Optimized Automated Sequencing with Pellet Paint® NF Co-precipitant

By: Mark McCormick
Novagen, Inc.

Pellet Paint® NF Co-Precipitant is a visible, non-fluorescent, dye-labeled carrier for use in alcohol precipitation of nucleic acids. Unlike the original Pellet Paint® CoPrecipitant formulation,1,2 Pellet Paint® NF Co-Precipitant is labeled with a non-fluorescent dye. The pelleted material from alcohol precipitation steps can be easily visualized as a dark blue pellet. The resuspension of precipitated material can also be confirmed by the dissolution of the dark blue pellet.

With the incorporation of a non-fluorescent dye, Pellet Paint® NF Co-Precipitant avoids interference with procedures involving fluorescent detection such as fluorescent sequencing, quantitative PCR, and the preparation of FISH probe materials.

One important application for which Pellet Paint® NF Co-Precipitant was specifically designed is the precipitation of BigDye cycle sequencing reactions. Dye-labeled terminators are easily and rapidly removed with any of the standard precipitation methods in the ABI PRISM® user protocols.3 The only modifications made to these protocols are the reduction or elimination of pre-incubation following alcohol addition and the reduction of centrifugation times. The sequencing reaction products are efficiently precipitated and excess dye-terminators remain in the supernatant. Pellet Paint® NF does not inhibit the sequencing reaction and can be added prior to thermal cycling. To avoid extra sample processing steps, the carrier can be added directly to reaction master mixes or to template, primer, or other reaction components. Pellet Paint® NF has no detectable effect on the incorporated signal strength or sequencing accuracy, as illustrated in Figure 1. In addition, the carrier effect of the co-precipitant allows users to recover more of the sequencing products resulting in higher signal strength. In Table 1, the average signal strengths of reactions purified with Pellet Paint® NF are compared to the standard protocol.

 

Cycle sequencing with Pellet Paint®

Figure 1. Cycle sequencing with Pellet Paint® NF Co-precipitant. 100 ng ABI Control Template and 1.6 pmol primer in a 10 μL BigDye terminator cycle sequencing reaction performed in 8 well MicroAmp® reaction tubes including Pellet Paint® NF Co-Precipitant. Following thermal cycling, reactions A & B were purified by addition of ethanol and centrifugation for 10 min at 3000 × g according to the Pellet Paint® NF protocol. Reaction C was precipitated with ethanol and centrifugation for 45 min at 3000 × g according to the ABI protocol. Samples were drained by inversion and the plate was spun at low force in an inverted position. The samples were resuspended in 20 μL of Template Suppression Reagent and run on an ABI PRISM® 310 automated sequencer fitted with a long capillary. Sequence data were nearly identical. The reads extended beyond 700 bases.

 

Table 1. Sequence signal strength comparison

Sequencing reaction treatment Precipitation method G signal >average A signal average T signal average C signal average
Carrier addition after sequencing 60% ethanol, spin 3,000 × g 10 min, invert 727 647 827 553
Carrier addition prior to sequencing spin 2 min 1052 884 1159 652
No carrier addition standard ABI protocol, 60% ethanol 568 508 646 387
  • BigDye Cycle Sequencing reactions were performed in parallel using the control template and primer supplied by the manufacturer. Reactions were thermocycled in MicroAmp® Trays for 25 cycles using a four minute extension. Sequencing reactions were precipitated with ethanol using the indicated protocol. Reactions were suspended in deionized formamide, denatured and run on an ABI PRISM® 310 sequencer. The injection times and electrophoresis conditions were identical for all reactions.

Increased signal strength can be advantageous when sequencing small amounts of template. The use of Pellet Paint® NF does not require the addition of any salts beyond those already present in the standard BigDye Ready Reaction Mix. Sodium Acetate is provided for other precipitation applications where the addition of salt may be required.

Pellet Paint NF has many potential applications beyond fluorescent cycle sequencing including quantitative PCR, fluorescent probe-based assays or any application where nucleic acids are analyzed with a fluorescent reporter. However, users are cautioned that initial empirical testing is recommended to confirm compatibility with applications other than cycle sequencing.

Pellet Paint NF Co-precipitant is rigorously treated to eliminate contamination by RNA, DNA, proteases and nucleases. Preparations are tested for ribonuclease, exonuclease, endonuclease and compatibility with fluorescent cycle sequencing.

 

Materials

     

References

  1. McCormick, M. (1995) inNovations 4a, 10–11.
  2. McCormick, M. (1996) inNovations 5, 10.
  3. ABI PRISM® BigDye Terminator Cycle Sequencing Ready Reaction Kit Protocol (1998) The Perkin-Elmer Corporation, Foster City, CA.