HOPE( s) for Qantitative Environm ental Microbiological Studies Wen-Tso Liu Division of Environmental Science & Engineering Molecular Biological Tools (MBT) Sample collection & Challenges Nucleic Acid based concentration Whole cell based large - types of targets volume? yes Conc. sample - QA/QC - DNA/RNA - Cell fixation - Reproducibility extraction - DNA/RNA - Reliability Sample quantification - Ease of use preparation/ secondary - Sensitivity/specificity purification concentration no - Quantitation - lowest amount/conc. - FISH • q-PCR • NA-based • q-rt PCR - 1-bp • immuno-assay cell - Quantitation - Fingerprinting lysis - MAR • DGGE - Resolution (phylogeny) - SIP • T-RFLP Detection/ • LH-PCR - Time analysis - Detection System • microarray • Optical microscope - Cost • RAMAN scope - Sequencing - Safety • Flow Cytometry • Clone library • Metagenomics nucleic acid/ Intact cell - lab use v.s. on-site protein/metab detection tools olite based detection Other “Omics” - clone library v.s. fingerprinting methods AST seminar in Thailand, 11-12 Mar 08 1
A A (2) PCR with a C C fluorescently labeled (1) DNA extraction forward primer Mixture of microbial DNA Mixture of microbial DNA B B 5’ 3’ Environmental sample Environmental sample 3’ 5’ 3’ 5’ 3’ 5’ Image analysis Image analysis 3’ 5’ Cluster analysis Cluster analysis 3’ 5’ Outline of Outline of Principal- Principal -component analysis component analysis Fluorescent Fluorescent PCR products PCR products Web- -based database analysis based database analysis Web http://rdp.cme.edu of a specific gene of a specific gene http://rdp.cme.edu T- - RFLP RFLP T http://hermes.campus.uidaho.edu http://hermes.campus.uidaho.edu http://trflp.limnology.wisc.edu http://trflp.limnology.wisc.edu ( Liu et al., 1 9 9 7 ) ( Liu et al., 1 9 9 7 ) http://www.oardc.ohio- http://www.oardc.ohio -state.edu/trflpfragsort state.edu/trflpfragsort/ / (3) Restriction enzyme digestion (6) Data analysis � 1200 bp 300 bp 5’ 3’ B Fluorescent intensity (RFU) 1200 bp 1100 3’ 5’ 1100 bp (4) Separation fragments 1100 bp 800 bp 400 bp 5’ 3’ by electrophoresis C A 3’ 5’ 600 bp 600 1200 600 bp 400 bp 800 bp 3’ 5’ (5) Detection of 300 bp 3’ 5’ fluorescent fragments 100 bp 100 bp Cut at specific site Cut at specific site Typical electropherograms electropherograms of of Typical Fragments on gel after Fragments on gel after T T- -RFLP fingerprint RFLP fingerprint electrophoresis electrophoresis AST seminar in Thailand, 11-12 Mar 08 Constraints of current molecular quantitative techniques The difficulty to conduct multiplexing analysis is the most severe bottleneck for launching large- scale investigation of microbial abundances. Novel quantitative approach • Easy • Fast • Multiplexing AST seminar in Thailand, 11-12 Mar 08 2
Hierarchical Oligonucleotide Primer Extension (HOPE) (Wu and Liu, NAR, 2007) Acknowledgement - Wu Jer-Horng - Hong Pei-Ying - NUS funding Outline - concept - hierarchical primer design - specificity - sensitivity - multiplexing AST seminar in Thailand, 11-12 Mar 08 PCR amplicons (e.g., 16S rRNA gene) Purification 10 min Sequence specific fluorescence tag labeling by incorporating dye-terminators to 3’end of primers Dye-Terminators Primer: 5’ --- 3’ (Fluorophore-labeled ddNTPs) P1 A ddATP P2 30~45 T P3 ddTTP min C Pol DNA Polymerase G ddCTP Total time required: 2~3 hr ddGTP G T T A C C A G C A T Pol C T Pol 3’ Pol G A 5’ Target a C Pol G Pol 5’ Target b 3’ G C G Pol 3’ C Pol 5’ Target c C Clean up dye-terminator-tagged products 30-70 min C T 3’ G A 5’ Target a C G 3’ G C 5’ Target b C 3’ G 5’ Target c C Measure labeled primers by gel-based DNA sequencer Internal standards 40 min P1* P2* Intensit P3* y Size AST seminar in Thailand, 11-12 Mar 08 3
Hierarchical Oligonucleotide Primer Extension - mini-sequencing or single base extension Dye-Terminators DNA (Fluorophore-labeled ddNTPs) Oligonucleotide Polymerase Probe ddATP A ddTTP T ddCTP C ddGTP G A T G C G C C 3’ 5’ Target G Capillary Electrophoresis-Laser Induced Fluorescence (CE-LIF) Detection 1 st cycle C Internal standards Intensity 2 nd cycle C C n cycle Size AST seminar in Thailand, 11-12 Mar 08 Hierarchical Oligonucleotide Primer Extension (HOPE) Dye-Terminators Oligonucleotide DNA Polymerase (Fluorophore-labeled ddNTPs) Probe ddATP A P1 ddTTP T polyTs ddCTP C P2 ddGTP G P3 Capillary Electrophoresis-Laser Induced A T Fluorescence (CE-LIF) Detection G C Internal G P1 standards C C T P2 3’ G 5’ Target a A Intensity P3 A T C G 5’ Target b 3’ G C A T G C C 3’ G C 5’ Target c Size AST seminar in Thailand, 11-12 Mar 08 4
Principle of HOPE C , final concentration of the extended primer, C p 1 = Co , p 1 × E 1 × n C o , the initial concentration of target template E , the extension efficiency, a constant C p 2 = Co , p 2 × E 2 × n n , the number of thermal cycles C p 3 = Co , p 3 × E 3 × n E 1 , E 2 , E 3 , constant, but E 1 ≠ E 2 ≠ E 3 Calibrated E 2 / E 1 = a C p1 =aC p 2 =bC p 3 E 3 / E 1 = b AST seminar in Thailand, 11-12 Mar 08 Hierarchical probes P1 P3 P2 Template Ideal Actual C p1 =C p2 =C p3 C p1 =C p2 =C p3 1.5 1.5 Factors to synchronize Calibrated 1 1 C p1 =aC p2 =bC p3 0.5 0.5 AST seminar in Thailand, 11-12 Mar 08 5
Primer Design: Fecal Bacteroides as a model system EUB338 (79.5% for C*, 9.9% for T*) EUB338Ia (91.6% for T*) Escherichia coli Indicator for fecal pollution BAC303 (C*) o obligate anaerobes Bacteroides fragilis (C*) o abundant in the faeces of warm- BTH274 BTH584 Bacteroides blooded animals (T*) thetaiotaomicron (C*) o predict recent fecal contamination Bacteroides vulgatus o distinguish fecal pollution sources from human and livestock. Bacteroides distasonis Bifidobacterium Collinsella Eubacterium Low GC AST seminar in Thailand, 11-12 Mar 08 Probe name Specificity Binding length Poly dA length Total length EUB338Ia Bacteria domain 17 0 17 BAC303 Bacteroide-Prevotella group 17 5 22 BTH274 B. thetaiotaomicron 17 15 32 B. fragilis BTH584 B. thetaiotaomicron 20 16 36 60000 Fluorescence intensity (rfu) EUB338Ia BTH274_15a BTH584_16a 50000 BAC303_5a 40000 30000 20000 10000 0 Size (nt) 0 10 20 30 40 50 60 70 80 90 AST seminar in Thailand, 11-12 Mar 08 6
Conversion factor = constant? 8 Conversion factor (v.s EUB338Ia) BAC303-5a BTH274-15a BTH584-16a 6 4 2 0 100 1000 10000 20000 Probe-to-target molar ratio (Fixed probe concentration but varied template concentrations) AST seminar in Thailand, 11-12 Mar 08 Specificity - Target vs. non-target AST seminar in Thailand, 11-12 Mar 08 7
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