dLUTE SEQ™ is an unique DNA sequencing reagent developed by Nucleics allowing nanolitre scale DNA sequencing reactions to be performed using conventional microlitre scale fluidics equipment and protocols. dLUTE SEQ provides the following benefits over existing approaches to save on DNA sequencing premix chemistry:
- Sequencing reactions require as little as 25 nanolitres of ABI BigDye DNA sequencing premix reagent. This equivalent to a 1:320 dilution!
- Reaction substrates are maintained at the optimum concentration resulting in longer reads and more robust results when sequencing DNA.
- Avoids the need for new high precision liquid handling equipment.
Figure 1. The two phase states of dLUTE SEQ. Left. Set-up state. The aqueous phase (pink) is dispersed throughout the inert phase. Right. Reaction state. The aqueous reaction-phase (red) has separated from the inert phase (clear).
Modern DNA sequencers, such as the Applied Biosystems 3100, 3130 and 3730, and the Amersham Biosciences MegaBACE 1000 and 4000, require a tiny fraction of the labeled sequencing fragments created by a full scale DNA sequencing reaction. Given this, combined with the high cost of sequencing reagent, has led many sequence centers to reduce sequencing costs by scaling down the reaction volumes and/or by using large dilutions of the premix reagent. Using this approach many core facilities have been able reduce the amount of DNA sequencing premix chemistry used to as little as 1/128th that required in full scale reactions, although 1 in 16 to 1 in 32 is more typical.
While these approaches can offer very significant cost savings, precision limitations in the fluidics equipment used to set up the reactions (especially the robotics pipetting equipment) has prevented precise matching of the reaction scale to the high sensitivity of the DNA sequencing instruments. Even when it possible to pipette sub-microlitre volumes accurately, it is impossible to cycle sequence the sequencing reactions without excessive evaporation. Furthermore, the use of very high reagent dilutions has a very negative impact on trace read lengths and reaction success rate. This is due to the enzymatic DNA sequencing reaction becoming substrate limited – basically the polymerase is starved of nucleotides and is unable to complete the sequencing extension. Because of all these problems, most sequencing reactions are currently performed at scales far in excess of the true sensitivity of the DNA sequencing instruments.
The dLUTE SEQ sequencing reagent avoids these problems by using a novel two state formulation chemistry (see Figure 1). In the first state (the setup state), the aqueous sequencing reagents are dispersed in a large volume of the inert component of the dLUTE formulation. This effectively dilutes the aqueous reagents and enables pipetting with standard equipment. The dLUTE SEQ DNA sequencing reaction is then induced by heat to enter the second (reaction) state. In this state, the aqueous phase separates from the inert phase forming a small “bubble” (in which the DNA sequencing reaction takes place) overlain by the inert phase.
Feature and benefits of dLUTE SEQ
Improved sequencing data
The small volume of a dLUTE SEQ reaction provides true scaled reaction conditions (i.e. the concentration of the sequencing chemistry is the same in both an undiluted standard reaction and a dLUTE SEQ reaction). The dLUTE SEQ mix maintains the sequencing DNA polymerase with the optimal nucleotide and dye labeled terminator concentrations. This avoids the “ski-slope” effect (i.e. rapid fall off in signal strength) observed with excessive chemistry dilutions (Figure 2A). The dramatic increase in large DNA sequencing product signal can be seen with the dLUTE SEQ reactions, even when using only one quarter (0.1µl) the amount of sequencing chemistry (Figure 2B).
Figure 2. Raw sequencing traces of (A) Standard diluted standard and (B) dLUTE SEQ DNA sequencing reaction with 0.1µl of BigDye v3.1 in a total volume of 2.5µl (0.25µl aqueous). Both reactions were purified by alcohol precipitation and analyzed on an ABI 3700 DNA sequencer.
The dLUTE SEQ sequencing reagent allows sequencing reactions to be performed in volumes far below the accuracy of the conventional pipetting equipment. For example, an effective 0.25µl sequencing reaction (using 0.1µl of BigDye premix) can be created using an easily handled 2.5µl volume of dLUTE SEQ. In addition, modest DNA sequencing chemistry dilutions can be used enabling as the use of as little as 25 nanolitres of BigDye per reaction.
Ease of implementation
The implementation of dLUTE SEQ into existing production pipelines is straightforward and demands only minor modification to existing protocols. The DNA sequencing chemistry and oligo primer are simply mixed with the dLUTE SEQ reagent to form a stable homogenous, mixture. This mix is aliquoted onto the DNA sequencing template using standard microlitre-scale robotic equipment. In the subsequent thermocycling a temperature induced separation of the two phases occurs and the DNA sequencing reaction proceeds in the small aqueous volume. The inert phase overlays the small reaction volume and prevents sample evaporation. Finally, the dLUTE SEQ sequencing reaction is cleaned using any of a number of compatible methods including SPRI beads, size-exclusion spin columns or plates, or via a simple modified alcohol precipitation protocol.
dLUTE SEQ CF
A new formulation of dLUTE SEQ (dLUTE SEQ CF) has recently been developed for small scale users and core facilities. This version provide dilution to an effective 1:40 level and is easier to set up and works with a wide range of template concentrations and quality. To find out more about dLUTE SEQ CF please visit the dLUTE SEQ CF page or visit our dLUTE SEQ users forum.
We are currently developing a new formulation of dLUTE SEQ and until it is released we will no longer sell the older version.
The dLUTE SEQ DNA sequencing reagent is shipped directly from Australia by express courier. The estimated delivery times are:
- Australia – 2 working days
- Japan, Taiwan and South East Asia – 5 working days
- USA and Europe – 6 working days
- Rest of the world – 10 working days
Returns and Refunds Policy