Huong Workshop Taxonomic Identification 9.2014
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Transcript of Huong Workshop Taxonomic Identification 9.2014
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Taxonomic identification of pureendophytic strains
Huong Minh Nguyen PhDINSTITUTE OF BIOTECHNOLOGY
Vietnam Academy of Science and Technology
Natural Product Research with Endophytic Fungi WorkshopSeptember 23, 2014
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An overall workflow
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Advantages of taxonomic identification using DNA markers
DNA-based methods are culture- independent, quick and unbiased Amplification efficiency of PCR-based method allows for the use of
minimum starting fungal materials, making this method much moresensitive and affordable
Development of new sequencing technology creates large genomedatabases for diverse and accurate comparison
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DNA Isolation (1)
Grind 1g ofmycelium in
liquidnitrogen
Add 750l of lysisbuffer to 1g of
mycelium ground inliquid nitrogen
Lysis buffer: 50 mM Tris-HCl, 50 mMEDTA, 3% SDS, 1% 2-mercaptoethanol(add just before use)
Vortexmixture,
incubate at65C for 1
hour
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DNA Isolation (2)
Centrifuge at4000 rpm for5min at RT,
transfer aqueousphage to a new
tube
Add 700l of SEVAG,vortex mixture then
centrifuge for 12000 rpmin 10min, transfer upperphase to a new tube
SEVAG:chloroform:isoamylalcohol, 24:1
Add 20 l of3M NaOACand 1 vol ofisopropanol,mix well
Shouldobserve DNAprecipitation
Separatecellular debrisfrom DNA
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DNA Isolation (3)
Centrifuge at12000 rpm for3min at RT,discard
supernatant
Add 300l of EB topellet with 1l of100mg/ml RNAse,incubate at 65C for
15min
This step is to cleanupcontaminated RNA
Add 250l of 7.5MAlOAc, centrifugeat 12000 rpm for5min then transfertop layer to a new
tube
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DNA Isolation (4)
Pellet DNA byadding 1 vol ofisopropanol then
centrifuge at 12000rpm for 2min at RT
Wash pellet with 1 volof 70% EtOH twice,collect pellet by
centrifuge at 12000rpm for 1min
Resuspend pellet in50l of TE buffer,store at -20C until
use
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DNA Isolation (5)Previous results: Isolation of Xanthomonas sp. and Saccharomyces sp.genomic DNA
Contaminated RNA
Treatment withRNAse
X S
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PCR Amplification and Sequencing (1) The choice of target fragments for amplification can affect the efficiency
of amplification and the accuracy of comparison Common targets for fungal taxonomic identification include 18S small-
subunit ribosomal DNA (rDNA), 28S large-subunit rDNA or internaltranscribed spacer (ITS) regions
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PCR Amplification and Sequencing (2) PCR reaction component for amplification of fungal ITS region :
Component Volume/ 50l reactionDDW To 50l10x Pfu Buffer 510mM dNTPs mix 2.520 pmol primer mix 1DNA template 0.5 1Pfu (2 - 4U/l) 0.5
94C2min
94C30sec 52 - 57C
30sec
72C30s 1m
72C5min 4C
X 35
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PCR Amplification and Sequencing (3) 0.8 1.5% agarose gel is
used to check for the successof PCR amplification
Successful amplified productsare cleaned-up using PCRPurification kit (ThermoScientific) and an appropriateamount of cleaned-upsamples are sent forsequencing
Sacch
aromy
ces sp
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ITS3-4 ITS1-4
Sacch
aromy
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Phell
inuss
p.
Phell
inuss
p.
Phell
inuss
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ITS3-4
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PCR Amplification and Sequencing (4)Previous results: Sequencing of Xanthomonas sp. 16S rRNA amplified targetusing IBT Sequencing Service
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PCR Amplification and Sequencing (5)Genomic sequence comparison: using databases such as NCBI Genome Database
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PCR Amplification and Sequencing (6)Taxonomic identification: build phylogenetic tree for isolated fungal strainswith closely related species
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(Endophytic) Fungal Taxonomic Identification Remaining Challenges and Future Prospects
Limited database: as of February 2014, the National Center for BiotechnologyInformation (NCBI) Genome database listed 57 complete fungal genomescompared with >2700 bacterial genomes
Genomic sequence of sexual (teleomorph) and asexual (anamorph) forms ofa fungus are often annotated as different taxa in many databases, causingcomplication in identification
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THANK YOU