Nucleic acids constitute two of the three main components of the central dogma of life and are thus pivotal to molecular biology studies. Manipulation of nucleic acids is essential for advancements in almost all domains of biology. Workflows that require handling or manipulation of nucleic acids involve multiple liquid handling steps, including reagent transfers, normalization, sample pooling, and serial dilutions. There are critical steps at which even minor liquid handling errors can significantly impact the accuracy and reliability of the results.
Nucleic acid extraction is an essential step in most molecular biology workflows and depends heavily on the efficiency of the liquid handling method. There are multiple approaches for the extraction of nucleic acids, and all of them rely on lysing the cells and separating proteins and membranes from the nucleic acids. This is accomplished using differential solubility and binding affinities. The same approaches are used to purify the nucleic acids in workflows where specific unwanted enzymes and nucleic acid molecules are to be separated from the required nucleic acid.
All methods used for nucleic acid extraction and purification involve multiple repetitive pipetting steps, which are time and labour-intensive for large number of samples. As throughput requirements increase, liquid handling automation becomes a necessity. Therefore, the accuracy, precision, dead volume, and contamination risk of the liquid handling system determine the yield and purity of nucleic acids as well as the method’s cost-effectiveness.
Conventional nucleic acid isolation relies on organic extraction. Cells are lysed with a lysis buffer and mixed with phenol–chloroform–isoamyl alcohol (PCI) to form an organic and aqueous phase. After centrifugation, cell debris and proteins partition into the organic phase, while nucleic acids remain in the aqueous phase, which is then collected. This method ensures purity but is limited by manual aqueous-phase collection, centrifugation, and throughput, making it labour-intensive.
The next advancement involves the use of affinity membranes and beads that bind nucleic acids and release them upon a change in pH. The fundamental process consists of four steps: Lysis, Binding, Washing, and Elution. These methods can be automated to enhance throughput. However, the affinity membrane-based techniques require a centrifugation step, which necessitates either manual intervention or the use of specially designed vacuum chambers.
The magnetic bead-based approach, on the other hand, uses magnetic racks to collect the magnetic beads attached to nucleic acids. The use of a magnetic rack in situ enables automated liquid handling (ALH), making it the most suitable approach for high-throughput and low-volume samples. These workflows require ALH systems capable of handling viscous magnetic-bead suspensions, which can challenge some automated dispensers, and must support integration with magnetic racks.
The basic workflow of magnetic bead-based nucleic acid purification
In addition to nucleic acid extraction, other workflows primarily focus on nucleic acids. Among them is ‘in vitro RNA synthesis’, which is a cornerstone for applications such as mRNA vaccine production, RNA-based therapeutics, CRISPR-based approaches, and other molecular biology studies. Liquid handling is crucial in in vitro transcription (IVT) as precise transfer and mixing of reagents ensures optimal RNA synthesis. The basic principle is in vitro transcription from a DNA template, followed by post-transcriptional changes and purification. As an example, mRNA production requires 5’ and 3’ modifications, template linearization, polymerase degradation, and purification. Accurate and precise liquid handling throughout these steps ensures efficient RNA production, minimizes errors, reduces costs and waste, and enhances yield and reliability.
Liquid handling steps in in vitro RNA synthesis
The ALH systems from Formulatrix, including the Mantis® and Tempest® tipless non-contact dispensers, along with the F.A.S.T.™ and FLO i8® PD positive displacement liquid handlers, provide precise and accurate transfer of small volumes, ensuring consistent results across experiments. Designed for versatility, they are compatible with magnetic racks and magnetic bead handling, making them ideal for processing a wide range of nucleic acid workflows.
Our ALH systems feature user-friendly programming interfaces that facilitate the easy definition and automation of complex protocols. With hardware designed for compatibility with robotic arms and API integration capabilities, these systems enable seamless connectivity with other laboratory instruments and software, supporting comprehensive workflow automation and efficient data synchronization.
Formulatrix Liquid Dispensers
Formulatrix Liquid Handlers
| Liquid Handling Features | Mantis | Tempest | F.A.S.T. | FLO i8 PD |
|---|---|---|---|---|
| Technology | Microdiaphragm pump | Microdiaphragm pump | Positive displacement | Positive displacement |
| Precision (CV) | < 2% at 100 nL | < 3% at 200 nL | < 5% at 100 nL | < 5% at 0.5 µL |
| Liquid Class Compatibility | Up to 25 cP | Up to 20 cP | Liquid class agnostic | Liquid class agnostic |
| Throughput | Low to medium | Medium to high | Medium to high | Low to medium |
| Contamination Risk Mitigation | Non-contact dispensing with isolated fluid path | Non-contact dispensing with isolated fluid path | Disposable tips | Disposable tips, Non-contact dispensing support, and HEPA filter module |
| Hold-Up Volume (µL) | ~6 | ~48 (per chip) | Close to zero | Close to zero |
Table: Formulatrix ALH systems
Webinars
Automate the Smart-seq3 protocol with the Mantis to increase throughput, reduce dead volumes, and eliminate pipetting steps.
Learn how to reduce costs, minimize waste, and optimize qPCR assays by tipless non-contact dispensing using the Mantis liquid dispenser.
Application Notes
Deliver precise, reliable, and consistent liquid handling, ensuring optimal bead-to-sample ratios for optimal DNA extraction with the Mantis automated liquid dispenser.
Discover how the Mantis provides researchers with a flexible workstation for setting up and executing various protocols in minutes with enhanced reproducibility.
