User manual

16 | Droplet Digital
™
PCR Applications Guide
For best results, restriction digestion of your DNA sample outside of the amplicon
region is recommended. We have extensively investigated the use of endonucleases for
fragmentation and found that a wide range of enzymes with 4-base and 6-base recognition
sites perform satisfactorily for this purpose. The benefits of predigestion can be achieved
with a wide range of enzyme concentrations. Considerations should be taken into account
in the choice of enzyme for a particular locus:
1. The enzyme should not cut within the PCR target sequence itself.
2. It is best to use an enzyme that is insensitive to methylation to avoid incomplete
fragmentation due to methylation of the target DNA.
3. In some instances, it is best to digest the target copy to the smallest size fragment that
fully contains the amplicon footprint — preferably less than a few hundred base pairs.
4. If added at a relatively high concentration, some restriction enzyme buffers can result
in a significant change in salt concentrations of the reaction mix. To avoid this, always
digest in the lowest possible volume and mix with water before adding the digested
DNA to the reaction mix.
Adding DNA to the Reaction Mix
The recommended dynamic range of the QX100 system is from 1 to 120,000 copies/20 µl
reaction. There are about 120,000 copies in 400 ng of human DNA, assuming
1 copy/haploid genome. To estimate the number of copies/ng of DNA for your organism
you must know the mass or the number of base pairs in the genome (see formula below).
If the experiment entails quantifying samples known to have extremely high amounts of target
molecules (such as next-generation sequencing [NGS] libraries), plan to reduce the starting
sample accordingly. If the target copy number/genome is unknown, we recommend that you
determine the optimal starting amount by doing four tenfold dilution series of each sample at
the expected digital range. By assaying the four data points above and below the expected
digital range, you ensure that one of the data points is within the optimal digital range.
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 × 10
9
bp (haploid):
m = (3.0 × 10
9
bp) (1.096 × 10
–21
g/bp)
m = 3.3 × 10
12
g or 3.3 pg
Designing Droplet Digital
™
PCR Experiments