A well-designed and optimized ddPCR reaction yields highly reproducible and robust results. Before running a ddPCR experiment, it is important to understand the goal or expected outcome of the experiment, as different types of experiments require different sample preparation methods, different amounts of input target DNA, and different types of data analysis. Here, we discuss the fundamental experimental parameters to consider in designing a ddPCR assay. Many factors, such as amplicon selection and primer and probe design, will be familiar to researchers experienced in designing qPCR assays, while others are unique to the ddPCR system. This section explores ddPCR assay design, selecting target sequences, designing primers and probes, and sample preparation for a ddPCR assay.
Related Topics: Droplet Digital™ PCR (ddPCR™) Technology, Absolute Quantification of PCR Targets with the Droplet Digital PCR System.
PCR parameters play an important role in obtaining accurate results. A successful PCR reaction requires efficient and specific amplification of the product. Because the properties of both the primers and the target sequence can affect amplification efficiency, care must be taken when choosing a target sequence and designing primers.
Target amplification in a ddPCR system follows the same principles as in qPCR and regular PCR assays. Primer and probe hybridization kinetics, as well as polymerase processing, follow similar patterns whether amplification is in a 20 µl bulk reaction or in nanoliter droplets. This similarity may not be obvious at first glance, but at a molecular level, the relative numbers of reactants are still large; in a nanoliter-sized drop, there are approximately 540 million molecules of each primer (900 nM) and 150 million probe molecules (250 nM).
Digital PCR assays focus on the end-point analysis of each partition (droplet) to generate quantitative data. As such, amplification efficiency plays a smaller role in the outcome of the results than in qPCR; nevertheless, a proper assay should be designed and sometimes optimized to allow for clear and crisp differentiation of positive and negative droplets.
When choosing a region of the target for amplification, follow these guidelines:
When designing primers for a chosen target sequence, follow these guidelines:
Verify the specificity of the primers for the target sequence using tools such as the Basic Local Alignment Search Tool (BLAST). Ensure there are no SNPs (single nucleotide polymorphisms) within the primer sequences.
A number of free online resources are available to facilitate primer design, including the Primer3 website (Whitehead Institute for Biomedical Research, MIT). Commercially available programs such as Beacon Designer Software (Premier Biosoft International, Palo Alto, CA) aid in both primer design and target sequence selection.
The QX200™ Droplet Digital PCR system provides absolute quantification of target DNA or RNA molecules with EvaGreen or probe-based digital PCR assays. The advantages of using hydrolysis probes include high specificity, a high signal-to-noise ratio, and the ability to perform multiplex reactions.
When designing probes, follow these guidelines:
The QX200 system is compatible with FAM dye and either HEX or VIC as a secondary dye. Using these dyes together in the same reaction, in which each is used to label a different target-specific hydrolysis probe, enables multiplex experiments and the quantification or detection of up to two targets per sample.
The quality of sample preparation can impact droplet digital PCR results. For best results observe the following guidelines:
Sample concentration can also affect results. The recommended dynamic range of the QX200 ddPCR system is 1–100,000 copies per 20 µl reaction.
To help determine copy number per genome, collect the following information:
m = n (1.096 × 10–21 g/bp)
where m is the genome mass in grams, and n is the genome size in base pairs.
The following example calculates the mass of the human genome using the Celera Genomics estimate of 3.0 × 109 bp (haploid):
m = (3.0 × 109 bp) (1.096 × 10–21 g/bp)
m = 3.3 × 1012 g or 3.3 pg
The example is relevant to any gene that is present at the usual frequency of two copies per diploid genome, such as RPP30, and provides a basis for performing a digital screening experiment to determine the optimal digital range.
For sample DNA loading, follow these guidelines:
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