Lower costs and increased speeds have led researchers sequencing genes and genomes to generate an enormous amount of data that can provide a better understanding of diseases. This abundance of data suggests a need for additional, repetitive studies to determine the importance of small variations in a large number of components, and this is where the benefits of automating basic molecular biology techniques using relatively inexpensive robots integrated with familiar research tools become clear.
Background
Molecular biology research is guided by the concept of the central dogma. Information stored in DNA is transcribed into messenger RNA, which is translated into specific proteins through a simple code consisting of three nucleotides for each amino acid. The gene expression process has two steps: the transcription of DNA into mRNA and the translation of mRNA into protein (see Figure 1). The procedures described here ultimately involve some aspect of this central dogma.
Figure 1 – a) When a gene is expressed, its portion of the chromosome is exposed and copied into a strand of messenger RNA (mRNA) (b) which is then translated into proteins at the ribosome.Molecular biology research is aimed at answering two basic questions:
- Which genes are being expressed in various types of cells under normal circumstances, and what is the role of the corresponding proteins?
- What has gone wrong in a specific disease? Are genes under- or over-expressing? Are mutated proteins ineffective or overeffective?
Once these questions can be properly understood, it is possible to pursue mechanism-based approaches to medical intervention.
Basic workflow
Figure 2 shows a simplified view of the key processes often carried out in molecular biology laboratories. Biological samples can include blood, plant material, bacteria or the product of cloning experiments. In each case, DNA is isolated from these samples. When the amount of DNA is too small for additional work, it is amplified by PCR. This DNA can be sequenced or ligated into DNA vectors as part of the cloning process. These vectors are incorporated into modified bacteria (transformation) or eukaryotic cells (transfection), and typically contain reporter genes such as those coding for fluorescence or antibiotic resistance and which are used to identify successful clones. The desired clones are then selected and grown in bioreactors to produce biological samples that often need to go through the cycle again.
Figure 2 – Basic molecular biology workflow.Why automation?
Automation of laboratory work is usually justified because it reduces human error, facilitates better allocation of human resources, provides increased precision and reproducibility, enables automatic digital storage of results and offers greater throughput.
Greater throughput is particularly relevant because it offers the ability to carry out the same process numerous times, and to optimize a protocol to evaluate the effect of slight variations in any number of experimental parameters. This leads to improved understanding of biological mechanisms in normal and disease states. While it is critical that microplate and liquid handling robots are mechanically precise, it is also important that the controlling software is simple to use and easily modified to carry out optimization and exploratory procedures.
Automating the basic workflow
Following the sequence depicted in Figure 2, the basic steps are given in Tables 1–5, along with a list of all relevant instrumentation.
Table 1 – DNA/RNA/protein isolation
Table 2 – PCR: amplify small amounts of DNA to make it easier (possible) to study
Table 3 – DNA sequencing: determine the exact sequence of a gene
Table 4 – DNA synthesis: create a synthetic gene from component parts (oligonucleotides)
Table 5 – Cloning: incorporate modified DNA into a cell and produce colonies of modified cells
In the DNA/RNA/protein isolation procedure, shown in Table 1, pure DNA is isolated from the cell. A variety of protocols are available to do this, but each involves the same four basic steps: lysis, adsorption, washing and elution. The details vary, depending on the source material and the expected concentration of DNA.
Going beyond the basic workflow
Table 6 – Instruments referenced*
This article covers the most basic procedures commonly encountered in the molecular biology laboratory. Each procedure has a common sequence of steps and is therefore amenable to this type of general analysis. A number of other procedures that are not mentioned can be carried out on the same basic set of equipment, that is, automated pipettor, liquid dispenser, plate washer, shaking nest and heating/cooling nest. The instruments referenced in this article are listed in Table 6.
Key to true flexibility is the software. Laboratory automation software should provide:
- A simple user interface that allows mapping of the desired protocol
- The ability to manipulate user-defined variables to support protocol optimization and complex experiments conducted in order to understand the effect of small changes in a gene’s sequence, or the concentration of a component
- Error checking and the ability to communicate problems to the user
- The ability to talk to, configure and run a wide variety of equipment (plug-ins)
- The ability for users to create custom plug-ins for their own instruments.
Another important element required for greater throughput is a robotic arm, which permits a wider range of instruments to be used in the same protocol and enables the user to replenish sample plates, pipet tips, filter plates, etc. A simple workcell for carrying out DNA isolation at low throughput and at higher throughput with the addition of a robotic arm is shown in Figure 3.
Figure 3 – a) Simple workcell for carrying out DNA isolation. The addition of the robotic arm (b) makes it possible to include a UV reader and a thermocycler in the same protocol.This article is far from a comprehensive review of automated molecular biology, and each topic could be the subject of a detailed review. Hopefully this overview will inspire researchers to automate their work.
Alan H. Katz, Ph.D., is CSO, Hudson Robotics, Inc., 10 Stern Ave., Springfield Township, N.J. 07081, U.S.A.; tel.: 973-376-7400; e-mail: [email protected]; www.hudsonrobotics.com