Genetic diagnostics Flashcards
DNA extraction
To perform an analysis, first the DNA must be extracted from the cell nucleus and purified.
This procedure involves the disruption of cell membranes by chemical exposure to a detergent.
This is typically followed by purification using centrifugation or other methods for removal of cell debris, proteins, and enzymes, leaving DNA in solution.
DNA analysis method
Conventional genetic assay protocols use four fundamental chemical procedures for the analysis of DNA:
- Chemical amplification
- Fluorescence and staining visualization
- Electrophoretic Separation
- Hybridization
Chemical amplification
DNA samples are often present at concentration levels that are too low for any direct test. Therefore, chemical amplification is used to increase the concentration of the sample.
The amplification basically consists of a set of reactions that allow a DNA molecule or fragment to duplicate. Further amplification is hence obtained by repeating the procedure.
Several schemes can be used for amplification, but all of these use powerful enzymes.
Enzymes are “miracle worker” protein catalyst molecules that can manipulate and modify DNA strands present in every living organism. In particular, polymerase enzymes assemble complementary strands of DNA from a single-strand fragment. This enzyme scans single strands of DNA and, starting from a specific location, captures matching nucleotides from solution and connects them to the single strand, assembling the complementary strand one base at a time like a zipper.
Molecular replication takes place when a double-stranded fragment is first thermally separated (or
denatured) into two strands followed by the polymerase action. This procedure is known as the
polymerase chain reaction (PCR).
A typical amplification uses the high-temperature resistant Taq polymerase enzyme extracted from a heat-resistant microorganism (Thermus aquaticus) mixed with the unknown DNA sample (or template), an adequate supply of nucleotides and primers that determine the starting point of the replication.
The amplification begins by denaturing of DNA at 95°C, yielding single strands of DNA.
The temperature is next decreased, permitting the attachment of the matching primers to the single strands (annealing) and the enzyme action (extension). At the end of this cycle, two double strands of DNA are formed.
The cycle can then be repeated 20 to 50 times (30 typical) to provide a very large amplification factor. The success of the reaction depends on the correct composition of components, precise temperature control, particularly during the denaturing and annealing phases, the nature of the reactor walls, and the presence of contamination, which can inhibit the enzymatic action. After the completion of cycles, the concentration of template increases by a factor: F=(1+E(n))^n where E is an efficiency factor, function of n. For n<20, E =circa 1, but for n>20, the efficiency drops.
Conventional PCR amplifications are made in macroscopic thermal cyclers accomodating multiple reaction vials. Macroscopic cyclers require roughly 90 min to complete the amplification therefore, faster thermal cyclers are desirable.
Specifications for a PCR system
The specifications for a PCR system are determined by the thermal cycle requirements.
Denaturing and annealing each require only a few seconds once the sample has reached the correct temperature.
The extension time depends on the specific polymerase and on the length of the DNA segment being amplified. If Taq Polymerase is used, with an extension rate of 50 base-pairs per second (bp/s), and the segment to be amplified is 500 base pairs (bp), then 10 seconds of extension time is required.
Therefore, in aggregate, the total reaction time required for a PCR cycle is about 15 seconds.
In a macro-scale instrument, which is a bench-top instrument employing standard 96-well plate for molecular biology, the actual cycle time is dominated by the temperature ramps, up and down. A total of 65 seconds is required for the various temperature ramps during one cycle, while only 15 seconds is required for performing the reactions.
This fact alone is enough to motivate the search for a microfabricated implementation of PCR, because thermal masses and response times can be made smaller than for corresponding macro-scale devices, with the result that temperature-ramp times can be reduced. Reduction in cycle time translates into instrument throughput.
PCR batch microdevices
Over the past decade, the miniaturization of DNA assays has been investigated by several groups using a broad range of fabrication technologies and materials. While the construction for many of these devices is often rudimentary, these simple devices serve well as microscale protocol demonstrators. A number of micromachined devices have been developed to accomplish faster amplification cycles by basically reducing or eliminating the large thermal mass present in macroscopic systems. There are two fundamentally different approaches to creating microfabricated PCR systems: BATCH and CONTINUOUS FLOW.
Northrup has reported a bulk-micromachined silicon PCR chamber developed at Lawrence Livermore National Laboratories. This device consists of a microwell cavity structure formed in a silicon substrate by anisotropic etching. The well bottom is a thin silicon nitride membrane with polysilicon heaters on the underside. This type of structure is essentially the same used for many bulk micromachined pressure sensors; hence, it can be fabricated cheaply. The well lead is a glass slide bonded to the top. Due to its small thermal mass this structure can be heated at rated of 15°C/s. A 20-cycle amplification in a 50 microL microwell was carried out toughly four times faster than in a conventional cycler with a much lower power budget.
PCR Batch microdevices: PDMS (da sapere???)
PDMS PCR chip with 1064 reaction chambers in a 31,5mm × 25,7 mm chip.
In order to make the reagents flow inside the chips smoothly, and vent the air bubbles out of as many chambers as possible, column chambers and inclined sub-channels have been designed.
The reaction chambers are designed in column with the diameter of 460 microm and the depth of 200 microm. The volume of each chamber is calculated to be about 30 nl. The interval between the two chambers is 800 microm.
The capillary force and syringe pressure draw the reagents into the reaction chambers in the loading process. The outlet ports were designed open to air, therefore the air inside the chambers and the redundant liquid can be vented out easily in the unloading process.
Extraction+Purification+Batch PCR
- Dna Extraction + purification +PCR:
- Polymeric
- Simple Layout
- No Surface Functionaliz. or Passivation
- No chaotropic salts, magnetic beads, …
- No Elution to purify.
*Amplification of gene involeved in cistic fibrosys: 1 Dna ladder 2 standard PCR 3 negative control 4 PCR in chip
*Amplification of gene for hemocromatosis:
1 PCR in chip
2 Dna ladder
PCR continous flow microdevices
-A radically different continous-flow design has been reported by Kopp et al., in which a sample flows from one fixed-temperature zone to the next, executing the PCR cycle along the way.
-PDMS
A completed flow-through thermocycling chip made of PDMS has been produced and mounted to the silicon part of the silicon-glass thermocycler with integrated thin-film transducers.
The PDMS easily adheres to all smooth surfaces forming a very close connection supporting heat
dissipation.
-Micro peristaltic pump
The micro peristaltic pump is actuated by 3 piezo-discs located in a recess etched in the Pyrex. The fluid inlet and outlet holes are etched into the Pyrex wafer together with the pump membrane. Six wire
bonding holes are also etched into the Pyrex wafer to provide electrical access. The PCR is to be achieved by introducing the reactant droplet of 1 µl
into the inlet hole then, by driving the micro bi-directional pump, the reaction droplet is to be moved back and forth between these three reaction chambers at 90°C, 72°C and 55°C.
The position of the droplet is detected and controlled as a result of an optical signal detected by a pn-diode integrated in the chip as the droplet meniscus scatters the illumination light.
-Continuous flow microreactors:
Continuous flow microreactors with an annular microchannel for cyclical chemical reactions were
fabricated by either bulk micromachining in silicon or by rapid prototyping using EPON SU-8.
Fluid propulsion in these microchannels was achieved using AC magnetohydrodynamic (MHD)
actuation. This integrated micropumping mechanism obviates the use of moving parts by acting locally
on the electrolyte, exploiting its inherent conductive nature. Both silicon and SU-8 microreactors were
capable of MHD actuation, attaining fluid velocities of the order of 300 microm s^-1 when using a 500 mM KCl electrolyte. A polymerase chain reaction (PCR) protocol was implemented. The cyclical nature of the PCR chemistry suggests that a circular, or annular, channel could be used to conduct a thermal cycle every revolution. Accordingly, temperature zones were provided to enable a thermal cycle during each revolution. With this approach, fluid velocity determines cycle duration.
Batch & Flow Comparison
PCR continous flow
Pros:
• very good temperature control
• no risk of overshoot,
• it requires a rather simple control system (three fixed temperature zones)
Cons:
• it is not as flexible as the batch system because the relative times spent in each zone must be designed into the chip (of course supposing a constant flow velocity, otherwise the control will be much more complicated).
For a different ratio of times in the various zones, a differently designed chip is required.
PCR batch
Pros:
• flexibility in programming the cycle
Cons:
• requires a more sophisticated control system
• because of the relatively larger chamber size,
has heat-transfer time delays that are substantially larger than for the continuous-flow system
Fluorescence and staining visualization
The presence of DNA fragments is commonly detected by introducing in the mixture a suitable label molecule that binds to the fragment.
Early labeling methods used radioactive 32P incorporated in the fragment nucleotides. Most modern labeling methods use fluorophore dyes that emit light when bound to DNA under external excitation.
Light-emitting labels are extremely sensitive, permitting the detection of individual molecules in femtoliter samples. Hence, these dyes are almost universally used for the visualization of DNA fragments.
Intercalating dyes such as ethidium bromide (EtBr) fluoresce when excited by ultraviolet (UV) light only
when bound between two nucleotides in double-stranded DNA. Since a fragment can accommodate one intercalating label per base pair, a single DNA molecule can contain hundreds of fluorophores and emit a strong signal. Intercalating dyes affect somewhat the migration of fragments during separations; therefore, for best accuracy, sometimes a single fluorophore is attached at the end of the molecule.
Light emission can be excited in several ways. Conventional fluorescent labels require the excitation from UV light; hence, the emission signal must be separated from the excitation using filters and dichroic mirrors. Light emission can also be excited chemically and by electrochemical reactions.
Electrochemiluminescence (ECL) tagging methods use a large bright Ru(bpy)32+ end label that emits light in the presence of an
electrochemical reaction. This technique is becoming increasingly important as it is regarded as the most sensitive tagging method.
Electrophoretic separation
Electrophoresis is a technique used for separating DNA fragments of different sizes from a mixture.
DNA fragments in solution are negatively charged; therefore, they drift under the presence of an applied field E with velocity v=mu_i(Ni)E where mu_i is the fragment mobility. The mobility depends on the type of mobile phase and the fragment size Ni. Therefore, if the mixture is introduced as a single band at a starting point in a mobile phase, the fragments are separated into bands composed of different sizes of DNA as they drift in a “race track” fashion.
The fragment separation is DeltaL = Deltamu·E·t, where t is the drift time. The fragment position along the track and the band pattern are indicative of the fragment size and the number of fragments present in the mixture.
DNA fragments hold uniform charge to mass ratio, making mu independent of size in liquid phases; therefore, an auxiliary molecular sieve is needed for the separations.
The sieving medium is typically an entangled polymer matrix in the form of a gel. The mobility of DNA fragments in gels is roughly inversely proportional to the logarithm of the fragment size, and the type of sieving matrix depends on the fragment size range.
Both linear and cross-linked polymers are used.
Agarose, for example, is used for large fragments (>3
kb), while a denser matrix like polyacrylamide is used for shorter fragments. Both double-and single-stranded DNA fragments can be separated with gels. The resolution of single-stranded fragments is higher than that of double strands.
Microfabricated devices. electrophoresis devices
These devices consist of two crossing
perpendicular channels.
The first channel defines the sample injection
plug and the second separates the sample.
These channels are made by wet etching two 10- microm-deep crossing grooves on Pyrex glass wafers.
Platinum electrodes are then deposited and
patterned, and channels are then sealed with a
top glass wafer with access holes bonded to the
substrate. After introduction of the mobile phase, the sample is placed in the lower reservoir. A low voltage is then applied across the vertical capillary, forming a long plug that fills the capillary with
sample (with no separation). Next, the vertical capillary voltage is turned off, and a high voltage is applied across the horizontal capillary, moving the plug of sample at the intersection forward and resulting in a high-resolution separation.
Separation channels have been fabricated on silicon substrates with on-chip detectors which eliminate the need of expensive readout optics and open the road to low-cost disposable devices.
Hybridization techniques
Hybridization is the term used for the hydrogen bonding of two complementary single strands of DNA, thus forming a duplex. This renaturation process occurs at specific temperature and salinity conditions.
In hybridization-based analyses, one of the strands is known (a DNA probe) and the other unknown. The hybridization bond is specific since it occurs only when there is a match of complementary strands. The presence of a double strand in the mixture (detected by fluorescence) is indicative of a match;
hence hybridization serves as a sequence detection mechanism.
DNA probes are immobilized to a rigid surface by a linker molecule. DNA probes can be either synthetic oligonucleotides or longer DNA fragments typically arranged in array form.
There are some difficulties associated with array hybridization techniques. Hybridization is never perfect, especially for short duplexes, and single base mismatches can occur. Oligomer probes that are self-complementary can fold on themselves or
bind with neighbors.
The arrayer
The arrayer is a device with a number of pins that are first dipped into the well plate, drawing out a small bit of each solution on each pin.
The pins then “print” the chip by releasing the solution onto a glass slide.
The gene chip now contains spots, each containing the DNA for one gene.
The slides to be spotted are placed on the trays and the robotic arm of the arrayer moves over each slide. A computer keeps track of the position of each gene spot.
Microarray scanning
Hybridization arrays have been constructed on polypropylene, glass and silicon.
Reading of these arrays is typically done using confocal epifluorescence microscopes with cooled and intensified CCD’s.
Detection of hybrids is especially difficult as the size of the array pixels continues to shrink.
