BigDye Terminator Cycle Sequencing Kits

Both the BigDye Terminator Plus and BigDye Terminator v3.1 kits use the same fluorescent dye (Z dye set v3.1) attached to the dideoxy terminators, and they have similar dNTP/ddNTP ratios.

In contrast, the BigDye Terminator v1.1 Cycle Sequencing Kit uses a different dye set (E dye set v1.1) and has a different dNTP/ddNTP ratio compared to the BigDye Terminator Plus and BigDye Terminator v3.1 kits.

From a performance perspective, the BigDye Terminator v1.1 kit is typically used to improve sequence resolution close to the primer (5′ region), particularly when used with POP-6 polymer.

The BigDye Terminator Plus and BigDye Terminator v3.1 kits are generally more suitable for larger templates, providing improved 5′ to 3′ signal balance and longer read lengths.

Additionally, the BigDye Terminator Plus kit, when used with POP-7 polymer and BigDye Hi-Res M13 primers, delivers exceptional 5′ resolution and high-quality basecalling within the first few bases downstream of the M13 priming site.

BigDye Terminator Plus is designed to deliver high-quality sequencing results while maintaining the same workflow used in standard Sanger sequencing. It can be implemented as a direct replacement without requiring changes to protocols, instruments, or analysis methods.

BigDye Terminator Plus is ideal when high-quality sequencing results are required across a wide range of template types, including challenging regions such as GC-rich sequences or templates with secondary structure. It preserves the long read lengths typically achieved with standard Sanger sequencing while providing more robust performance in difficult sequence contexts.

BigDye Terminator Plus supports flexible reaction setup, including reduced reaction volumes where appropriate, while maintaining consistent sequencing performance.

It can also be used in cycle sequencing protocols optimized for speed, including shorter extension times, when appropriate.

HiRes primers have the same nucleotide sequence as standard M13 primers but include a chemical modification that improves resolution at the 5′ end. This modification enhances basecalling quality within the first few bases downstream of the priming site, where standard primers typically show reduced resolution. The modification is proprietary.

Standard M13 primers remain fully compatible with BigDye Terminator Plus and provide reliable performance for most applications. HiRes primers are used when improved 5′ sequence resolution is required.

When using BigDye M13 HiRes primers, it is recommended to use the BDTv3direct.mob mobility file (for example, KB_3600_SeqStudioFlex_POP7_BDTv3direct.mob, depending on the instrument and setup). This mobility file is specifically optimized for use with HiRes primers.

While the standard BDTv3.mob file is compatible, using the BDTv3direct.mob file provides improved peak spacing and more accurate base alignment, particularly at the beginning of the sequence. This results in enhanced resolution of the first bases after the primer and improved overall data quality.

No changes to the sequencing workflow are required. Users can continue with standard conditions and simply select the appropriate mobility file during the plate setup or data analysis to achieve optimal results.