Primer Information for the Detection of Wolbachia, D. immitis, WO
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Primer Information for the Detection of Wolbachia, D. immitis, WO phage in mosquitoes Basic Information: The genomic DNA extracted from mosquitoes must be aliquoted into at least 4 tubes! Work with only one at a time. Be sure to record that number and the dilution date in your lab notebook! Repeated freezing and thawing causes DNA degradation! All PCR reactions should be VT=25 l All primers and dNTPs must be diluted into working stocks. The primer and dNTP working stocks should be a large enough volume to do several experiments. It is a good idea to aliquot the primers and dNTP working stocks into at least 3 tubes to guard against contamination or degradation. Use only one at a time and record the number and dilution date in your lab notebook! Your professor will add Taq to the PCR reactions. The amount of primers will vary depending on the primer set used. dNTPs (deoxynucleotides) should be at 200 M final in the tube. In other words use 1 l of a 5mM dNTP working stock for a 25 l PCR reaction. The PCR cycling conditions will vary depending on the primer set used. Primer Information: Many of these primers sequences and conditions have been obtained from the literature base. Some have been designed in house or obtained from Dr. Werren at U of R. As of 7/16/12, the conditions for the wsp primers (wol81F and wol691R), Wolbachia 16srRNA (16SwspecF and 16SwspecR) and D. immitis 16SrRNA (di16SrRNAF and di16SrRNAF) have been workedout using positive controls. The others are still in the works. Students will want to work first with the ones that are known to produce bands with positive controls. Accession numbers are the numbers given by Genbank to indicate a specific gene sequence. Prelab Assignment: Be sure you know how to do these things and what they mean, because it will be on the next exam!! Activity #1- Go to www.pubmed.com and cut and paste the accession # for Wolbachia 16S rRNA gene from the information below into the box at the top of the pubmed homepage. Select “nucleotide” from the drop down menu at the left of the box and click on “search”. You will see pages with the following information. A- Write below the number of nucleotides that encode the: 1) gene 2)protein from the gene Do you think they will always be the same number? Why or why not? Activity #2-Repeat Activity #1 for the D. immitis 16S rRNA gene Write down below the first 4 amino acids in single letter format as shown on the site and then using Figure 2 retrieve the full names of those amino acids. Circle names of the ones that like water. A- Write below the number of nucleotides that encode the: 3) gene 4)protein from the gene Do you think they will always be the same number? Why or why not? B- Write down below the first 4 amino acids in single letter format as shown on the site and then using Figure 2 retrieve the full names of those amino acids. Circle names of the ones that like water. Figure 1- The Pubmed Page for Accession# DQ900649.1 Wolbachia endosymbiont of Culex pipiens surface protein precursor (wsp) gene, partial cds GenBank: DQ900649.1 FASTA Graphics Go to: LOCUS 2006 DEFINITION precursor DQ900649 602 bp DNA linear BCT 24-DEC- Wolbachia endosymbiont of Culex pipiens surface protein (wsp) gene, partial cds. ACCESSION DQ900649 VERSION DQ900649.1 GI:119658040 KEYWORDS . SOURCE Wolbachia endosymbiont of Culex pipiens ORGANISM Wolbachia endosymbiont of Culex pipiens Bacteria; Proteobacteria; Alphaproteobacteria; Rickettsiales; Anaplasmataceae; Wolbachieae; Wolbachia. REFERENCE 1 (bases 1 to 602) AUTHORS Djadid,N.D. and Zakeri,S. TITLE Identification of Wolbachia spp. in Iranian Culicidae species JOURNAL Unpublished REFERENCE 2 (bases 1 to 602) AUTHORS Djadid,N.D., Daneshinia,N., Gholizadeh,S. and Zakeri,S. TITLE Characterization of wsp gene in Wolbachia species from Iran JOURNAL Unpublished REFERENCE 3 (bases 1 to 602) AUTHORS Djadid,N.D. and Zakeri,S. TITLE Direct Submission JOURNAL Submitted (15-AUG-2006) Biotechnology Department, Pasteur Institute of Iran, Pasteur, Tehran 1316943551, Iran FEATURES Location/Qualifiers source 1..602 /organism="Wolbachia endosymbiont of Culex pipiens" /mol_type="genomic DNA" /host="Culex pipiens Tg1567" gene /db_xref="taxon:363538" sequence /country="Iran: Langrood district, Guilan province" /note="PCR_primers=fwd_name: 81F, rev_name: 691R" coding sequence gene <1..602 /gene="wsp" CDS <1..602 amino acid sequence of the protein encoded by the gene /gene="wsp" /codon_start=2 /transl_table=11 /product="surface protein precursor" /protein_id="ABL87105.1" /db_xref="GI:119658041" Each letter represents one amino acid (see Figure 2) /translation="GPISDEETSYYVRLQYNGEVLPFKTRIDGIEYKKGTEVHDPLKASFMAGGAAFGYKMDDIR VDVEGLYSQLNKNDVSGATFTPTTVANSVAAFSGLVNVYYDIAIEDMPITPYVGVGVGAAYISNPSEASAVKDQK GFGFAYQAKAGVSYDVTPEIKLFAGARYFGSYGASFNKEAVSATKEINVLYSAVGAEAGVAFNF" gene DNA sequence ORIGIN 1 61 121 181 241 301 361 421 481 541 601 // tggtccaata tttacctttt tcctttaaaa cagggttgat atttactcca ttacgatata agcatatatc tgcttatcaa tggtgctcgt taaagagatc tt agtgatgaag aaaacaagaa gcatctttta gttgagggac acaactgttg gcgattgaag agcaatcctt gcaaaagctg tattttggtt aatgtccttt aaactagcta ttgacggcat tggctggtgg tttactcaca caaacagtgt atatgcctat cagaagctag gtgttagtta cttatggtgc acagcgctgt ctatgttcgt tgaatataaa tgctgcattt actaaacaaa ggcagcattt cactccatac tgcagttaaa tgatgtaacc tagttttaat tggtgcagaa ttgcaatata aaaggaaccg ggttataaaa aacgacgtta tcaggattgg gttggtgttg gatcaaaaag ccagaaatca aaagaagcag gctggagtag atggtgaagt aagttcatga tggacgatat gtggtgcaac ttaacgttta gtgttggtgc gatttggttt aactctttgc tatcagctac cgtttaattt Figure 2- Structure of the 20 Amino acids (http://www.bing.com/images/search?q=amino+acid+structure&view=detail&id=0690722C0F6619C8155ADA8C8D2570AD54E1D0B5&first=1) Target species: Wolbachia Target gene: wsp gene (Wolbachia surface protein gene) - supposedly supergroup A and B specific. However, DNA alignments brings that into question. (Simoes et al Molecular Ecology Resources (2011) 11:567-572 and supplemental information). Gene function: Surface protein Accession number: DQ900649 Forward primer name: wol81F Annealing temperature: 52°C Forward primer length: 24 Forward primer sequence: 5’ – TGG TCC AAT AAG TGA TGA AGA AAC – 3’ Reverse primer name: wol691R Annealing temperature: 52°C Reverse primer length: 20 Reverse primer sequence: 5’ – AAA AAT TAA ACG CTA CTC CA – 3’ USE 5 pmoles/25 l reaction Cycling Conditions: 95°C – 1 min 92°C – 30 sec 52°C – 40 sec 35X 72°C – 90 sec 72°C – 10 min Size of Product: ~610bp Sample product: Wolbachia endosymbiont of Culex pipiens surface protein precursor (wsp) gene, partial cds Accession number: DQ900649 1 tggtccaata agtgatgaag aaactagcta ctatgttcgt ttgcaatata atggtgaagt 61 tttacctttt aaaacaagaa ttgacggcat tgaatataaa aaaggaaccg aagttcatga 121 tcctttaaaa gcatctttta tggctggtgg tgctgcattt ggttataaaa tggacgatat 181 cagggttgat gttgagggac tttactcaca actaaacaaa aacgacgtta gtggtgcaac 241 atttactcca acaactgttg caaacagtgt ggcagcattt tcaggattgg ttaacgttta 301 ttacgatata gcgattgaag atatgcctat cactccatac gttggtgttg gtgttggtgc 361 agcatatatc agcaatcctt cagaagctag tgcagttaaa gatcaaaaag gatttggttt 421 tgcttatcaa gcaaaagctg gtgttagtta tgatgtaacc ccagaaatca aactctttgc 481 tggtgctcgt tattttggtt cttatggtgc tagttttaat aaagaagcag tatcagctac 541 taaagagatc aatgtccttt acagcgctgt tggtgcagaa gctggagtag cgtttaattt 601 tt Literature source: Gavotte, L., H. Henri, R. Stauthamer, D. Charif, S. Charlat, M. Bouletreach, and F. Vavre. 2007. A survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia. Molecular Biology and Evolution 24(2): 427-435. Target species: Wolbachia Target gene: 16S rRNA gene - from the Werren lab at University of Rochester Gene function: Protein synthesis Accession number: JN100083 Forward primer name: 16SwspecF Annealing temperature: 55°C Forward primer length: 21 Forward primer sequence: 5’ – CAT ACC TAT TCG AAG GGA TAG – 3’ Reverse primer name: 16SwspecR Annealing temperature: 55°C Reverse primer length: 21 Reverse primer sequence: 5’ – AGC TTC GAG TGA AAC CCA TTC– 3’ USE 25 pmoles/25 l reaction Cycling Conditions: 94°C – 2 min 94°C – 30 sec 55°C – 45 sec 35X 72°C – 90 sec 72°C – 10 min Size of Product: ~400bp Sample product: Wolbachia endosymbiont of Curculio elephas strain 1594E 16S ribosomal RNA gene, partial sequence Accession number: JN100083 1 ggtgttgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccacaacgag 61 cgcaaccctc atccttagtt accatcaggt aatgctgggg actttaagga aactgccagt 121 gataaactgg aggaaggtgg ggatgatgtc aagtcatcat ggcccttatg gagtgggcta 181 cacacgtgct acaatggtgg ctacaatggg ctgcaaagtc gcgaggctaa gctaatccct 241 taaaagccat ctcagttcgg attgtactct gcaactcgag tacatgaagt tggaatcgct 301 agtaatcgtg gatcagcatg ccacggtgaa tacgttctcg ggtcttgtac acactgcccg 361 tcacgccatg ggaattggtt tcactcgaa Literature source: Obtained from Werren Lab, University of Rochester Target species: Wolbachia Target gene: wsp gene (Wolbachia surface protein gene) - supposedly supergroup C and D specific. Gene function: Surface protein Accession number: AJ252062 Forward primer name: wolWSPintF Annealing temperature: 50°C Forward primer length: 21 Forward primer sequence: 5’ – TAG YTA CTA CAT TCG CTT GCA - 3’ Reverse primer name: wolWSPintR Annealing temperature: 50°C Reverse primer length: 22 Reverse primer sequence: 5’ – CCA AYA GTG CYA TAA AGA AC - 3’ USE 25 pmoles/25 l reaction Cycling Conditions: 94°C – 3 min 94°C – 45 sec 50°C – 45 sec 35X 72°C – 90 sec 72°C – 5 min Size of Product: ~710bp Sample product: Wolbachia endosymbiont of Dirofilaria immitis sp gene for surface protein Accession number: AJ252062 1 atgcattata aaaagttttt ttcagcaact gctttagtgg cgttgctaag tttatcaaat 61 gctgcttttt caggtcctat tggtccaata gatgatgaag agactagcta ctacattcgc 121 ttgcagtaca acagtgaatt tttacctttt aaaacggaag ttgatggcgt tataggtgct 181 aaaaagaaga gtgcaaatgc taatgtaact actgaccttt ataaagcttc ttttatggct 241 ggtggtagtg cttttggtta taagatggac gatattaggg ttgatattga aggtctttac 301 tcgcaactga gtagagatat atttgaaaca gctcctactc cagcaattgc agataactta 361 aatgcacttt caggattagt taacgtatat tatgatgtat caattgaaga tatgcctgtt 421 actccatata ttggtattgg tgttggcgca gcatatctta gcaatcctct agcaacaaaa 481 gttacaggtg ataaggaata tggatttggt tttgcttatc aggcaaaagc tggtatcagt 541 tatgatgtaa ctccagagat taaactcttt gctggagctc gctattttgg ctcttatggt 601 gctaaatttg ataaaccagt tactgaaaat gatcagcagc ctattaaaga cggaattaaa 661 gttctttatg gcactgttgg cacagaagct gggttaatgt ttaattttta g Literature source: Bazzacchi, C., W. Jamnongluk, S.L. O’Neill, T.J.C. Anderson, C. Genchi, and C. Bandi. 2000. wsp gene sequences from the Wolbachia of Filarial nematodes. Current Microbiology 41: 96-100. Target species: Dirofilaria immitis (Heartworm) Target gene: 16S rRNA Gene function: Protein synthesis Accession number: FJ765450 Forward primer name: di16SrRNAF Annealing temperature: 50°C Forward primer length: 21 Forward primer sequence: 5’ – TAT GTT ACC TTA ATT GGT TGC- 3’ Reverse primer name: di16SrRNAR Annealing temperature: 50°C Reverse primer length: 21 Reverse primer sequence: 5’ – GAA GAC ATT CTT GGC AAA TGC - 3’ USE 25 pmoles/25 l reaction Cycling Conditions: 94°C – 2 min 94°C – 45 sec 50°C – 45 sec 40X 72°C – 90 sec 72°C – 10 min Size of Product: ~250bp Sample product: Dirofilaria immitis isolate CN-8 16S ribosomal RNA gene, partial sequence Accession number: FJ765450 Note: Primers created using above sequence. Below sequence is an expected sequence based on primers. 1 tatgttacct taattggttg catatagtag ctagtaagtt taccttgaaa aaattagagt 61 gctcaatgcg ggcttaatgc ctgaatactc gtgcatggaa taataaaata ggatcttggt 121 tctattttgt tggttttctg atctaagata atggttaaga gggacggacg ggggcattcg 181 tatcgctacg tgagaggtga aattcttgga ccgtagcgag acgtacaact gcgaaagcat 241 ttgccaagaa tgtcttc Literature source: Created at JCC Target species: Phage WO Target gene: orf7 Gene function: Nucleoprotein, not much known yet Accession number: AB036652 Forward primer name: WOorf7F Annealing temperature: 57°C Forward primer length: 23 Forward primer sequence: 5’ – CCC ACA TGA GCC AAT GAC GTC TG - 3’ Reverse primer name: WOorf7R Annealing temperature: 57°C Reverse primer length: 30 Reverse primer sequence: 5’ – CGT TCG CTC TGC AAG TAA CTC CAT TAA AAC - 3’ USE 25 pmoles/25 l reaction Cycling Conditions: 94°C – 5 min 92°C – 60 sec 57°C – 90 sec 35X 72°C – 75 sec 72°C – 10 min Note: As of 7/16/12 these have been tried only once with crickets Size of Product: ~350bp Sample product: Bacteriophage WO gene for capsid protein, partial cds, clone:wCauA-4 Accession number: AB036652 1 aaagtctgga aagcttacaa aaagaagtag gccgactata tgaaatgttt gtgcagctaa 61 tagcaagaaa cagaggtctt tcaattgaaa agattcgatc aacagaagca ggtctatatt 121 ttggcgagaa agcagtagaa ataggtcttg cagatggagt tacaacattt tttgaattta 181 ttaacaaagg agaaaatact atgaataaac aaactacaac tgacctagaa actgataatt 241 taactaagta tcgtactgaa gttgttgaat taatacgttt atgtaactta tcacgaatgc 301 cagagaaaat aggaaaattt attgagcagg gcgtaagtgt tgagcaagca agggag Literature source: Guruprasad, N.M., L. Mouton, Sumithra, and H.P. Puttaraju. 2011. Survey of Wolbachia and its phage WO in the Uzifly Exorista sorbillans (Diptera: Tachinidae). Current Microbiology 63: 267-272. Target species: Wolbachia Target gene: ftsz - supposedly pan Wolbachia Gene function: Cell division microtubule protein Accession number: AJ276501 Forward primer name: wolftsZuniF Forward primer length: 20 Forward primer sequence: 5’ – GGY AAR GGT GCR GCA GAA GA - 3’ Reverse primer name: wolWSPintR Annealing temperature:, 55°C for 2nd 5 cycles, 50°C for last 25 cycles Reverse primer length: 18 Reverse primer sequence: 5’ - ATC RAT TCC AGT TGC AAG - 3’ USE 25 pmoles/25 l reaction Cycling Conditions: 94°C – 1 min 94°C – 30 sec 60°C – 30 sec 5X 72°C – 2 min 94°C – 30 sec 55°C – 30 sec 5X 72°C – 2 min 94°C – 30 sec 50°C – 30 sec 25X 72°C – 2 min 72°C – 5 min Note: May need optimization. As of 7/16/12 these have been tried only once with crickets, but did not work Size of Product: ~710bp Sample product: Wolbachia endosymbiont of Onchocerca volvulus partial ftsZ gene for cell wall protein FtsZ Accession number: AJ276501 1 attgaagaaa ttatggaaca tataaaagat agtcatatgc tctttattac agcaggaatg 61 ggtggtggta ctggtacagg tgcagcacca gtgattgcga aagcggcaag ggaagcaaga 121 gctgcggtta aagataaaat gttaaaagag aaaaagatat tgactgttgg ggttgtaacc 181 aagccatttg actttgaagg tgtacgtcgt atgcgcattg cagaactcgg acttgaagaa 241 ttgcaaaaat atgtagatac actaattgtt attccaaacc aaaacttatt tagaattgcc 301 aatgaaaaaa ctacatttgc cgatgcgttt agacttgctg ataatgttct gcatattggc 361 atcagaggag taactgactt aatggttatg ccaggactca ttaatcttga ctttgctgat 421 ataggaacag tgatgaacga aatgggtaaa gcaatgattg gtaccggaga agcagagggg 481 gaagatagag cagttaccgc tgcagaggct gcaatatcta atccattgct cgataatatg 541 tcaatgaaag gtgcacaggg aatattaatt aatattactg gtggtgagga catgactcta 601 tttgaagttg atgctgcggc gaatagagtg cgtgaagagg tagatgaaga cgcaaatata 661 atatttggtg ctacttttga tcaggcaatg gaaggtaaag ttagagtttc t Literature source: Casiraghi, M., T.J.C. Anderson, C. Bandi, C. Bazzocchi, and C. Genchi. 2001. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 122: 93-103. Target species: Dirofilaria immitis (Heartworm) Target gene: COI Gene function: Respiration protein Accession number: AJ271613 Forward primer name: diCO1intF Annealing temperature: 52°C Forward primer length: 19 Forward primer sequence: 5’ – TGA TTG GTG GTG GTT TTG GTA A - 3’ Reverse primer name: diCO1intR Annealing temperature: 52°C Reverse primer length: 21 Reverse primer sequence: 5’ – ATA AGT ACG AGT ATC AAT ATC - 3’ USE 25 pmoles/25 l reaction Cycling Conditions: 94°C – 2 min 94°C – 45 sec 52°C – 45 sec 40X 72°C – 90 sec 72°C – 10 min Note- We tried with heartworm (+) canine blood. As of 7/16/12 these primers have not worked Size of Product: ~650bp Sample product: Dirofilaria immitis mitochondrial partial COI gene for cytochrome oxidase subunit I Accession number: AJ271613 1 ttgatgttgc ctttgatatt gggtgcccct gaaatggctt ttcctcgtgt taatgcttta 61 tctttttgga ttacttttgt tgcgttgttg atggtttatc aatctttttt tattgggggg 121 ggtcctggga gtagttgaac tttttatcct cctttgagtg tagagggtca gcctgagtta 181 tctttggata gaatgatttt aggtcttcat actgttggta ttggttcttt attaggtgct 241 attaatttta tggttactgt tcagaatata cgttctactg ctgtaacttt agatcagatt 301 agtatgtttg tttgaacttc ttatttaact tcttttttat tggtattgtc agtgcctgtt 361 ttggctggtt ctttattatt tttgttgttg gatcgtaatt ttaatacttc tttttatgat 421 gctaataagg ggggtaatcc tttattgtat cagcatttgt tttggttttt tggacatcct 481 gaggtttatg ttattatttt accggtgttt gggattgtta gtgaatgtgt tttatttttg 541 actgataagg atcgtttgtt tggccagact agtatgactt ttgcttctat ttggattgct 601 gtattgggga cttctgtttg gggtcatcat atgtatacag ctggtttg Literature source: Casiraghi, M., T.J.C. Anderson, C. Bandi, C. Bazzocchi, and C. Genchi. 2001. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 122: 93-103.
