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

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

5. Cycle sequencing

6. ABI DNA Sequencer The ABI DNA Sequencer is an automated instrument designed for analyzing fluorescently-labeled DNA fragments by Capillary electrophoresis. Capillary electrophoresis 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. Sample Tube DNA - - Electrode Capillary DNA - - Laser Inlet Buffer Capillary -+ Outlet Buffer Sample tray Detection window (cathode) (anode) Data Acquisition

7. 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. Mixture of dye-labeled P CR products from multipl ex PCR reaction CCD Panel (with virtual filters) Argon ion LASER Color Separation Fluorescence ABI Prism spectrograph Size Separation Sample Interpretation Sample Injection Sample Separation Sample Detection Sample Preparation Capillary

8. 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. Dichroic Mirror Capillary Holder Microscope Objective Lens Laser Shutters Laser Filter Diverging Lens Capillary Long Pass Filter Re-imaging Lens Focusing Mirror CCD Detector Diffraction Grating Argon-Ion L aser

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

10. Observation Possible Cause Solution Poor quality template Weak, noisy signal. Contaminated Template: Template DNA must be free of residual etha nol and salts. And also free of cellular compo nents such as RNA, proteins, polysaccharide s, and chromosomal DNA Re-precipitate template Large stop peaksDegraded DNARe-isolate DNA Multiple, overlapping peaks More than one template present Double pick of two colonies Clean up PCR products Re-isolate DNA Primer related problems No sequence generatedInsufficient templateIncrease amount of template ContaminantClean up template Insufficient primerIncrease amount of primer Primer has no annealing site Poor primer design Incorrect sequence Re-design primer Noisy data with weak signalNot enough DNAUse more DNA in reactions Primer anneals poorlyRe-design annealing temp Noisy data with good signal strengthContaminated templateClean up template Multiple priming sitesRedesign primer Multiple primers with PCR productsPurify PCR template Too much DNAUse less DNA DNA Sequencing data Troubleshooting

11. “ Thank you ”