PCR of rbcLin Scutellariasp. Phylogenetics/006-DNA... · 2013-04-15 · PCR of rbcLin...
Transcript of PCR of rbcLin Scutellariasp. Phylogenetics/006-DNA... · 2013-04-15 · PCR of rbcLin...
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PCR of rbcL in Scutellaria sp.
S. st
rigill
osa
1
S. st
rigill
osa
2
S. peki
nensi
s va
r. a
lpin
a
S. in
dic
a
Isodon inflexs
us
500bp
1,000bp
1,500bp
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DNA sequencing
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Sanger methodSanger and Gilbert shared a Nobel prize in 1980
Classical one-dye (or isotope) four-lane systemhttp://www.youtube.com/watch?v=6ldtdWjDwes
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Acrylamide Gel Electrophoresis with S35 for Sequencing
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Gel dryer
IntensifyingCacette
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Four-dye one-lane system using four different florescence dyes
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1990’~2000’ABI 377: Gel-type Automatic sequencer
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Current sequencing service machine ABI 3730: Capillary-type Automatic sequencer
ABI 3100
http://www.youtube.com/watch?v=SRWvn1mUNMA&feature=related
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Next generation sequencing (NGS)Pyro-Sequencing
1) Emulsion PCR2) Sequencing by synthesis (SBS)3) Microtiter plate
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Three Big Companies
Roche GS-FLX 100M bp / runIllumina Solexa 80G bp X 4 /runABI SOLid 200G bp /run
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50 Million Clusters Per Flow Cell
20 Microns
100 Microns
Solexa Sequencing
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Sequencing request to the service company:e.g., MACROGEN
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Input order information
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Input sample and primerinformation
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Download sequencing result
RESULT
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Sequence editing using SEQUENCHER
Open .ab1 files
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Sequence of one “.ab1” files
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Chromatogram of one “.ab1” files
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rbcLAssemble four sequences from four reactions
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A “Contig” is generated rbcL
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Heterogeneous parts should be “edited”
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Now heterogeneous parts are “edited”
“Primer” sequences should be deleted.
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Save edited sequencesas text file with FASTA format
FASTA format:
>taxa name1sequence…
>taxa name2sequence…
>taxa name3sequence…
…
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Sequence comparison and phylogenetic analysis using MEGAOpen FASTA file in the MEGA
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Translate DNA sequences into amino-acid sequences.
Adjust “frame”!
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Do sequence “alignment”
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Now sequences are well aligned
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Change aligned AA sequences into DNA sequences
Now it is ready to do analyses Change the matrix into MEGA format
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Result of phylogenetic analyses
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Scutellaria strigillosa (참골무꽃)
Scutellaria indica (골무꽃)
Scutellaria pekinensis var. alpina(수골무꽃)
Isodon inflexsus (산박하)
This is a result of your sequencing data!