Primer Information for the Detection of Wolbachia, D. Immitis, WO Phage in Mosquitoes

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 freezingand 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 at200 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 worked-out 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 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

FASTAGraphics

Go to:

• Features
• Sequence

LOCUS DQ900649 602 bp DNA linear BCT 24-DEC-2006

DEFINITION Wolbachia endosymbiont of Culex pipiens surface protein precursor

(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"

/db_xref="taxon:363538"

/country="Iran: Langrood district, Guilan province"

/note="PCR_primers=fwd_name: 81F, rev_name: 691R"

gene <1..602

/gene="wsp"

CDS <1..602

/gene="wsp"

/codon_start=2

/transl_table=11

/product="surface protein precursor"

/protein_id="ABL87105.1"

/db_xref="GI:119658041"

/translation="GPISDEETSYYVRLQYNGEVLPFKTRIDGIEYKKGTEVHDPLKASFMAGGAAFGYKMDDIRVDVEGLYSQLNKNDVSGATFTPTTVANSVAAFSGLVNVYYDIAIEDMPITPYVGVGVGAAYISNPSEASAVKDQKGFGFAYQAKAGVSYDVTPEIKLFAGARYFGSYGASFNKEAVSATKEINVLYSAVGAEAGVAFNF"

ORIGIN

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

//

Figure 2- Structure of the 20 Amino acids (

Target species: Wolbachia

Target gene: wsp gene (Wolbachia surface protein gene)

- supposedly supergroup A and B specific. However, DNA alignments brings that into question.

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 5pmoles/25l reaction

Cycling Conditions:95°C – 1 min

92°C – 30 sec

52°C – 40 sec35X

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 25pmoles/25l reaction

Cycling Conditions:94°C – 2 min

94°C – 30 sec

55°C – 45 sec35X

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 25pmoles/25l reaction

Cycling Conditions:94°C – 3 min

94°C – 45 sec

50°C – 45 sec35X

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 25pmoles/25l reaction

Cycling Conditions:94°C – 2 min

94°C – 45 sec

50°C – 45 sec40X

72°C – 90 sec

72°C – 10 min

Size of Product: ~250bp

Sample product:Dirofilaria immitis isolate CN-8 16S ribosomal RNA gene, partialsequence

Accession number:FJ765450

Note: Primers created using above sequence. Below sequence is an expected sequence based on primers.

1 tatgttacct taattggttg catatagtag ctagtaagtttaccttgaaa aaattagagt

61 gctcaatgcg ggcttaatgc ctgaatactc gtgcatggaa taataaaata ggatcttggt

121 tctattttgt tggttttctg atctaagata atggttaaga gggacggacg ggggcattcg

181 tatcgctacg tgagaggtga aattcttgga ccgtagcgag acgtacaact gcgaaagcat

241 ttgccaagaatgtcttc

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 5pmoles/25l reaction

Cycling Conditions:94°C – 5 min

92°C – 60 sec

57°C – 90 sec35X

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 25pmoles/25l reaction

Cycling Conditions:94°C – 1 min

94°C – 30 sec

60°C – 30 sec5X

72°C – 2 min

94°C – 30 sec

55°C – 30 sec5X

72°C – 2 min

94°C – 30 sec

50°C – 30 sec25X

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 25pmoles/25l reaction

Cycling Conditions:94°C – 2 min

94°C – 45 sec

52°C – 45 sec40X

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.