eBook: Optimizing Results from Nucleic Acid Isolation - 12

Table 1 lists several of these technologies and approaches to NGS sample prep.

sample-to-sample basis that becomes second nature to researchers.

Solution-based sample
preparation methods

As such, phenol-chloroform-based sample preparation is better suited for low-throughput, high-yield
needs, rather than high-purity or throughput required by sequencing labs.

Probably the most well-known and widely used solution-based method for nucleic acid isolation is phenol-chloroform DNA extraction. Although this method uses hazardous chemicals, when handled correctly
it yields high-quality DNA at a low cost. The approach
is common in research laboratories, where students
and post-doctoral researchers perform phenol-chloroform extractions as part of daily routine.

An alternative solution-based method that is more
suited to automation involves Phi29 DNA polymerase-based DNA amplification. This approach faithfully amplifies limited samples, such as those from single cells, using a one-tube, one-temperature format.
The high-fidelity Phi29 DNA polymerase is active at
30°C, does not require thermal cycling, and can produce micrograms of DNA from picograms of starting
material. Phi29 relies on isothermal amplification,
and can be used for both circular (or circularized)
DNA template and linear DNA template (Figure 2).

However, the approach is not so amenable to
high-throughput applications. It requires careful
handling and could have phenol (an inhibitor) carryover. Also, although modern liquid handling robots are quite sophisticated, they cannot judge and
achieve a balance between yield and purity on a

Commercial kits using Phi29 polymerase, such as
GenomiPhi™ and TempliPhi™ DNA amplification kits,

Table 1. Overview of technologies and approaches to NGS sample preparation
Type

Example method
or chemistry
Phenol-chloroform
(organic solvent)

Solution-based

Solid-phase

Phi29-based
amplification

Silica spin column

Key points
* Produces high yield and quality DNA.
* Relies on hazardous chemicals requiring careful handling.
* Amplifies DNA without the need for thermal cycling.
* Used for Whole Genome Amplification.
* Highly processive and proofreading polymerase.
* Uses chaotropic buffers to bind positively charged silica membrane with negatively
charged DNA.
* Robust and can be automated.

Silica coated
magnetic beads

* High-throughput isolation of nucleic acids from a range of sample types.
* No need for centrifugation or vacuum systems.
* Robust and amenable to automation.
* High-throughput purification of mRNA.

Magnetic bead-based
Oligo(dT) surface
chemistry

* Specific sequence capture using intermediate oligonucleotide with complementary
target sequence.
* No need for centrifugation or vacuum systems.
* Robust and amenable to automation.

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