07-16-12 Journal Meeting

1. 07-16-12 Journal Meeting DNA Sequencing & ABI DNA Sequencer Jung-Yun Ko

2. CONTENT • DNA Sequencing • Sequencing experiment step • ABI DNA Sequencer • DNA Sequencing data Troubleshooting

3. DNA Sequencing DNA sequencing experiments determine the order of the bases in a DNA sample. Fluorescently labeled dyes are attached to ACGT extension products in DNA sequencing reactions. Dye labels are incorporated using either 5´-dye labeled primers or 3´-dye labeled dideoxynucleotide terminators. Polymerases are used for primer extension. The sequencing reaction sample tubes are placed in a tray in the instrument’s autosampler. The autosampler successively brings each sample into contact with the cathode electrode and one end of a glass capillary filled with polymer. An anode electrode at the other end of the capillary is immersed in buffer. A portion of the sample enters the capillary as current flows from the cathode to the anode. This is called electrokinetic injection. The end of the capillary near the cathode is then placed in buffer. Current is applied again to continue electrophoresis. When the nucleotides reach a detector window in the capillary coating, a laser excites the fluorescent dye labels. Emitted fluorescence from the dyes is collected by a CCD camera. The data collection software collects the light intensities from the CCD at particular wavelength bands and stores them on the computer as digital signals for processing.

4. Sequence experiment step 2. Set up sequencing reaction 1. Purify PCR product 3. Perform cycle sequencing 5. Read order of terminators (DNA sequence) 4. Resolve sequence fragments

5. Cycle sequencing

6. Laser Capillary Detection window - + (cathode) (anode) Outlet Buffer Inlet Buffer DNA- - - Data Acquisition Sample tray ABI DNA Sequencer The ABI DNA Sequencer is an automated instrument designed for analyzing fluorescently-labeled DNA fragments by Capillaryelectrophoresis. Capillaryelectrophoresis is the movement of charged molecules through a solution in an electrical field. It is used to separate DNA fragments by size. The instrument functions the same way for both sequencing and GeneScan applications. The main differences between these applications is in the preparatory chemistries used, and the type of analysis performed on the resulting data. Capillary Electrode DNA- Sample Tube

7. Sample Separation Sample Detection Sample Injection Argon ion LASER To use the instrument, DNA fragments labeled with up to four different fluorescent dyes are combined and loaded into one lane on a vertical slab Capillary . This feature permits run times and sample resolution to be optimized according to the type of analysis, either base calling (DNA sequencing), molecular sizing in base pairs, or molecular quantitation. Once the samples are loaded, voltage is applied, causing the fragments to electrophorese through the Capillary and separate according to size. A laser beam continuously scans across the Capillary. The laser excites the fluorescent dyes attached to the fragments, and they emit light at a specific wavelength for each dye. Size Separation ABI Prism spectrograph Color Separation Fluorescence Capillary CCD Panel (with virtual filters) Mixture of dye-labeled PCR products from multiplex PCR reaction Sample Preparation Sample Interpretation

8. CCD Detector Diffraction Grating Focusing Mirror Re-imaging Lens Long Pass Filter Argon-Ion Laser Capillary Holder Capillary It is then separated according to wavelength by a spectrograph, charge coupled device (CCD) camera, so all four types of fluorescent emissions can be detected with one pass of the laser. The data collection software collects the light intensities from the CCD at particular wavelength bands and stores them on the computer as digital signals for processing. Laser Shutters Microscope Objective Lens Laser Filter Dichroic Mirror Diverging Lens

9. Mechanical pump (with polymer) Detection window Capillary array Oven Fan Lower gel block Polymer bottle Outlet buffer reservoir Sample tray Autosampler Inlet buffer reservoir

10. DNA Sequencing data Troubleshooting

11. “ Thank you ”