Taq-polymerase catalyzed cycle sequencing using fluorescent-

Thursday, November 20, 2003

Description
One of the major problems in DNA cycle sequencing is that when fluorescent primers are used the reaction conditions are such that the nested fragment set distribution is highly dependent upon the template concentration in the reaction mix. We have recently observed that the nested fragment set distribution for the DNA cycle sequencing reactions using the fluorescent labelled terminators is much less sensitive to DNA concentration than that obtained with the fluorescent labelled primer reactions as described above. In addition, the fluorescent terminator reactions require only one reaction tube per template while the fluorescent labelled primer reactions require one reaction tube for each of the four terminators. This latter point allows the fluorescent labelled terminator reactions to be pipetted easily in a 96 well format. The protocol used, as described below, is easily interfaced with the 96 well template isolation and 96 well reaction clean-up procedures also described herein. By performing all three of these steps in a 96 well format, the overall procedure is highly reproducable and therefore less error prone.

Procedure
1. Place 0.5 ug of single-stranded or 1 ug of double-stranded DNA in 0.2 ml Robbins PCR tubes.

2. Add 1 ul (for single stranded templates) or 4 ul (for double-stranded templates) of 0.8 uM primer and 9.5 ul of ABI supplied premix to each tube, and bring the final volume to 20 ul with ddH2O.

3. Centrifuge briefly and cycle as usual using the terminator program as described by the manufacturer (i.e. preheat at 96oC followed by 25 cycles of 96oC for 15 seconds, 50oC for 1 second, 60oC for 4 minutes, and then link to a 4oC hold).

4. Proceed with the spin column purification using either the Centri-Sep columns or G-50 microtiter plate procedures given below.

Terminator Reaction Clean-Up via Centri-Sep Columns
1. Gently tap the column to cause the gel material to settle to the bottom of the column.

2. Remove the column stopper and add 0.75 ml dH2O.

3. Stopper the column and invert it several times to mix. Allow the gel to hydrate for at least 30 minutes at room temperature. Columns can be stored for a few days at 4C; longer storage in water is not recommended. Allow columns that have been stored at 4 C to warm to room temperature before use. Remove any air bubbles by inverting the column and allowing the gel to settle. Remove the upper-end cap first and then remove the lower-end cap. Allow the column to drain completely, by gravity. (Note: If flow does not begin immediately apply gentle pressure to the column with a pipet bulb.)

4. Insert the column into the wash tube provided.

5. Spin in a variable-speed microcentrifuge at 1300 g for 2 minutes to remove the fluid.

6. Remove the column from the wash tube and insert it into a Sample Collection Tube.

7. Carefully remove the reaction mixture (20 ml) and load it on top of the gel material. If the samples were incubated in a cycling instrument that required overlaying with oil, carefully remove the reaction from beneath the oil. Avoid picking up oil with the sample, although small amounts of oil (<1 ml) in the sample will not affect results. Oil at the end of the pipet tip containing the sample can be removed by touching the tip carefully on a clean surface (e.g., the reaction tube). Use each column only once.

8. Spin in a variable-speed microcentrifuge with a fixed angle rotor, place the column in the same orientation as it was in for the first spin--this is important because the surface of the gel will be at an angle in the column after the spin.

9. Dry the sample in a vacuum centrifuge. Do not apply heat. Do not overdry. If desired, reactions can be ethanol precipitated.

Terminator Reaction Clean-Up via Sephadex G-50 Filled Microtiter Format Filter Plates
The following protocol was developed at the C. Elegans Genome Sequencing Center at Washington University, St. Louis, Missouri, was conveyed to us by Dr. Richard Wilson, and now has been modified to take advantage of the Millipore 45 ul Column Loader (cat. # MACL 096 45). Additional information about this procedure also is available at the Millipore web site.

Preparation of Sephadex G-50 containing Microtiter Filter Plates:

1. Add dry Sephadex G-50 to the Millipore (cat.# MACL 096 45) 45 ul Column Loader.

2. Remove the excess of resin from the top of the Column Loader with the scraper supplied.

3. Place MultiScreen HV Plate (Millipore MAHVN4550) upside-down on top of the Column Loader.

4. Invert both MultiScreen HV Plate and Column Loader.

5. Tap on top of the Millipore Column Loader to release the resin.

6. Using a multi-channel pipettor, add 300 ul of ddH2O to each well to swell the resin. and let stand at room temperature for 3 hours.

7. Once the minicolumns are swollen in MultiScreen plates, they can be sealed with saran wrap and stored in the refrigerator at 4 deg C for several weeks. A batch of plates also can be stored in the refrigerator at 4 deg C for several weeks in a sealed plastic container with a damp towel to assure the plates are kept moist.

8. When needed, the matrix containing filter plate is taped over a microtiter plate and centrifuged for 2 minutes at 1500 RPM in a Beckman GS-6R to pack the columns and to remove any access buffer.

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Taq-polymerase catalyzed cycle sequencing using fluorescent-