Automation in Genomics High-throughput purification of nucleic acids from biological samples. Valentina Gualdi Operational Scientist PGP

Transcription

1 Automation in Genomics High-throughput purification of nucleic acids from biological samples Valentina Gualdi Operational Scientist PGP OVERVIEW Nucleic acid purification technologies general aspects Genomic DNA from plant Magnetic beads Genomic DNA from blood column system Genomic DNA from plasmid/cosmid column system E-gels 2 1

2 NA extraction, quantification and normalization LiHa: 8 channel with disposable tips RoMa arm 3 Hotels: for storage of different types of labwares (MPT, Deep-well ) Barcode reader: to scan barcodes on labware during transfer actions Te-VacS: vacuum separation module for fully automated NA extraction (96-well plate) Thermo-shaker: for automation of incubation step Tecan Genios reader: for DNA and RNA quantification and normalization via fluorescence and absorbance measurement 3 BIOLOGICAL SAMPLES and NUCLEIC ACID PURIFICATION TECHNOLOGIES Starting material: Every kind of liquid samples (serum, blood, DNA/RNA, homogenized tissues, bacterial cultures) Different liquid classes Different volumes Organized in 96 MPT or deep-well Different kits for different applications: Magnetic beads (DNA) Plant tissues Blood low volume (max 20-50µl) Column systems under vacuum (DNA and RNA) Plasmid/cosmid/BAC Blood 200µl Homogeneized tissues Bacteria (gram+ and gram-) Food Plant (RNA) Blood (RNA) Viral DNA/RNA 4 2

3 DNA from PLANT TISSUE MAGNETIC BEADS SYSTEM - 1 Add Magnetic beads/lysis buffer mixture Prepare the mixture fresh just prior to use Mix well before each addition Mix lysate and beads 5 DNA from PLANT TISSUE MAGNETIC BEADS SYSTEM - 2 Deep-well: fresh leaf disks or seeds + Lysis Buffer Mixer Mill (different times and speeds for each sample type) Centrifuge deep-well x g for 10 Transfer cleared lysate from each sample in a U- bottom plate Transfer lysate without disturbing the pelletted debris Aspirate at liquid level + off-set with tracking; slowly Dispense at z-max Mix by pipetting Incubate 5 room T 6 3

4 DNA from PLANT TISSUE MAGNETIC BEADS SYSTEM - 3 Transfer the plate on the 96 Magnetic Separation Device (MSD) for 1 Different magnetic separation device types Discard the liquid by pipetting/aspiration Aspirate at z-max, slowly, excess volume 7 DNA from PLANT TISSUE MAGNETIC BEADS SYSTEM - 4 Remove plate from the MSD and add wash solution Resuspend Magnetic beads by mixing seconds Don t discard tips. Useful for other wash steps 8 4

5 DNA from PLANT TISSUE MAGNETIC BEADS SYSTEM - 5 Transfer plate on the 96 MSD for 30 Discard liquid by pipetting/aspiration Remove plate from the MSD and perform 2 wash steps Multi-dispensing After the last wash dry particles at room temperature for 5 9 DNA from PLANT TISSUE MAGNETIC BEADS SYSTEM - 6 Remove plate from the MSD and add TE buffer (or Nuclease-free water) Resuspend magnetic particles and incubate room T 5 Place the plate on the MSD for 1 and transfer purified DNA to a new stock plate Liquid class with off-set V bottom plates allow maximum DNA ricovery 10 5

6 CONCLUSIONS MAGNETIC BEADS SYSTEM - QUANTITY AND QUALITY Yield vary from 100 ng to 2 µg pure DNA Tested with barley, rice, maize DNA average ~1.6µg ADVANTAGES Eliminates lengthy incubation steps and use of hazardous organic solvents Binding of nucleic acids to magnetic beads in solution Increased binding kinetics and binding efficiency During wash steps particles are completely resuspended Enhancing contact, enhancing removal of contaminants, increased NA purity DISADVANTAGES Automation more difficult (right choice of liquid classes) Long Time (120 minutes for 96 samples) Use of Te-MO or MCA to reduce time 11 DNA from BLOOD COLUMN SYSTEM - 1 Dispense proteinase K to samples: Up to 200µl blood Fresh or frozen EDTA, citrate, heparin Also buffy coat or cultured cells Avoid contamination Z-dispense 12 6

7 DNA from BLOOD COLUMN SYSTEM - 2 Buffer BQ1 to lyse samples: Mix by pipetting Incubate room T shaker 1250rpm Room T or higher T Alternative lysis protocol Add less buffer BQ1 incubation step - add BQ1/Ethanol - mix 13 DNA from BLOOD COLUMN SYSTEM - 3 Add Ethanol 100%: Mix 3-5 times High speed pipetting (400µl/s) Transfer to Blood Binding Module (columns) Excess volume Module on Te-vacs; right choice of spacer 14 7

8 DNA from BLOOD COLUMN SYSTEM - 4 Overlay lysate with buffer B5 Just 8 tips Avoid contamination Z-dispense very slow (50 µl/s) 15 DNA from BLOOD COLUMN SYSTEM - 5 Apply vacuum to bind DNA to silica membrane: Optimization of vacuum pressure and time Check for clogging of binding plates Increase time and strenght of vacuum User prompt to verifiy clogging of binding plates: If clogging manually remove supernatant to avoid crosscontamination 16 8

9 DNA from BLOOD COLUMN SYSTEM different wash steps: Same tips to reduce cost Z dispense Check for clogging user prompt Usually less problem during wash steps if no prloblem in binding step Optimaztion of strenght and time of vacuum Apply vacuum to dry Blood Binding Module (10 ) Manually dry columns by placing module on a paper sheet before drying step Important to remove all residual washing buffer (ethanol) 17 DNA from BLOOD COLUMN SYSTEM - 7 Robotic Manipulator to transfer binding plate 18 9

10 DNA from BLOOD COLUMN SYSTEM - 8 Elution step: Critical step Equilibrate elution buffer at 70 C Move binding module on elution plate Right choice of spacer to avoid cross contamination 1 or 2 elution steps Better to dispense EB directly to the bottom of each well Apply vacuum just after at least 5 incubation time room T Centrifuge instead vacuum to increase yield Dispensed elution buffer (µl) Recovered volume vacuum (µl) Recovered volume centrifuge (µl) CONCLUSIONS BLOOD - COLUMN SYSTEM - QUANTITY AND QUALITY Up to 20 µg of pure genomic DNA from 200 µl whole blood Average ~4 µg (swine) A 260/280 between Average ~1.79 (swine) ADVANTAGES Also blood treated with EDTA, citrate or heparin Automation very easy Less critical steps (choice of liquid class) Shorter time than magnetic beads system ~60 for 96 samples DISADVANTAGES Higher risk of cross contamination if clogging 20 10

11 DNA from PLASMID/COSMID COLUMN SYSTEM - 1 Cultivation of cells at 37 C LB medium + antibiotic 8-channel pipet to inoculate culture in 96 deep well plates Overnight Constant shaking ( rpm plasmid; rpm for large low-copy constructs) in 96 deep well plates Gas-permeable sealer 21 DNA from PLASMID/COSMID COLUMN SYSTEM Induction step for large low-copy constructs (COSMID) 11

12 DNA from PLASMID/COSMID COLUMN SYSTEM - 3 Pellet bacteria by centrifugation 2500g x 10 Discard supernatant Remove as much medium as possible Paper towel Resuspend bacterial cells Buffer F1 + RNase A Mix by pipetting 23 DNA from PLASMID/COSMID COLUMN SYSTEM - 4 Lyse bacterial cells Buffer F2 Mix by pipetting Room T x 5 Bacterial suspension become clear as cell lysis occurs Don t vortex Shearing of genomic bacterial DNA Neutralize lysate Addition of buffer F3 Cell debris precipitate with SDS Visible white precipitate 24 12

13 DNA from PLASMID/COSMID COLUMN SYSTEM - 5 Transfer lysates Clear crude lysates Apply moderate vacuum (400mb) Typically sufficient <5 No clogging 25 DNA from PLASMID/COSMID COLUMN SYSTEM - 6 Precipitate DNA Isopropanol Liquid class Mix by inverting the block Centrifuge 2500g x 1h 4 C 26 13

14 DNA from PLASMID/COSMID COLUMN SYSTEM - 7 Remove supernatant and wash pellet Ice-cold ethanol 70% Centrifuge 2500 g x 10 4 C 27 DNA from PLASMID/COSMID COLUMN SYSTEM - 8 Resuspend DNA DNase free water Overnight +4 C Discard supernatant Paper towel Dry pellet Heat incubation shaker Ethanol inhibits enzymatic reactions 28 14

15 CONCLUSIONS PLASMID/COSMID - COLUMN SYSTEM - QUANTITY AND QUALITY Average yield 8µg (plasmids), 1µg (BAC) COSMID with induction Average ~8-9 µg ; good quality A 260/280 average 1.88 ADVANTAGES Automation very easy No critical steps No clogging Short time 30 for robot script DISADVANTAGES Long centrifugation steps Not fully automated 29 - TOTAL RNA EXTRACTION (1) - Avoid contamination with RNase Column-based system Different suppliers (wide choice) Different biological samples Animal tissues, blood, bacteria, plant Automation easy Clogging: strenght and time of vacuum; lysis of samples Approx. Times for 1 plate Good yield 0.3-5µg/mg tissue DNase treatment to avoid DNA contamination (not all kits) Sometimes gdna contamination No use of phenol:chloroform Β-mercaptoethanol in some kits Not necessary 30 15

16 - TOTAL RNA EXTRACTION (2) - Magnetic beads system Less market choice Some kits just for low volume Different biological samples Animal tissues, blood, bacteria, plant Automation more difficult Liquid classes Approx. Times 1h for a 96-well plate Average yield 2µg 10 5 cells No good results in our laboratory DNase treatment to avoid DNA contamination 31 - CHECK QUALITY of DNA E-Gels SYSTEM (1) Pre-cast agarose gels E-gels 96 1% Agarose or 2% Agarose SYBR safe, ethidium bromide or no DNA gel stain Mother E-base for electrophoresis Daughter E-base and E-holder for robotic loading Advanatges Fast and safe No need to prepare agarose gels and buffers No handling of ethidium bromide Compatible with robotic system Li-Ha and Te-Mo No need loading buffer 32 16

17 - CHECK QUALITY of DNA E-Gels SYSTEM (2) E-Gel 96 gels Each one contains 96 sample lanes and 8 marker lanes Dry, disposable, UV-transparent cassette Lane numbers and barcode fluorescent Standard format designed for high-throughput agarose electrophoresis Sample volume 10-20µl; fill all wells Run time 12 Resolution: 1.6cm run length 1% gel 1 Kb-10 Kb 2% gel 100bp-2 Kb 33 - CHECK QUALITY of DNA E-Gels SYSTEM (3) 34 17

18 - CHECK QUALITY of DNA E-GELS SYSTEM (4) Load Water and DNA in the slope of the wells 35 - CHECK QUALITY of DNA E-GELS SYSTEM (5) Loading of E-gels with Te-Mo 36 18

19 - CHECK QUALITY of DNA E-GELS SYSTEM (6) E-editor 2.02 software for Windows Reconfigure digital images Reconfigures wells into a side-by side format 37 CONCLUSIONS ADVANTAGES OF AUTOMATION of NUCLEIC ACID PURIFICATION Traceability barcodes export files txt, excel different labwares coordinates and volumes log-files and error-files High-throughput 96 samples in 1h Precision Reproducibility, repeatibility, low rate of errors Low risk of contamination External Cross contamination 38 19