Large Construct Isolation

Introduction

Large construct isolation from off-target fragments is becoming important in DNA research. Long read sequencing technology is being used to characterize amplicons that are >5 kbp in size, and gene synthesis technology improvements continue to increase the maximum length of assembled constructs.   Ranger® Technology from Coastal Genomics enables automated, gel-based size selection of constructs from 50 bp to over 20 kbp in size. Up to 96 samples can be processed in a single run on the NIMBUS Select (Hamilton Robotics), which comes equipped with Ranger Technology. The system has the capacity to handle high throughput demand for large construct isolation, and can improve reliability of assays or pipelines that require purified large constructs.

Experiment

Proxy samples composed of both 15 kbp and 20 kbp NoLimits constructs (Thermo Scientific) were mixed with TE buffer to produce a 1X concentration of 40 ng/μl of each band (total NoLimits concentration = 80 ng/μl). This 1X Proxy Stock solution was used to produce four aliquots of varying NoLimits amounts:

  1. Aliquot 1: 25 ul of 1X Proxy Stock (1 μg of each NoLimit construct)
  2. Aliquot 2: 12.5 ul of 1X Proxy Stock, 12.5 ul TE (500 ng of each NoLimit construct)
  3. Aliquot 3: 6.25 ul of 1X Proxy Stock, 18.75 ul TE (250 ng of each NoLimit construct)
  4. Aliquot 4: 3.12 ul of 1X Proxy Stock, 21.88 ul TE (125 ng of each NoLimit construct)

Each aliquot was mixed with 5 kbp & 10 kbp marker Dual Dye loading buffer (custom marker pairs commercially available from Coastal Genomics), and the resulting solutions were loaded onto the NIMBUS Select. Size selection targets were defined through Ranger Software to target recovery of the 20 kbp fragment.

Results

Size Selection Run

The NIMBUS Select generated electropherograms for each aliquot prior to the size selection (Fig. 1). By the middle of the run, the 20 kbp construct had separated from the 15 kbp construct for most aliquots. Figure 1 Fig. 1Mid-run electropherogram traces of Aliquot 1(A), Aliquot 2 (B), Aliquot 3 (C) and Aliquot 4 (D). A 5 kbp and 10 kbp Dual Dye marker pair (red) was used for sizing purposes. The 1X Proxy Sample is represented by the blue trace.

Isolation of 20 kbp Construct

The 20 kbp constructs were recovered in 200 μl of running buffer (Note: Concentration of extraction volumes down to 25 μl can be automated with filter plates on the NIMBUS Select). A sample from each extraction was then loaded onto the NIMBUS Select workstation and run again to assess the rejection of the contaminating 15 kbp construct. Analysis of the isolates was conducted with the Ranger Analytics Application (Fig. 2). Relative enrichment of the 20 kbp from the 15 kbp construct exceeded 90% for all Aliquots (Table 1). Figure 2 Fig. 2: Ranger Analytics characterization of size selected fractions from Aliquot 1 (A), Aliquot 2 (B), Aliquot 3 (C) and Aliquot 4 (D). Enrichment of the signal from the 20 kbp construct was indicative of successful isolation. Note that the Dual Dye markers at 5 and 10 kbp are also represented.   Table1 Table 1: The magnitude of the NIMBUS Select’s enrichment of the 20 kbp construct target from the 15 kbp contaminant was characterized by comparison of relative peak heights between the 15 and 20 kbp construct signals found in Fig. 2.

Recovery

Our 1X Proxy Stock contained 15 and 20 kbp NoLimits constructs at concentrations of 40 ng/μl/construct. Size selection recovery yields for the four Aliquots with varying amounts of the 1X Proxy Stock were assessed via the dsDNA HS Assay on the Qubit Fluorometer (Life Technologies) (Table 2). Intrinsic recovery yields ranged from a low of 66% in Aliquot 1 up to as high as 85% in both Aliquots 3 and 4. table 2 Table 2: Qubit characterization of concentration of extracted 20 kbp isolates from all Aliquots. The 20 kbp input amount for each Aliquot was derived from the amount of 1X Proxy Stock added (see “Experiment”). Intrinsic yield is a measure of the amount of 20 kbp construct recovered compared to the amount of 20 kbp construct that was used as input.

Discussion

The NIMBUS Select with Ranger Technology is a good option for isolation of large constructs from off-target fragments. This experiment assumed suboptimal conditions in which a closely-sized off-target fragment was present at a mass that was equivalent to that of the target. Under these conditions, Ranger Technology was able to:

  • Process DNA samples of a high mass
    • Up to 2 ug of total DNA now validated
  • Ensure high enrichment of the target construct
    • As high as 30 fold increase in target:contaminant ratio
  • Maintain high intrinsic recovery yields
    • 85% intrinsic recovery
  • Conduct size selections in a high throughput fashion
    • 192 large-fragment size selections can be conducted in a single work day

The NIMBUS Select is an effective option for groups looking to reduce costs associated with processing off-target fragments. Gel-based size selection is critical to this aim for large fragment applications, and Ranger Technology is the only high throughput, fully automated solution for this requirement.