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Today House Keeping –CARC –Course evaluation on-line soon, –Primer order –Final report (manuscript style) Continue RT-PCR, PCR Phylogeny, COI 15min.ppt.

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Presentation on theme: "Today House Keeping –CARC –Course evaluation on-line soon, –Primer order –Final report (manuscript style) Continue RT-PCR, PCR Phylogeny, COI 15min.ppt."— Presentation transcript:

1 Today House Keeping –CARC –Course evaluation on-line soon, –Primer order –Final report (manuscript style) Continue RT-PCR, PCR Phylogeny, COI 15min.ppt fungal sequences

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6 PARASITES AND SNAIL BIOLOGY “identity, possibilities” phylogenetics “intentions” transcriptomics PCR rDNA/mito Bioanalyzer DNA-free, direct sequencing gel electrophoresis nanodrop spec Sequence ID (BLAST) editing Phylogenetics electrophoresis RT-PCR gel CTAB Trizol TA cloning, B/W screening M13 sequencing Primer design, walking Qiagen plasmid extraction Restriction digests DNA RNA GenBank submission + Sevilleta snails

7 RNA DNA Central Dogma of Molecular Biology A Reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. It is mainly associated with retroviruses. RT inhibitors are widely used as antiretroviral drugs. RNase H specifically degrades only the RNA in RNA:DNA hybrids, it is commonly used in molecular biology to destroy the RNA template after first-strand complementary DNA (cDNA) synthesis by reverse transcription,molecular biology complementary DNAreverse transcription

8 Non-covalent bonds Experimental treatment (denaturation) Gel electrophoresis, all fragments resolve as one HIDDEN BREAK PROTOSTOME rRNA 18S 28S DNA RNA Deuterostomes Most protostomes

9 Protocol on-line http://biology.unm.edu/cmadema/ThermoScriptRTPCR.pdf

10 MM-denat MM-RT

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12 https://lifescience.roche.com/shop/PrintView?langId=-1&storeId=15016&articleId=68045

13 Primers: LBP/BPI Physalysin PGRP Positive control: eIF2a MM add template and the correct primer mix

14 Start RT- PCR Groupimmune genecontrol(spans intron?) 1LBP/BPIeIF2a 2Physalysin 3PGRP 5LBP/BPI 6Physalysin 7PGRP 8LBP/BPI 9Physalysin 10GRPeIF2a (sizes known from predicted transcripts)

15 Phylogenetic analysis

16 COI sequence >PS1COI GGTCANCNNAATCATAAAGATATTGGAACATTATATTTAATTTTTGGGATCTGGTGTGGATTGGTCGGTA CTGGTTTAAGCTTGTTAATTCGTTTGGAATTAGGAACATCTCTTGTACTGTTGGATGAACATTTTTATAA TGTAATTGTTACAGCACATGCTTTTGTAATGATTTTTTTTATAGTTATACCTATAATAATTGGAGGGTTT GGGAATTGAATAGTACCTATATTAATTGGTGCTCCCGATATAAGATTTCCTCGAATAAATAATATAAGTT TTTGACTTTTACCGCCTTCATTTATCTTATTATTATGTAGGTCTATAGTTGAGGGTGGAGTTGGAACTGG GTGAACTGTTTACCCCCCTCTATCAGGACCTGTAGCTCACTCTGGTTCATCAGTAGATCTTGCTATTTTC TCATTACACTTAGCTGGGTTATCATCTATTCTAGGTGCTATTAATTTTATCACTACCATTTTTAATATAC GTTCTCCTGGTATTACACTGGAACGAATAAGCTTATTTGTTTGATCGGTGTTAATTACTGCATTTTTATT ATTATTGTCATTGCCTGTTTTAGCAGGGGCTATTACTATACTATTAACTGATCGAAATTTTAATACTAGG TTCTTTGATCCAAGAGGAGGGGGAGACCCTATTCTATATCAGCATCTATATCTGANNATTGGTCANCANN NANNT >PS2COI ANTTGGTCNNNNNATCATAAAGATATTGGAACATTATATTTAATTTTTGGGATCTGGTGTGGCTTGGTC GGTACAGGCTTAAGCTTGTTAATTCGTTTGGAATTAGGAACATCTCTGGTACTATTGGATGAACATTTT TATAATGTAATCGTTACAGCACATGCTTTTGTAATGATTTTTTTTATAGTTATACCTATAATAATTGGA GGCTTTGGGAATTGAATAGTACCTATATTAATTGGTGCTCCCGATATAAGCTTTCCTCGAATAAATAAT ATAAGATTTTGACTATTACCCCCTTCATTTATCTTATTATTATGCAGGTCTATAGTTGAGGGTGGGGTT GGAACTGGGTGAACTGTTTATCCCCCACTATCAGGACCTGTAGCTCACTCTGGTTCATCAGTAGATCTG GCTATTTTCTCATTACACTTAGCTGGGTTATCATCTATTTTAGGTGCTATTAATTTTATTACTACAATT TTTAATATACGTTCTCCTGGTATTACACTGGAACGAATAAGCTTATTTGTTTGATCAGTGTTAATTACT GCATTTTTACTATTATTGTCATTGCCTGTTTTAGCGGGGGCTATNNCCNTACTATTAACTGATCGAAAT TTTAATACTAGGTTCTTTGATCCAAGAGGAGGGGGAGATC >PS4COI GGTCAACCAATCATAAAGATATTGGAACATTATATTTAATTTTTGGGATCTGGTGTGGATTGGTCGGTA CTGGTTTAAGCTTGTTAATTCGTTTGGAATTAGGAACATCTCTTGTACTGTTGGATGAACATTTTTATA ATGTAATTGTTACAGCACATGCTTTTGTAATGATTTTTTTTATAGTTATACCTATAATAATTGGAGGGT TTGGGAATTGAATAGTACCTATATTAATTGGTGCTCCCGATATAAGATTTCCTCGAATAAATAATATAA GTTTTTGACTTTTACCGCCTTCATTTATCTTATTATTATGTAGGTCTATAGTTGAGGGTGGAGTTGGAA CTGGGTGAACTGTTTACCCCCCTCTATCAGGACCTGTAGCTCACTCTGGTTCATCAGTAGATCTTGCTA TTTTCTCATTACACTTAGCTGGGTTATCATCTATTCTAGGTGCTATTAATTTTATCACTACCATTTTTA ATATACGTTCTCCTGGTATTACACTGGAACGAATAAGCTTATTTGTTTGATCGGTGTTAATTACTGCAT TTTTATTATTATTGTCATTGCCTGTTTTAGCAGGGGCTATTACTATACTATTAACTGATCGAAATTTTA ATACTAGGTTCTTTGATCCAAGAGGAGGGGGAGACCCTATTCTATATCAGCATCTATATCTGATATTAT TGGTCACACCANNNNTTTAA >SS1COI TTGGTCAACCAATCATAAAGATATNGGAACATTATATTTAATTTTTGGGATCTGGTGTGGCTTGGTCGGT ACAGGCTTAAGCTTGTTAATTCGTTTGGAATTAGGAACATCTCTGGTACTATTGGATGAACATTTTTATA ATGTAATCGTTACAGCACATGCTTTTGTAATGATTTTTTTTATAGTTATACCTATAATAATTGGAGGCTT TGGGAATTGAATAGTACCTATATTAATTGGTGCTCCCGATATAAGCTTTCCTCGAATAAATAATATAAGA TTTTGACTATTACCCCCTTCATTTATCTTATTATTATGCAGGTCTATAGTTGAGGGTGGGGTTGGAACTG GGTGAACTGTTTATCCCCCACTATCAGGACCTGTAGCTCACTCTGGTTCATCAGTAGATCTGGCTATTTT CTCATTACACTTAGCTGGGTTATCATCTATTTTAGGTGCTATTAATTTTATTACTACAATTTTTAATATA CGTTCTCCTGGTATTACACTGGAACGAATAAGCTTATTTGTTTGATCAGTGTTAATTACTGCATTTTTAT TATTATTGTCATTGCCTGTTTTAGCGGGGGCTANNNNCNTNCTATTAACTGATCGAAATTTTAATACTAG GTTCTTTGATCC >SS2COI NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNCGGTACAGGCTTAAGCTTGTTAATTCGTTTGGAATTAG GANNNNNNNNNNNANTATTGGATGAACATTTTTATAATGTAATCGTTACAGCACATGCTTTTGTAATGAT TTTTTTTATAGTTATACCTATAATAATTGGAGGCTTTGGGAATTGAATAGTACCTATATTAATTGGTGCT CCCGATATAAGCTTTCCTCGAATAAATAATATAAGATTTTGACTATTACCCCCTTCATTTATCTTATTAT TATGCAGGTCTATAGTTGAGGGTGGGGTTGGAACTGGGTGAACTGTTTATCCCCCACTATCAGGACCTGT AGCTCACTCTGGTTCATCAGTAGATCTGGCTATTTTCTCATTACACTTAGCTGGGTTATCATCTATTTTA GGTGCTATTAATTTTATTACTACAATTTTTAATATACGTTCTCCTGGTATTACACTGGAACGAATAAGCT TATTTGTTTGATCAGTGTTAATTACNGNANNNNTNTTATTATTGTCATTGCCTGTTTTNNNNNNNNNNNN NNNNNNNNNTTAACTGATCGAAATTTTANT Physid snails (Radix sp, Lymnaeid?) What species acuta, anatina, virgata ?

17 Includes: Physids anatomical + sequence ID Outgroups: Planorbids and Lymnaeids

18 Add sequences to the dataset: Physella acuta A and B COI sequence (from complete genomes), include the Radix swinhoei COI sequence (complete genome NC_026707.1), Radix sp. COI MOTU5 mitochondrion, complete genome (KP098539.1) Popsetplus.txt Align, Trim to remove primers, close relatives and achieve equal sequence length New dataset for phylogenetic analysis snailCOI.txt Root tree with outgroup “Stagnicola” or “Planorbella“ What species group do “our” physids belong to?

19 “Radix” Physella A Physella B Radix Biomphalaria Physid 16S, COI

20 The mitochondrial gene order of P. acuta compared to other sequenced Pulmonate. Phylogeny arranged on molecular designed tree using 18S, 16S and CO1 genes as done by Dayrat, et. al., 2001. Protein-coding and rRNA genes were color coded to emphasize gene patterns and rearrangements. Negative stand genes are underlined and remain consistent throughout Pulmonates. Dark lines indicate conserved gene regions found in P. acuta compared to B. glabrata, as considered to have a conserved mitochondrial gene arrangement.

21 http://sunsite.ualberta.ca/Projects/Aquatic_Invertebrates/?Page=19

22 sinistral dextral LymnaeidPhysidPlanorbid For shells with spire, check aperture right (dextral) or left (sinistral) Planorbids have a sinistral shell, coiled in one plane, sunken spire New Mexico has several species each of Lymnaeids and Physids Shell length: Lymnaeids ≤ 2.5 cm, Physids ≤ 1 cm Planorbids include Gyraulus spp. (adults < 5 mm shell diameter) and Planorbella (Helisoma) duryi, P. (H) trivolvis (large shells) Freshwater pulmonate snail family-level identification based on shell morphology Gyraulus sp. Planorbella sp. For additional info see: http://www.mkohl1.net/Lymnaeidae.html, http://www.mkohl1.net/Physidae.html, http://www.mkohl1.net/Planorbidae.htmlhttp://www.mkohl1.net/Lymnaeidae.html http://www.mkohl1.net/Physidae.htmlhttp://www.mkohl1.net/Planorbidae.html

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