Microfluidics for DNA analysis
1. Introduction about microfluidics for DNA analysis
Microfluidics presents numerous advantages for biological analysis:
- Whole biological process integrated and thus simplified for the end-users
- High-throughput, multiplexed and highly parallel assays
- Faster analyses due to the shorter reactions and/or separation times
- Portable devices for point-of-care applications
- Low reagent consumptions
- Global cost reduction per analysis
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All these advantages have been extensively exploited for cellular and molecular biology. This review presents the main applications of microfluidics for this latter domain and its numerous techniques for DNA analysis.
2. Microfluidic DNA extraction and purification
Nucleic acids assays obviously required to use purified nucleic acids from biological cells.
DNA extraction basically requires two steps:
- Cell lysis, i.e. disrupting cells membrane and nucleus membrane to liberate their content.
- DNA purification from the unwanted sample like membrane lipids, proteins and RNA and eventually the different reagents used during the previous step.
a. Cell Lysis and its application in microfluidic devices
Cell lysis may be done by several methods:
- Chemical lysis
- Thermal lysis
- Ultrasonic lysis
- Electrical lysis
- Mechanical lysis
Chemical lysis is the most common laboratory bench method because of its ease of use with no specific equipment.
This method can be used for on-chip lysis but reagents are rather stocked off-chip and then injected in the device with external flow system (e.g. pressure controller) to perform an on-chip lysis (1,2), which complicates the making of all-integrated lysing systems.
Thermal lysis is a convenient method for PCR lab-on-chip devices since the required thermal system for lysis can also be used for the following PCR steps (3).
Indeed, several systems integrating these thermal lysis and PCR steps on-chip have been previously developed (4,5). Thermal lysis simply consists in exposing cells to high temperatures. Most of the time, thermoelectric materials are integrated on the chip.
From Privorotskaya et al., Lab on Chip, 2010 (6)
Ultrasonic lysis in microfluidic devices presents the advantage to avoid local heating in pulse-mode and thus maintain protein structure while enabling a simple integration (unlike chemical lysis).
Ultrasonic lysis uses ultrasound to oscillate cavitation within cells until they disintegrate. Microfluidic devices use mainly integrated piezoelectric transducers to generate this ultrasound (7,8).
Electrical lysis use high electrical fields that can disrupt cells membrane and thus induce their lysis. Thanks to the easy integration of electrodes, electrical lysis is a common technique used in microfluidics devices (9,10).
Nevertheless, this method presents some drawbacks like the possible water electrolysis at high voltage and thus the bubble formation.
Interestingly, electrodes can also be used for cell dielectrophoresis for upstream cell sorting (11) (see our other review about label-free cell sorting).
From Medimoon website (12)
Mechanical lysis can be carried out in many forms in microfluidic devices. It is possible to integrate sharp micro-nano objects within the microchannels to damage the cell membranes and the lyse cells (13). Pressure forces can be used as well by using for example highly pressurized quake membranes compressing cells until their lysis (14). Magnetic beads can be used as well to lyse cells by making them beat with a rotating external magnet (15). It can be observed here that mechanical lysis requires quite specific setups which may complicate their integration in more complete devices like lab-on-chip systems.
From Quora website (16)
b. DNA purification and its applications in microfluidic devices
Most of microfluidic DNA purification techniques have adapted the macroscopic method made in extraction columns and using the DNA adsorption on silica beads under certain buffer conditions (highly ionic and with an adapted pH). After this DNA adsorption, DNA is released from silica particles with an elution buffer (low ionic buffer as water for example).
Within microfluidic devices, silica beads can thus either be blocked by mechanical obstacles (17) or silica-coated magnetic beads (18) to easily block these particles with a magnet for the washing and elution phases.
Other types of functionalization can be used for the magnetic beads (e.g. nucleotides (19)).
Isotachophoresis used with pressure-driven flow in capillaries can also be used to efficiently isolate DNA (20).
Briefly, isotachophoresis uses the different ionic mobilities between the DNA, a “slow” and a “fast” electrolyte.
Electrolytes must be chosen so that DNA has an intermediate DNA mobility and will thus focus at the interface when an electric field is applied.
A pressure driven counter-flow can be used to control the position of the focusing interface. Thus, DNA can easily be extracted from crude samples (21).
From Standford Microfluidics Laboratory Website (22)
3. Microfluidic PCR, qPCR & Molecular detection
Once, DNA has been extracted and purified from biological samples and cells, it now can be used for DNA analysis. The most common DNA analysis is the Polymerase Chain Reaction or PCR which is the specific exponential amplification of a DNA sequence.
PCR has numerous applications, some directly and other indirectly implied in other molecular techniques like sequencing for example (see paragraph 5). One of the direct PCR applications is obviously the cloning of DNA sequences (e.g. gene). It can help to make detectable low amounts of DNA (e.g. for pathogen detection, like bacteria or virus).
a. PCR principle and its applications in microfluidic devices
PCR requires several reagents in a common mix:
- DNA sample: the original DNA from which a specific sequence will be amplified.
- Primers: a pair of short nucleotides sequences (10-20 base pairs) corresponding to the partial sequences at the two extremities of the specific amplified DNA sequence. They will thus determine the specific amplified sequence.
- Nucleotides or more precisely deoxynucleotide triphosphates (dNTP): basically, the 4 types of DNA bases (A,T,C,G) which will be used to synthesize the new DNA amplicons (i.e. the new DNA samples resulting from the PCR process).
- DNA polymerase: an enzyme that enables to create the new DNA strand from single-stranded DNA as template by assembling specifically the nucleotides.
Once the mix is prepared, the PCR basically consists in temperature cycles between 2 or 3 different temperatures for each PCR phases described in the schema above:
- Denaturation: this step is carried out at 94-98°C and causes DNA melting, i.e. the separation of the DNA strands yielding two separate single-stranded DNA.
- Annealing: this step is carried out at 50-65°C to allow the annealing of the primers to the single-stranded DNA templates at the specific locations corresponding to the two extremities of the specific amplified DNA sequence. Annealing phase can be made at the same temperature as the extension phase to make a PCR using only 2 different temperatures.
- Extension: this step is usually carried out around 70°C. The temperature mainly depends on the DNA polymerase used since it corresponds to the phase where the DNA polymerase binds to the primer/single-strained DNA complex to synthesize the new complementary DNA strand by incorporating the surrounding nucleotides.
The importance of PCR in genomic analysis leads to the development of numerous microfluidic devices for PCR. Indeed the miniaturized volume and the high surface to volume ratio enable rapid thermal transfer for rapid and integrated PCR (23,24).
It should be mentioned that PCR also requires a subsequent analysis mainly carried out by electrophoresis to check that amplicons size corresponds to the expected PCR products and thus many works have been made to integrate PCR and electrophoresis on-chip. (25,26). Microfluidic electrophoresis is discussed in the following paragraph.
Eventually, integrated microarrays can be used as well since they enable to check specifically PCR products and not just their molecular weight (27).
Interestingly, DNA processes like DNA extraction and post-PCR analysis carried out on-chip can also be combined to design total analysis systems and lab-on-a-chip devices (3,25,28).
PCR & Electrophoresis microfluidic system, From Kaigala et al., Analyst, 2010 (26)
b. qPCR principle and its applications in microfluidic devices
Quantitative PCR (qPCR) is also called real-time PCR but can easily be confused with reverse-transcript PCR (RT-PCR, for RNA analysis).
qPCR relies on the same principle than PCR instead that the PCR product is detected in real-time during the amplification process thanks to fluorescent reporters associated with adapted optical excitation and detector.
These fluorescent reporters can be either fluorescent intercalating agents like Sybr Green or specific fluorescent probes.
Intercalating agents react with double-stranded DNA which make this fluorescent DNA detection non-specific but cheaper. Therefore, this type of detection requires another step to verify the amplification specificity: a melting curve analysis. During this step, fluorescence is monitored while the temperature is increased. As the temperature is raised, DNA strands dissociate and thus the fluorescence, specific to double-stranded DNA, decreases. If the derivative of the fluorescence in function of temperature reveals a single-peak, the PCR reaction can be considered as specific. If several peaks appears, it means that non-specific PCR products have been unintentionally amplified (like primer dimers for example).
Melting curve analysis for qPCR with intercalating agent
Specific fluorescent probes are short nucleotide sequences, specific to the amplified DNA sequence, with fluorescent report at one end a quencher of fluorescence at the opposite end.
The probes anneal to the single-stranded DNA in the same time than primers, during the annealing phase and are degraded when they are reached by the DNA polymerase during the elongation phase. This type of detection is therefore highly specific without any further step.
Fluorescent probes mechanism
Compared to PCR, qPCR presents the advantage to be faster (automated detection during PCR), more sensitive, have a much broader dynamic range and have a better repeatability.
Since the development of qPCR and the first PCR microfluidic devices, many qPCR microfluidic devices integrating optical detection are therefore rather developed (29–31).
As an example, using Elveflow ultra-fast pressure controller and fluorescence reader and based on the Elveflow ultra-fast temperature control, ELVESYS developed an ultra-fast qPCR microfluidic system for the molecular detection of diseases like Anthrax and Ebola in less than 8 minutes with a detection efficiency identical to commercial systems that are 7 to 15 times slower (32).
This system uses the improved thermal conduction of liquids compared to air, enables to reach high thermalization speeds. The device also uses the advantage of a large surface to volume ratio inherent to microfluidics to enhance thermal exchanges. In addition to ultra-fast and sensible qPCR, the system uses monophasic samples and is compatible with a broad sample volume ranging from nanoliters to tens of microliter.
Elvesys system for ultra-fast microfluidic qPCR
c. Digital qPCR principle and its applications in microfluidic devices
With the development of microfluidics, emerged the field of digital microfluidics dealing with emulsion and droplets within microfluidic devices.
The precursor paper of Beer et al. (33) showed the interest of this technique to handle ultra-low amount of DNA within droplets and thus increase even more the detection limits obtained with qPCR. Indeed, with 10 pL droplets, a single DNA copy could be amplified. At the end of qPCR reaction, fluorescent droplets are counted and Poisson distribution is finally used to thus determine the absolute number of templates in the droplets.
From Beer et al., Analytical Chemistry, 2007 (33)
Now, digital qPCR is commercialized through several companies and is specially used for ultra-sensitive detection. Nevertheless, digital qPCR requires longer thermalisation times to obtain a homogenous PCR thermalisation between all the generated droplets if they are transfered in a PCR tube.
4. Microfluidic DNA Electrophoresis
Electrophoresis is the displacement of charged particles caused by a uniform electric field. Basically, since DNA is negatively charged due to its sugar-phosphate backbone, it will migrate towards the anode electrode in the presence of an uniform electric field. DNAs with higher molecular weights will migrate slower and thus DNAs with different molecular weights can be differentiated.
a. Electrophoresis applications
Like PCR in a lower extent, DNA electrophoretic separation has many applications.
One of the main applications is the verification of PCR results like mentioned in the previous paragraph. DNA must be stained with an intercalating agent like ethidium bromide or Sybr Green and can then be seen under UV light. With the electrophoretic separation, DNA is separated by size and compared to a calibrated molecular weight ladder to check the obtained molecular weight and compare it with the expected PCR result. Even if molecular weight is verified, the molecular sequences are not, which makes this type of detection less specific than qPCR with fluorescent probes.
Electrophoresis can also be used to analyze DNA molecules cut by restriction enzymes. These restriction enzymes cut DNA at specific restriction sites. The different DNA sequences can then be differentiated by electrophoresis and, depending on the equipment used, recovered from the electrophoresis substrate to use them as DNA sequence template for plasmid construction for example (plasmid is a circular DNA, often synthesized as cloning vector or for protein production).
Electrophoresis can also be used for DNA sequencing. The principle will be discussed in the next paragraph.
b. Microfluidic devices for electrophoresis
Electrophoresis was originally carried out in gels, mainly made in agarose (for longer DNA) and polyacrylamide (for shorter DNA). These gels are porous which explains they decrease DNA electrophoretic mobility according to DNA size/molecular weight.
Then electrophoresis was adapted to capillaries which present several advantages over gels. First, they enable to use much higher voltage and thus reduce the time of analysis (34). Secondly, thanks to their large surface to volume ratio, they dissipate effectively Joule heat which is important to not create temperature gradient and thus convection effect that could disturb electrophoretic effect. Also electrophoretic mobility depends on the temperature and thus a temperature gradient could cause spatial dependence of the mobility and deformation of the migrating zone (34,35).
With the advent of microfluidics, DNA electrophoresis was one the first molecular process to be integrated on a chip (36). This miniaturisation enabled to increase even more the process time to realize electrophoresis (e.g. sequencing of hundreds of bases in a few minutes (37), reduce reagent consumption and assemble on a chip other DNA process steps as mentioned before. Now, microdevices for DNA electrophoresis analysis have been commercialized.
It is interesting to note that pressure control can also be used in conjunction with electrokinetic force to inject DNA sample within the separation channel (38). This technique enables to avoid the injection disparities between species with different electronic mobilities (39).
Example of sample injection methods in electrophoresis microchip. (a) floating injection, (b) double-T mode. Adapted from Dorfman et al., Chemical Review, 2012 (34). Explanations about these injection methods can be found in this excellent review.
5. DNA Sequencing & Microfluidics
Sequencing is the analysis determining the nucleotide order of DNA sequences. First sequencing was developed based on the Sanger method.
Briefly, the Sanger method is based on PCR method but, instead of using only regular deoxynucleotides (dNTP, cf PCR paragraph), labeled dideoxynucleotides (ddNTP) are also added at much lower concentration. Unlike dNTPs, ddNTPs miss a 3′ hydroxyl group which implies that when they are added at the end of an elongated DNA strand, elongation cannot be continued. Thus by making a PCR reactions with all the different types of dNTPs (A,T,C,G) and with less ddNTPs, the ddNTPs being labeled with different fluorophores, DNA fragments with different lengths and different fluorophores corresponding to different nucleotide ends will be obtained. Then these DNA fragments will be discriminated by electrophoresis and the different fluorophores will enable to optically discriminate the different bases and thus sequence the DNA.
Sanger method principle
Microfluidics has been widely used to improve Sanger sequencing, especially because of the work made on the improvement of electrophoresis (37) and the integration of other sequencing steps (DNA purification and PCR) (cf previous paragraphs) (40).
Next-generation sequencing technologies correspond to techniques that enable high-throughput sequencing to reduce drastically its cost and duration. The developed systems uses globally micro-nanotechnologies, biophysical and biochemistry techniques rather than specifically microfluidics even if milli and microfluidics are intrinsically required to easily transport and exchange reagents within consumables.
Among the most famous next-gen sequencing systems, we can name Illumina and its sequencing by synthesis technhology, Life technologies and its Ion Torrent using semiconductor technologies, Pacific Bio with its single molecule real-time sequencing using zero-mode waveguides, Roche with its 454 sequencing system using pyrosequencing technology.
Nevertheless, microfluidics is more straightforwardly used in other systems that rely on the sequencing technic initially based for microarray (41), i.e. hybridization sequencing. Basically, a sequence to be identified is exposed to large set of short labeled probes (typically 5 to 10 nucleotides) and their hybridization is thus optically detected after having washed away the non-hybridized probes. Either the studied sequence or the probes can be fixed on the microarray.
The same principle has been applied by placing the different labeled probes within droplets and exposing them to a DNA sequence. The hybridization is then optically detected by scanning each droplet (42).
Microfluidic devices have been developed to automatically prepare the sequencing library (43), i.e. the randomly fragmented DNA sequences that will be then used for the following sequencing steps. Devices for automated library preparation are now also commercialized by sequencing companies.
6. Conclusion about microfluidics for DNA analysis
We saw that microfluidics found numerous important and critical applications for DNA analysis.
Due to the complex procedures and long analysis time required for DNA analysis, microfluidics enabled to integrate all the different steps on-chip and drastically reduce the analysis time.
Nevertheless, this technology still needs to be widely accepted in molecular biology laboratories since its cost is currently relatively expensive compared to regular methods. Therefore, it is now more used in laboratories requiring high-throughput analysis or very sensitive assays.
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For more tutorial about microfluidics, please visit our other tutorials here: «Microfluidics tutorials». The photos in this article come from the Elveflow® data bank, Wikipedia or elsewhere if precised. Article written by Timothée Houssin (May 2015).
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