AUTOMATED MOLECULAR BIOLOGY continued
Table 3 – DNA sequencing: determine the exact sequence of a gene Procedure
DNA preparation: Isolation, fragmentation and addition of reagents.
DNA sequencing: Run the sample through the sequencing instrument.
Data analysis: Frequently complex computa- tions determine a consensus sequence.
Automation approach(es)
Samples are obtained from the previous two methods and modified by reagents suitable to the chosen sequencer.
The sample plate is transferred to the sequencer and a predefined program is run.
Lab automation software sends input and receives output from the desired software program.
Table 4 – DNA synthesis: create a synthetic gene from component parts (oligonucleotides) Procedure
Automation approach(es)
Assembly planning: For long sequences, computer programs determine the best combination of DNA sequences to assemble.
Oligo purification: Oligos need to have protecting groups removed before they can be used.
DNA synthesis: For individual DNA sequences, oli- gonucleotides are connected in sequence.
Lab automation software sends input and receives output from the desired software program.
A series of liquid-handling steps add the various reagents to de-block and de-protect the products from the oligo synthesizer.
Liquid handling prepares the required oligos and transfers them to a gene assembly device.
Tool(s)
• Automated pipettor • Liquid dispenser
• DNA sequencer • DNA sequencing software
Tool(s) • Gene assembly software
• Automated pipettor • Liquid dispenser • Oligo synthesizer
• Automated pipettor • Liquid dispenser
Table 5 – Cloning: incorporate modified DNA into a cell and produce colonies of modified cells Procedure
Automation approach(es)
Ligation: DNA sequences are connected to produce a vector.
Transformation/transfection: Insert a vector into a bacterial cell (transformation), or eukary- otic cells (transfection). The cells are then placed in growth media.
Colony growth and plating: The modified cell is placed in growth medium and allowed to reproduce, and is then spread onto agar plates.
Colony picking: Select strains of transformed bacteria using co-expressed reporter proper- ties as a guide. Place small aliquots of viable colonies in pure growth media.
A series of liquid-handling steps add the DNA ligase to the appro- priately modified DNA fragments.
Most common is the heat shock approach in which the tempera- ture of the cells is suddenly raised in the presence of the DNA. Electroporation approaches are available.
This is one of the more difficult automation steps, and often requires user intervention for spreading Omni plates or Petri dishes, though this process has been automated.
An automated colony picker uses a camera to analyze growth. Common markers include antibiotic resistance, where zones of inhibition are identified; or green fluorescent protein (GFP), where the appropriate filters are used.
Tool(s)
• Automated pipettor • Liquid dispenser
• Automated pipettor • Liquid dispenser • Heating/cooling nest • Incubator
• Automated pipettor • Liquid dispenser • Incubator
• Colony picker • Automated pipettor • Liquid dispenser
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 mecha- nisms in normal and disease states. While it is critical that microplate and liquid handling robots are mechanically precise, it is also important that
AMERICAN LABORATORY 42
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.
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