14.6 DNA analysis techniques [DNA top]

As mentioned earlier, DNA is of enormous biological importance, and its analysis is central to many biological studies. These analytical techniques are often implemented in microfluidic chips, so we briefly discuss a number of DNA analysis techniques below.

14.6.1 DNA amplification

DNA has the unique property that it can be amplified using any of several amplification techniques, most notably thePCR reaction (polymerase chain reaction). PCR amplification requires (1) adding primers, i.e., DNA strands of known sequence that are designed to initiate replication of a specific DNA sequence; (2) adding other chemicals, such as polymerase and oligonucleotides; and (3) thermally cycling the DNA sample. When this is done properly, a single DNA strand can be amplified into millions of DNA strands, making detection rather simple. From a microfluidic standpoint, PCR requires that microchips meter reagents and cycle temperature in a controlled manner.

14.6.2 DNA separation

Macroscopically, DNA is typically separated by size using electrophoresis in agarose or polyacrylamide slab gels. This involves inserting a DNA sample (as well as a set of DNA of known lengths) onto the gel, applying an electric field, and staining the resulting separated DNA bands. Microscopically, the separation function of these gels is to force the DNA to follow a tortuous path that creates molecular elongation and causes the longer DNA to travel more slowly (see [151, 152]). Since the electrophoretic mobility of DNA is insensitive to contour length in bulk solution owing to the free-draining nature of DNA in bulk, the gel facilitates the separation by causing the elution time to be dependent on molecular elongation and confinement. In microfluidic devices, separations are performed on DNA in gels much like they are in bulk for any other set of molecules with distinguishable electrophoretic velocities. An example of a microdevice for PCR amplification and DNA separation is shown in Figure 14.12. Nanofluidic devices [150, 153, 154] have been used as well.

Sequencing requires different chemical reactions, but still involves electrophoretic separations on reaction products (see [155]).

Sanger sequencing

Sangersequencing of DNA uses (1) DNA replication with DNA polymerase and fluorescently-labeled nucleotides combined with (2) electrophoretic separation to infer the base sequence of the DNA. Many copies of the DNA to be sequenced are incubated with an oligomeric primer, DNA polymerase, and free nucleotides, both fluorescently-labeled (a small fraction) and unlabeled (the majority of the nucleotides). Four colors are used, one each for each type of nucleotide. The fluorescently-labeled nucleotides, owing to the fluorescent conjugation, terminate the polymerization process. Incubation, in this case, creates copies of DNA with a variety of lengths and one of four fluorophores– for each molecule, polymerization occurs until the fluorophore is attached, at which point polymerization ceases. Each fluorophore denotes what was the terminal base on the copy. For example, if a DNA oligomer of sequence ACTGATT is sequenced in the presence of unlabeled nucleotides A, C, T, G, and fluorescently-labeled nucleotides A*, C§, T†, and G‡, the possible oligomers are A*, AC§, ACT†, ACTG‡, ACTGA*, ACTGAT†, and ACTGATT†. These seven oligomers can then be separated electrophoretically in a gel, with the shortest oligomer eluting earliest and the longest eluting latest. By monitoring the colors of the eluted peaks, the sequence of the DNA can be inferred. Obviously, the speed and resolution with which the DNA molecules can be separated is closely related to the ability of Sanger sequencing to sequence DNA molecules. Figure 14.13 shows the steps in Sanger sequencing schematically.

14.6.3 DNA microarrays

DNA microarraysare essentially flat surfaces that have been functionalized with a large array (> 1000) of complementary DNA (cDNA) strands that correspond to different sections of DNA or RNA from an organism. By incubating a microarray with a solution from a biological system and an intercalating dye, fluorescent signal on each spot indicates how much DNA/RNA was present in the sample. Figure 14.14 shows a schematic of the process. From a fluid mechanical standpoint, DNA microarrays are a challenge because the diffusion timescale for transport and hybridization in a microarray can be quite large. Advanced low-Rechaotic mixing systems have been applied to increase hybridization efficiency [24, 25, 26, 27, 28].

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