Transcript Reverse Transcriptase PCR

Today
• House Keeping
–CARC
–Primer order
–Final report (manuscript style)
• Start RT(-PCR), PCR next class
• Precipitate fungal sequencing reactions
• RNA Quality Control
• 15min.ppt
• Phylogeny, COI
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,
Protocol on-line
http://biology.unm.edu/cmadema/ThermoScriptRTPCR.pdf
https://lifescience.roche.com/shop/PrintView?langId=-1&storeId=15016&articleId=68045
MM-denat
MM-RT
Start RT
• AMOUNTS OF RNA? (also from bioanalyzer)
• Calculate your volume for ~1000 ng RNA,
• if < 2ml, use 2ml, if > 9ml, use 9ml.
Group
1
2
3
5
6
7
8
9
10
DN
[ng/ml]
add x ml sample to YOUR MM-denat
154
+ DEPC water for 9 ml total
498
155
(0, work with group 6)
633
Diethyl pyrocarbonate (DEPC) is an
efficient, nonspecific inhibitor of RNases. It
is typically used to treat water and
762
solutions before working with easily
degraded RNA.
668
116
(0, work with group 9)
2413
https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nucleaseenzymes/tech-notes/rnase-and-depc-treatment.html
Start RT
• AMOUNTS OF RNA? (also from bioanalyzer)
• Calculate your volume for ~1000 ng RNA,
• if < 2ml, use 2ml, if > 9ml, use 9ml.
Group
1
2
3
5
6
7
8
9
10
(DN
[ng/ml]
154
498
155
(0, work with group 6)
633
762
668
116
(0, work with group 9)
2413
add x ml sample to YOUR MM-denat
8 + 1 ml DEPC water
2 + 7 ml DEPC water
8 + 1 ml DEPC water
2 + 7 ml DEPC water
2 + 7 ml DEPC water
2 + 7 ml DEPC water
2 + 7 ml DEPC water
9
9
9)
Cleanup extension products fungal
amplicon sequencing
TYPICAL OUTPUT FORMAT
Ratio = RN number
For quality!
http://www.nature.com/jid/journal/v127/n2/full/5700557a.html
Hidden break
Ishikawa. Evolution of ribosomal RNA. Comp Biochem Physiol B. 1977;58(1):1-7.
1. The G + C content of ribosomal RNA of animals seems correlated with the length of periods required for maturation of those
organisms. 2. In Protostomes of the animal kingdom, the size of the 28S rRNA molecule does not seem to correlate with the
evolutionary stage of the organism. 3. Aphids and water-fleas as well as some protozoa have the 18S rRNA with mol. wt of 0.9
x 10(6) against an overwhelming pressure of evolution to conserve the rRNA molecule of 0.7 x 10(6) daltons. 4. All the
Deuterostomes examined were distinguished from Protostomes by having the 28S rRNA's void of the hidden break at the
central point. 5. Aphids and nematodes are exceptional Protostomes in that they have the 28S rRNA's without the hidden
break. This was discussed in the light of the evolutionary stage of these organisms. 6. Molecular properties of chloroplast
rRNA seem to evidence for endosymbiotic origin of this organelle. Mitochondrial rRNA differs considerably from prokaryotic
rRNA with respect to molecular size and base composition.
PMID: 400949 [PubMed - indexed for MEDLINE]
van Keulen et al. Organization of the ribosomal RNA genes in Schistosoma mansoni.
Mol Biochem Parasitol. 1985 May;15(2):215-30.
The organization of the rRNA genes of Schistosoma mansoni has been determined by Southern blot analysis of genomic
DNA digested with restriction enzymes, by isolation of the entire repeat on a single fragment of about 11 kilobase pairs from a
genomic DNA library constructed in bacteriophage lambda and by characterization of three cloned EcoRI fragments which
span the entire repeat. The segments encoding both the large and small rRNA subunits have been identified using specific
cloned yeast rDNA fragments as probes and EcoRI, HindIII and BamHI restriction enzyme maps of the rRNA genes were
constructed. The ends of the RNAs have been precisely mapped on the genomic DNA by S1 protection experiments. Our
data indicate that the rRNA genes are present as a tight cluster. The total length of the rDNA repeat is about 10 kilobase
pairs. There appears to be no variation in the size of transcribed and non-transcribed spacer DNA. At the RNA level we have
characterized and mapped a small gap in the 28S RNA molecule. The interruption causes the RNA to dissociate into two
equal sized fragments when analyzed under denaturing conditions. PMID: 4010707 [PubMed - indexed for MEDLINE]
HIDDEN BREAK PROTOSTOME rRNA
18S
28S
DNA
Non-covalent bonds
RNA
Experimental treatment (denaturation)
Gel electrophoresis, all fragments resolve as one
Deuterostomes
Most protostomes
15min.ppt
Bioinformatics
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?
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