DyeEx Dye-Terminator Removal System

The DyeEx dye-terminator removal system is designed for fast and efficient removal of unincorporated dye terminators from sequencing reactions using a simple spin column procedure.

Features and benefits

• Fast procedure with only two short centrifugation steps
• Ready-to-use prehydrated gel-filtration material
• Efficient removal of any dye terminators, including BigDye terminators
• Spin column and 96-well formats available

Principle

The DyeEx procedure uses gel filtration to quickly and efficiently remove unincorporated terminators from sequencing reactions. Removal of dye terminators is important to prevent the unincorporated dye terminators from interfering with analysis of sequencing results. The DyeEx gel-filtration material consists of spheres with uniform pores and separates molecules according to molecular weight. When sequencing reaction mixtures are applied to DyeEx columns, dye terminators diffuse into the pores and are retained in the gel-filtration material, while labeled DNA fragments are excluded and recovered in the flow-through (see figure "DyeEx separation principle").

Procedure

Dye-terminator removal with DyeEx Kits is fast because the procedure is so simple (see flowchart below). A quick centrifugation step removes storage buffer from the columns, the sequencing samples are loaded, and a second centrifugation step removes unincorporated dye terminators. Samples are then ready for direct loading onto a capillary sequencer, or can be dried, redissolved, and loaded onto a sequencing gel.

Handling

The DyeEx 2.0 Spin Kit is used with a microcentrifuge, while the DyeEx 96 Kit is used with a suitable table-top centrifuge equipped with a rotor with swing-out buckets, such as the QIAGEN 96-Well-Plate Centrifugation System.

Applications

DyeEx Kits remove any type of dye terminator from 10–20 µl sequencing reactions including BigDye, dRhodamine dye, Rhodamine, DYEnamic ET, and WellRED terminators. After cleanup, the sequencing reactions can be separated on ABI PRISM 377, 373, 310, 3100, or 3700, MegaBACE 1000, or CEQ 2000 sequencers. Signal intensities are high, resulting in long read lengths (see figure "Long-Read Sequence after DyeEx Dye-Terminator Removal").

Long-Read Sequence after DyeEx Dye-Terminator Removal

Sequence of a 4.7 kb plasmid sequenced using the ABI PRISM BigDye Terminator Sequencing Kit and purified using the DyeEx 96 Kit.

* We recommend the DyeEx 96 Kit using the optimized protocol for the ABI PRISM 3100 and 3700, and the CEQ 2000 sequencers.

High-quality sequencing

DyeEx Kits are optimized for fast and convenient dye-terminator removal leading to highquality sequencing results. In contrast, sequencing-reaction cleanup by ethanol precipitation is very time-consuming (see Table "Comparison of DyeEx procedure and ethanol precipitation") and inefficient. The failure to remove dye terminators efficiently often leads to the appearance of dye blobs in sequencing data (see figure "Superior Sequencing Results Using DyeEx Kits A") making stretches of sequence unreadable (see figure "Superior Sequencing Results Using DyeEx Kits B"). Using DyeEx Kits for sequencing reaction cleanup ensures that the reactions loaded onto sequencing instruments are free of dye terminators. Without dye blobs the sequence is easily readable throughout (see figure "Superior Sequencing Results Using DyeEx Kits").

Comparison of DyeEx procedure and ethanol precipitation

n.a.: not applicable
* Does not include time required to dry down samples before gel loading (where applicable).
† Using the standard protocol.

Superior Sequencing Results Using DyeEx Kits

A  Gel images and  B  sequence profiles of QIAprep purified plasmids sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Kit and ABI PRISM 377 DNA Sequencer. Sequencing reactions were purified using the DyeEx 96 Kit or ethanol precipitated as recommended by the supplier of the sequencing kit as indicated. The plasmids sequenced were pUC21 (DyeEx 96 Kit, and ethanol precipitation, sequence profile) or plasmids from a genomic library (ethanol precipitation, gel image). Dye terminators present in ethanol-precipitated sequencing reactions cause dye blobs (indicated by arrows) resulting in unreadable sequences.