sub-)phylum category, the number of clones is shown along with the GenBank accession number of a representative clone(s), the total number of clones, the range of sequence identity scores relative to the nearest phylogenetic neighbors for all clones in that group, and the genus/organism represented. Clones corresponding to prokaryotic organisms are listed first, followed by eukaryotic organisms, and then chloroplasts.

Clone group
Representative
clone(s)
(accession #)
Number of clones
% Identity
Genus/organism represented
by closest phylogenetic
neighbor (accession #)

Prokaryote rRNA
16S rRNA
α Proteobacteria 58 total
1
NBFF62
(AY389855)
NBFF221
(AY389856)
32
99-100%
Gram-negative acidothermophile
YNP isolate Y008 (AY140238)
2
NBFF56
(AY389853)
NBFF314
(AY389844)
10
98-100%
Acidocella sp (D86510)
3
NBFF21
(AB006711)
6
99%
Acidophilum multivorum
(AY389838)
4
NBFF149
(AY389835)
3
99%
Acidiphilum sp. (AF376026)
5
NBFF87
(AY389860)
1
99%
Thiobacillus acidophilus
(D86511)
6
NBFF48
(AY389852)
1
99%
Yellowstone thermal soil PCR
clone (AF391980)
7
NBFF217
(AY389839)
2
97%
Geothermal spring PCR clone
(AF232921)
8
NBFF57
(AY389854)
3
93-94%
Acidosphaera rubrifaciens
(D86512)
β Proteobacteria 24 total
9
NBFF120
(AY389832)
NBFF 426
(AY389848)
24
97%
Thiomonas sp.
(AJ549220)
γ Proteobacteria 6 total
10
NBFF110
(AY389831)
3
97%

 

Frateuria sp. (AF376025)
11
NBFF192
(AY389836)
2
97%
Biofilter community PCR
clone (AY096180)
12
NBFF233
(AY389841)
1
95%
Legionella rubrilucens
(Z326643)
Firmicutes 50 total
13
NBFF81
(AY389857)
NBFF 323
(AY389845)
28
97-99%
Alicyclobacillus acidiphilus
(AB076660)
14
NBFF85
(AY389858)
4
97-98%
Alicyclobacillus
acidoterrestris
(AB042058)
15
NBFF11
(AY389830)
1
90%
Alicyclobacillus
cycloheptanicus
(AB042059)
16
NBFF266
(AY389842)
2
99%
Alicyclobacillus hesperidum
(AJ5449220)
17
NBFF1
(AY389829)
NBFF193
(AY389837)
2
98%
Alicyclobacillus sp.
(AF450135)
Alicyclobacillus sp.
(AB060164)
100%
18
NBFF130
(AY389834)
1
98%
Alicyclobacillus hesperidensis
(AJ133633)
19
NBFF343
(AY389846)
2
97%
Bacillus soli (AJ542513)
20
NBFF469
(AY389851)
3
97%
Bacillus niacini
(AB021194)
21
NBFF354
(AY389847)
NBFF462
(AY389850)
4
98-99%
Biofilter community PCR
clone (AY096175)
22
NBFF86
(AY389859)
NBFF123
(AY389833)
3
91-92%
Sulfobacillus disulfidooxidans
(U34974)
Aquificae 1 total
23
NBFF44
(AY389849)
1
99%
Hydrogenobaculum sp.
(AJ320225)
Acidobacteria 1 total
24
NBFF308
(AY389843)
1
97%
PCR clone from methanol
soil enrichment (AF200696)
Actinobacteria 1 total
25
NBFF223
(AY389840)
1
95%
Bacterium Ellin301
(AF498683)
23S rRNA
α Proteobacteria 106 total *
26
NBFF15
(AY389866)
NBFF16
(AY389868)
106
88-93%
Acetobacter xylinus (X89812)

A. europaeus (X89771)
γ Proteobacteria 1 total
27
NBFF164
(AY389881)
1
90%
Xanthomonas campestris
(AEO12540)
Firmicutes 7 total
28
NBFF50
(AY389877)
NBFF54
(AY389878)
NBFF291
(AY389872)
NBFF357
(AY389875)
7
90-93%
Bacillus sp. (X60981)

B. subtilis (D11460)

Lactococcus lactis (X64887)

B. licheniformis (X68433)

Aquificae 3 total
29
NBFF332
(AY389873)
3
75-80%
Aquifex pyrophilus
(APY15785)
Eukaryote rRNA
18S rRNA
Fungi 15 total
37

NBFF72
(AY389879)
NBFF96
(AY389865)
NBFF144
(AY389867)
NBFF155
(AY389867)
NBFF 211
(AY389869)
NBFF 251
(AY389870)

15
97-99%
Trichoderma (AY180319)

Hypocrea (AF510497)

Eupenicillium (U21298)

Paecilomyces (AB033528)

Aphanoascus (ABO15788)

Penicillium (AF510496)

28S rRNA
Fungi 1 total
38
NBFF108
(AY389862)
1
99%
Hypocrea lutea (ABO27384)
25S rRNA
Bryophyta 1 total
39
NBFF46
(AY389876)
1
90%
Diphyscium foliosum (AJ271116)
28S rRNA
Metazoa 1 total
40
NBFF113
(AY389863)
1
87%
Austrobilharzia variglandis
(AY157250)
Chloroplast Large Subunit rRNA
41
NBFF106
(AY389861)
NBFF121
(AY389864)
1
97%
Chlorococcum (L44123)
1
89%
Pylaiella (X61179)
Chloroplast Small Subunit rRNA
42
NBFF337
(AY389874)
1
89%
Odontella (Z67753)
* Most 23S rRNA cDNA clones were detected by dot blot screening of clones probing with a labeled oligonucleotide probe specific for Acetobacter 23S rDNA. However, the Acetobacter-like identity was verified by sequence analysis with a random sample (20%) of these clones.

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