296 7.6  High-Throughput Techniques

sequencing has been applied to multiple different cell types and has an enormous advantage

of enabling correlation of phenotype of a cell, as exemplified by some biophysical metric such

as the copy number of a particular protein expressed in that cell measured using some fluor­

escence technique (see Chapter 8) with the specific genotype of that one specific cell.

7.6.3  OPTICAL “OMICS” METHODS

As introduced in Chapter 2, there are several “omics” methods in the biosciences. Many of

these share common features in the high-​throughput technologies used to detect and quan­

tify biomolecules. Typically, samples are prepared using a cell lysate. This comprises either

growing an appropriate cell culture or preparing first cells of the desired type from a native

tissue sample using standard purification methods (see Section 7.4) and then treating the

cells with a cell bursting/​permeabilizing reagent. An example of this is using an osmotic­

ally hypotonic solution, resulting in the high internal pressure of the cell bursting the cell

membrane, which can be used with other treatments such as the enzyme lysozyme and/​or

various detergents to weaken the walls of cells from bacteria and plants that would normally

be resistant to hypertonic extracellular environments.

The cell lysate can then be injected into a microfluidics device and flowed through par­

allel detection chambers, typically involving a microplate (an array of ca. microliter volume

incubation wells, a standard design having 96 wells). A good example of this method is

FISH (see Chapter 3). Here, the biomolecules under detection are nucleic acids, typically

DNA. The microplate wells in this case are first chemically treated to immobilize DNA

molecules, and a series of flow cycles and incubation steps then occurs in these microplate

wells to incubate with fluorescently labeled oligonucleotide probes that bind to specific

sequence regions of the DNA. After washing, each microplate can then be read out in a

microplate reader that, for example, will indicate different colors of fluorescence emissions

in each well due to the presence or not of bound probe molecule to the DNA. This tech­

nique is compatible with different probes simultaneously that are labeled with different

colored fluorescent dyes.

FISH is a particularly power genomics tool. Using appropriate probes, it can be used

diagnostically in clinical studies, for example, in the detection of different specific types

of infectious bacteria in a diseased patient. Similar FISH techniques have also been used

to study the species makeup of biofilms (see Chapter 2), also known as the microbial

flora, for example, to use probes that are specific to different species of bacteria followed

by multicolor fluorescence detection to monitor how multiple species in a biofilm evolve

together.

Similar high-​throughput binding-​based assays can be used to identify biomolecules across

the range of omics disciplines. However, proteomics in particular use several complementary

techniques to determine the range of proteins in a cell lysate sample and the extent of the

interactions between these proteins. For example, mass spectrometry methods have been

developed for use in high-​throughput proteomics (see Chapter 6). These can identify a wide

range of protein and peptide fragment signatures and generate useful insight into the relative

expression levels of the dominant proteins in a cell lysate sample.

To determine whether a given protein interacts with one or more other protein, the sim­

plest approach is to use a biochemical bulk-​ensemble-​based pull-​down assay. Traditional

pull-​down assays are a form of affinity chromatography in which a chromatography column

is preloaded with a target protein (often referred to as bait protein) and the appropriate cell

lysate flowed through the column. Any physical binding interactions with the target protein

will be captured in the column. These are likely to be interactions with one or more other

proteins (described as prey protein or sometimes fish protein), but may also involve other

biomolecules, for example, nucleic acids. These captured binding complexes can then be

released by changing either the ionic strength or pH of the eluting buffer in the column, and

their presence determined using optical density measurements (see Chapter 3) on the eluted

solution from the column.