Download Reverse Transcriptase PCR

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
PARASITES AND SNAIL BIOLOGY
DNA
“identity, possibilities”
phylogenetics
RNA
“intentions”
transcriptomics
CTAB
Trizol
gel electrophoresis
nanodrop spec
Bioanalyzer
DNA-free,
PCR
rDNA/mito
TA cloning, B/W screening
electrophoresis
direct sequencing
Sequence ID (BLAST)
editing
+
Sevilleta snails
Phylogenetics
GenBank
submission
Qiagen plasmid extraction
Restriction digests
M13 sequencing
Primer design, walking
RT-PCR
gel
Central Dogma of Molecular Biology
DNA
RNA
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,
HIDDEN BREAK PROTOSTOME rRNA
18S
28S
DNA
Non-covalent bonds
RNA
Experimental treatment (denaturation)
Gel electrophoresis, all fragments resolve as one
Deuterostomes
Most protostomes
Protocol on-line
http://biology.unm.edu/cmadema/ThermoScriptRTPCR.pdf
MM-denat
MM-RT
https://lifescience.roche.com/shop/PrintView?langId=-1&storeId=15016&articleId=68045
Primers:
LBP/BPI
Physalysin
PGRP
Positive control:
eIF2a
MM
add template
and the correct primer mix
Start RT-PCR
Group
immune gene
control (spans intron?)
1
2
3
5
6
7
8
9
10
LBP/BPI
Physalysin
PGRP
LBP/BPI
Physalysin
PGRP
LBP/BPI
Physalysin
GRP
eIF2a
(sizes known from predicted transcripts)
eIF2a
Phylogenetic analysis
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 ?
Includes:
Physids anatomical + sequence ID
Outgroups: Planorbids and Lymnaeids
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?
“Radix”
Physella A
Physella B
Physid
16S, COI
Biomphalaria
Radix
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.
http://sunsite.ualberta.ca/Projects/Aquatic_Invertebrates/?Page=19
Freshwater pulmonate snail family-level identification based on shell morphology
Lymnaeid
Physid
Planorbid
Planorbella sp.
Gyraulus sp.
dextral
sinistral
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)
For additional info see: http://www.mkohl1.net/Lymnaeidae.html,
http://www.mkohl1.net/Physidae.html, http://www.mkohl1.net/Planorbidae.html