Automated liquid handling instruments are essential component for a wide range of high throughput screening laboratories encompassing disciplines such as biochemistry, immunology, molecular biology, medicinal chemistry, and crystallography. Such automated pipetting instruments allow increased throughput and efficiency, eliminating tedium and the potential for error associated with manual pipetting.
As a result of the ever increasing number of chemical compounds and molecular targets available for lead discovery, the strain on both scientific and financial resources continues to steadily increase. Alongside the introduction of new technologies to cater for emerging fields such as epigenetics; recent advances in PCR and cloning technologies, next generation sequencing (NGS), are making molecular biology research more accessible for high throughput screening studies. This in turn is placing an additional demand on scientific resources and costs.
Assay miniaturization and automation
With the drive to reduce costs and increase throughput the move towards higher density plate formats such as 384-and 1,536-well plates, has enabled greater amounts of data to be obtained per screen, reducing the volume of costly reagents and often scarce samples required. As the assay volume decreases in proportion to increasing well density, manual pipetting of multiple aliquots and samples in even 96- and 384-well plates can be taxing and time consuming with mistakes being easily made. As such manual dispensing of samples in 384 and 1,536 microplates is unfeasible, open to sample tracking error as well as pipetting inaccuracies at these lower volume ranges. Furthermore, a number of reagents employed in the molecular biology field of are stored and dispensed in highly viscous solutions such as 30-40% glycerol for enzymes in NGS or the LCP solution for membrane protein crystallography. Pipetting of such viscous solutions can prevent accurate and reproducible dispensing especially in the nanoliter and microliter (low) volume range.
TTP Labtech’s mosquito portfolio of nanoliter liquid handling instruments are capable of rapid and accurate sample volume dispensing from 1,200nL down to 25nL, regardless of viscosity (Figure 1). With positive displacement pipetting technology and disposable tips to ensure zero cross contamination, users can quickly swap between applications without labware or system set up changes. mosquito can accurately dispense into and between 96-, 384- or 1,536-well plates for plate reformatting over a wide range of commercially available plate types.
The number of nanoliter liquid handling applications is expanding rapidly with new uses being continually introduced. mosquito pipetting instruments are regularly employed for the preparation of stock solutions, direct dispensing into plates, plate to plate transfer and compound screen hit picking, biological assay set-up, rtPCR and qPCR preparation, cell culture seeding and handling, as well as for crystal screen set-up (mosquito Crystal and LCP).
More recently, mosquito HTS has been employed for the successful assay miniaturization of both cloning and next generation sequencing techniques employing Gateway technology (Invitrogen) and the Nextera sequencing kit (Illumina). In these studies final volumes were reduced from the recommended protocol to 1 µL significantly reducing the cost of both clonase and tagmentation enzymes (Figure 2).
The gentle pipetting and mixing action of mosquito, reduces the potential for frothing of viscous enzymes often observed with both manual pipetting and other automated pipetting systems, minimizing the risk of reduced enzyme activity during DNA fragmentation and ligation stage of enzyme based NGS applications such as the Nextera sequencing kit (Illumina) (Figure 3). In addition, gentle pipetting action of mosquito reduces the risk of unwanted DNA fragmentation and loss in such studies.
The need for intermediate volume dispensing
The drive to increase laboratory efficiency with automation and miniaturization needs to be balanced with existing laboratory protocols and equipment where significant investment and scientific expertise has already been made. As a number of academic screening centers and pharmaceutical companies have set up existing instrumentation which use volumes around the 100 to 500 µL range in 96 or 384 well plate screening assays. In addition a number of current molecular biology applications, such as rtPCR or qPCR, RNAi screening and SNP genotyping protocols still regularly require volumes around and above the 100µL volume range. Integration of automated nanoliter volume dispensing would require significant outlay and re-validation of assays.
mosquito HV is an addition to TTP Labtech’s liquid handling portfolio, offering the versatility to perform highly accurate microlitre pipetting in the 0.5 to 5 µL volume range, whilst still retaining all of the unique features of the mosquito HTS such as high precision, high speed, accurate automatic dispensing (pipetting accuracy within 2% and c.v. less than 3.5 across the volume range) (Figure 2).
The ability of both mosquito HTS and mosquito HV to accurately dispense clonase and NGS sequence kit enzymes which are in solution containing 40% glycerol across the volume range 50 nL to 5 µL is demonstrated in figure 3.
Automated dispensing in the intermediate volume range allows easy integration into pre-existing screening protocols set up for high content screening and HTS cell based assays, making mosquito HV more suitable than nanoliter dispensers for a range of molecular biology applications. The disposable tips although larger are still able to reach to the bottom of wells, minimizing dead volume to under 50 nL per well.
For the study of the effect of small molecules on DNA and protein expression and structure, mosquito HV can also be successfully employed for near assay serial dilutions using standard plates where 2-8 µL of serially diluted compound are required. Using the mosquito serial dilution wizard, it is possible to quickly and easily define and set up a range of serial dilutions thereby eliminating time consuming and error prone manual programming effort (Figure 4).
Miniaturized assays provide a number of cost and time saving benefits for the research and drug discovery industries. Although assay miniaturizations enables reduces the amount of often costly enzymes or reagents and sample used in a screen, the most important aspects of assay miniaturization is the reduction of assay development times and faster screens, which enables more timely decision-making to helping companies move projects forward more rapidly. The decision to miniaturise assays needs to be offset against existing instrumentation outlay and assay development. The mosquito range of a liquid dispensing instruments are low cost, easy to use, providing automated pipetting for a range of assay types and sizes and for novel assay design in addition to being readily integrated into existing instrumentation to enhance laboratory throughput and efficiency.