Download OpenArray® Real-Time PCR System, Digital PCR Experiments

Chapter 2 Prepare and Perform the Digital PCR Experiments
Prepare the DNA samples
Determine the
optimal dilution
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In a digital PCR experiment performed on an OpenArray® System, gDNA or cDNA
samples are diluted down to a limiting quantity, such that most individual PCR
reactions in the through-holes of each subarray contain either zero or one target
molecules. The procedure for determining the optimal dilution for a sample differs
depending on whether or not the target copy number per genome of the sample is
known.
If the target copy number per genome of your samples is known, dilute the samples so
that, when aliquotted to a subarray, each through-hole reaction will contain
approximately 0.6 to 1.6 copies of the target sequence. For example, assuming 3.3 pg/
copy of a given gene are present per genome and a 33-nL through-hole volume, the
stock gDNA in a given sample would be diluted down to 60 pg/µL (0.06 ng/µL) in the
final reaction to give 0.6 copies per through-hole.
Copies/hole
Copies/µL
ng/µL
0.6
18.18
0.06
If the target copy number per genome is unknown, we recommend that you determine
the optimal dilution by loading a OpenArray® Digital PCR Plate with a three- or fourfold dilution series of the each sample at the expected digital range. By assaying three
to four data points above and below the expected digital range, you ensure that one of
the data points is within the optimal digital range.
How to determine the target copy number per genome
To help determine copy number per genome, collect the following information:
1. If the source or species of the gDNA is known but the genome size of the
organism of interest is unknown, refer to http://www.cbs.dtu.dk/databases/
DOGS/index.html to determine the size of the genome in question.
2. Once the size of the genome is known, determine the mass of the genome using
the following formula:
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 “normal” rate of two copies
per diploid genome, such as RNase P, and provides a basis to perform a digital
screening experiment to determine the optimal digital range.
Applied Biosystems OpenArray® Real-Time PCR System Digital PCR User Guide
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