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The invention concerns methods and instruments for fast, selective
replication of deoxyribo-nucleic acid (DNA) from biomaterial by the
well-known polymerase chain reaction (PCR), working in individual
duplication thermocycles.
The invention consists of extremely brief cycle times of only a few seconds
for the PCR reactions, generated, on the one hand, by PCR reaction
chambers constructed as a pattern of fine capillaries in close proximity
to heating and cooling elements and, on the other hand, by keeping the
flow rates in the capillaries to a minimum during the amplification phase
so that the polymerase reaction is not disturbed. In this way, the
temperature cycles in the reaction solution for the three temperature
phases of the PCR duplication can be optimally shortened in duration. The
capillary pattern can be simply produced by means of microfabrication
technology.
PRIOR ART
It is becoming more and more important for the medical care of patients
that analysis methods in genetic engineering are made available which work
very quickly. One example of this is the identification of infectious
microorganisms, which still requires days at present, but actually
requires treatment at the earliest possible stage, in the initial hours if
possible. More intense will be the demand for quick analysis during
examinations of tissue possibly affected by cancer or other disease during
surgery on the open patient by means of oncogenetic, virological or
bacteriological analyses. Here, a maximum analysis time of about ten
minutes is required. Mass spectrometry today provides very fast, highly
sensitive analysis methods for the size of amplified DNA segments.
Advances in matrix-assisted laser desorption and ionization (MALDI) make
it possible to analyze about 20 samples including the MALDI preparation,
the introduction of DNA MALDI samples into the mass spectrometer, the
MALDI analysis and the data evaluation up to presentation on the screen in
less than three minutes. The tissue cells and DNA extraction can be lysed
in less than two minutes.
This maximum of five minutes total for sample preparation and mass
spectrometry analysis stands in contrast to times of three hours for
classic PCR replication. Extreme reductions in these times are on the
horizon however. In one instrument, available commercially in the
meantime, this time has already been reduced to about 20 minutes. In a
recent publication (A. T. Woolley et al., "Functional Integration of PCR
Amplification and Capillary Electrophoresis in a Microfabricated DNA
Analysis Device", Anal. Chem. 68, 4081, December 1996), DNA in 20
microliters of reaction solution was amplified through 30 cycles in only
15 minutes in a miniature chamber made of polypropylene. Even this time
is, however, too long for a fast analysis in the above sense. The goal
must be to perform the PCR amplification in only two to three minutes.
As is known, DNA consists of two complementary chains made up of four
nucleotides, the sequence of which forms the genetic code. Each nucleotide
consists of a sugar (ribose), a phosphoric acid group and a base. Two
bases each are complementary to one another. Sugar and phosphoric acid
form the continuous chain of the DNA (or the so-called backbone), the four
characteristic bases are each lateral branches attached to the sugar. Both
complementary chains or single strands of DNA are coiled around one
another in the form of a double helix, whereby two complementary
nucleotides each are connected to one another via hydrogen bridges between
the bases and thus form a so-called double strand.
The basis for many analysis methods in genetics is the selectively
functioning PCR (polymerase chain reaction), a simple replication method
for specifically selected DNA pieces in a test tube, first developed in
1983 by K. B. Mullis (who received the Nobel Prize for this in 1993) and
which, after the introduction of temperature stable polymerases, went on
to unequalled success in genetic engineering laboratories.
PCR is the specific replication of a relatively short segment of
double-stranded DNA, precisely sought from the genome, in simple
temperature cycles. Selection of the DNA segment is through a so-called
pair of primers, two DNA pieces with about 20 bases length apiece, which
(described somewhat briefly and simply) encode the bilateral ends of the
selected DNA segment. Replication is performed by an enzyme called
polymerase, which represents a chemical factory in a molecule. The PCR
reaction takes place in aqueous solution in which a few molecules of the
original DNA and sufficient quantities of polymerase, primers,
triphosphates of the four nucleic acids (so-called "substrates"),
activators and stabilizers are present. In every thermal cycle, the DNA
double helix is first "melted" at about 95.degree. C., whereby both
strands are separated from one another. At about 55.degree. C., the
primers are then attached to complementary nucleotide sequences of the DNA
single strands ("hybridization"). At 72.degree. C. the double helixes are
reconstructed by elongation of the primers, done by the
temperature-resistant polymerase (e. g. taq-polymerase). Complementary
nucleotides are bonded, one after the other, to a specific end of the
primers to form two new double helixes. In this way, the selected DNA
segment is duplicated in principle between the primers. Therefore, over 30
cycles, around one billion DNA segments are generated from one single
double-strand of DNA as original material. (In a more exact description,
the shortening to the DNA segment between the primers only occurs
statistically with further replications).
The duration of time for a thermal cycle is practically only dependent on
the rate of heating up and cooling down, which is subsequently dependent
upon the volume of liquid, the dimensions of the chamber and the thermal
conductivity of the chamber walls and the reaction solution. For every
thermal stage, only a few seconds are necessary in principle, sometimes
even less. In the above cited article by Woolley et al., in which the PCR
amplification for 30 cycles only lasted 15 minutes, the following times
were required, for example, for the work in the three thermal stages: 2
seconds at 96.degree. C. for melting, 5 seconds at 55.degree. C. for the
primer attachment and 2 seconds at 72.degree. C. for reconstruction. The
remaining time of 21 seconds per cycle was used for the thermal
transitions.
The DNA melts almost instantaneously at a temperature a few degrees above
the "melting temperature." Analyses have shown that heating to this
temperature for one half second suffices for complete separation of all
double helix structures. Precise maintenance of the temperature is not
even especially critical here, as long as one remains above the melting
temperature but below a coagulation temperature. Hybridization also does
not need much time if the primers are available in sufficient
concentration. At an optimal concentration, about one to two seconds are
enough. For hybridization, the temperature is even less critical; it need
only remain under 60.degree. C. to proceed sufficiently fast. Optimal
conditions are at about 55.degree. C.
The growth of the attached primers into a complementary DNA molecule
through the polymerase, known as "reconstruction" in the following, has a
very high velocity. 500 to 1,000 bases can be bonded per second under
optimal thermal and concentration conditions by the polymerase. Since
generally only DNA segments of a maximum of 400 bases in length are
necessary for the analyses, two seconds are quite sufficient for this
reconstruction phase. For this process of reconstruction of a new double
helix, good maintenance of the optimal temperature is required in order to
achieve the high rate of reconstruction.
Theoretically, a PCR reaction cycle could thus be concluded in less than 5
seconds, under the precondition that heat can be introduced or removed up
to each sufficient thermal equilibrium in about 1/4 second each. One such
ideal thermal curve for a PCR cycle is shown in FIG. 1. The introduction
and removal of heat are the critical time-determining variables here. By
the addition of only one primer pair, uniform DNA segments can be
replicated. However, if several different primer pairs are added at the
same time, several DNA segments will also be replicated at the same time
("multiplexed PCR").
This type of multiplexed PCR is frequently used and often has special
advantages. For so-called "fingerprinting" for the identification of
individuals through DNA segments of variable length (methods of
"VNTR=Variable Number of Tandem Repeats" or "AMP-FLP=Amplified Fragment
Length Polymorphism"), it makes the analyses faster. Here through the
selection of primers, which determines the average molecular weight of the
DNA segments, the result can be achieved that the variations of molecular
weights for the DNA segments formed by the various primer pairs only
seldomly or never overlap. This type of multiplexed PCR requires an
analyzer which is capable of simultaneous measurement of a large range of
molecular weights. The method is particularly advantageous for the
identification of infectious organisms, since 20 types of bacteria (or
viruses, yeasts, molds) can be detected at the same time, for example,
with a single PCR replication.
The high sensitivity of modern measurement methods for the analysis of DNA,
for example the sensitivity of the above-mentioned mass spectrometric
measurements, permits the volume of reaction solution to be reduced while
maintaining the optimal concentration. Since on the one hand, for the same
initial amount of DNA, the reaction solution is then exhausted after a few
cycles (though on the other hand not very much amplified DNA material is
required for the analysis) the number of cycles can be reduced from the
normal amount of 30 to about 24 to 28. However, the time-saving due to
this is minimal. Possible reduction of the volumes suggests a solution
based on microfabrication technologies for a new PCR amplification method
such as has already been applied in the above cited article by Woolley et
al.
Also in the review article "Microfabrication Technologies for Integrated
Nucleic Acid", D. T. Burke, M. A. Burns and C. Mastrangelo, Genome
Research 7, 189 (1997), chambers manufactured using microfabrication
technology are presented for PCR amplification, without however giving any
indication of the achievable rates. Such chambers,
1,000.times.1,000.times.250 micrometers large here and made of a low
temperature polymer, nevertheless have the disadvantage that they can only
be emptied by extended rinsing with a washing liquid and thus force a
dilution of the amplified DNA when emptying.
Another obvious idea is to allow the reaction solution to run constantly
through a fine capillary which crosses three zones, kept stationarily at
the appropriate temperatures, on a microfabricated chip in a simple manner
for every cycle, whereby the standard temporal variation in the
temperature is replaced by a simple local variation in temperature. A
section of one such arrangement is shown in FIG. 3. A small dimension for
the capillary should then allow a rapid temperature change up to thermal
equilibrium.
Unfortunately, the flow in a capillary impairs the work of the polymerase
in the reconstruction phase. In a cylindrical capillary, a laminar flow
with a parabolic velocity profile generally prevails, whereby the average
velocity is doubled in the central axis of the capillary while it is zero
at the margin of the capillary. In a capillary with a square or
rectangular cross section, somewhat different conditions prevail, however
the differences are not decisive here. The flowing reaction solution is
therefore divided into sliding layers of differing velocity, while
adjacent molecules in different sliding layers move past one another. The
individual molecules are subject to shearing forces. Straight molecules
are aligned parallel to the direction of flow.
For a close-to-real average velocity of 2 millimeters per second in a
capillary 100 micrometers in diameter, two almost spherical molecules
which are in contact with one another on both sides of an imaginary
sliding surface, move past one another in one millisecond by about 8% of
their diameter on average. One millisecond corresponds to the minimum time
for the incorpo ration of a base. Molecules in the center of the flow do
not experience this sort of displacement. Molecules close to the wall of
the capillary experience a greater displacement. In this way, however, the
work of the polymerase which requires a calm, adjacent positioning of the
molecules on a millisecond scale, is greatly impaired. Increased errors
are the result and, with even greater displacement motion, the polymerase
work is even stopped.
The displacement motion of adjacent molecules increases for the same flow
in proportion to the third power of the reciprocal diameter of the
capillary. There is therefore a dilemma for the flow PCR: thinner
capillaries improve the temperature setting, however they extend the
distance, therefore necessitating an increased flow rate and thus
impairing amplification.
OBJECTIVE OF THE INVENTION
It is the objective of the invention to shorten the cycle time for the PCR
amplification of DNA to extremely short times of about four to six
seconds, and thus the entire PCR amplification to a time of two to three
minutes. Due to the extremely high sensitivity of modem analysis methods
for DNA (for example mass spectrometric measurements of the molecular
weight of amplified DNA segments), the volume for the reaction solution
can be limited to one microliter or even much less. It seems appropriate
to use microfabrication methods and instruments for these methods.
DESCRIPTION OF THE INVENTION
It is the basic idea of the invention to use, on the one hand, a pattern of
very fine capillaries in close proximity to heating and cooling elements
as a chamber system for the reaction solution in order to keep the heating
and cooling-down times for the reaction solution extremely low, while on
the other hand however keeping the flow rate for the reaction solution in
the capillaries during the reconstruction phase of the DNA double strand
using the polymerase as low as possible. The flow rate during the
reconstruction phase should never exceed ten times the maximum capillary
diameter prevalent there per second, while more favorable would be a
medium flow rate of less than five maximum capillary diameters per second.
The error rate for the reconstruction only approaches its minimum below a
medium flow rate which is less than double the diameter per second. The
maximum capillary diameter corresponds to the normal diameter for round
capillaries, for rectangular cross sections that of the diagonal.
A favorable, very fine capillary structure with closely adjacent heating
elements may be favorably produced using microfabrication technologies.
The low flow rate can be provided on the one hand (especially at a
constant flow of reaction solution through the capillary structure) by a
special design of the capillary net, on the other hand, the low flow rate
may also be produced by special methods of application with temporally
changeable flows of the reaction solution.
The advantage of a fine capillary structure is evident: the times for the
thermal transitions in the reaction solution may be kept very short This
advantage is however opposed by severe disadvantages: the extremely large
surface area of the chamber system disturbs the biochemical processes if
the surface even only minimally influences the affected molecules. Thus
for example a bare silicon surface immediately kills the activity of the
polymerase. Many plastics too have proven to be unsuitable for the PCR.
Even the same plastics from different manufacturers, for example the
normally favorable plastics polyethylene or polypropylene, have had
different types of effects on the PCR due to their varying qualities.
Therefore, the surface must very thoroughly be made completely inert
The activity of a surface can be almost completely eliminated by a thorough
coating. Coating methods for capillaries are known from chromatography,
especially from gas chromatography, which eliminate even the smallest
remnant of active surface. Particularly coatings with thread-shaped
molecules which are bonded monolaterally onto the surface ("chemically
bonded phases"), have generated thermally stable and extremely inert
surface coatings. Here, hydrophobic or hydrophilic, polar or nonpolar, fat
or water absorbent surface coatings can be generated which may also have
other characteristics within the depth of the layer. It is therefore a
further idea of the invention to use the known chromatographic coatings
for the deactivation of surfaces. Particularly for the coating of quartz
glass and glass surfaces on the interior of thin capillaries, explicit and
comprehensive formulas with descriptions of the necessary steps are
available. Silicon surfaces can be transformed by oxidation into quartz
surfaces.
Particularly for metal implants, stable coatings have been developed which
correspond to endogenous proteins and glycoproteins such as occur in cell
membranes. Such coatings may reduce the activities on the surfaces for
polymerase reactions in the present case, even if they are not yet
successful as implant coatings.
The micromanufacturing methods, however, also comprise the molding of
plastics in micromanufactured silicon forms. In this way as well,
capillary systems can be developed which may be used as reaction chambers.
It is therefore a further idea of the invention to use favorable polymers
such as low pressure polyethylene or polypropylene for the manufacture of
capillary systems. Since polymers normally possess poor thermal
conductivity characteristics, these may also be filled with thermally well
conducting nanopowders, for example with silver powder. These powders can
be produced with a particle diameter of about 10 to 1,000 nanometers. They
are excellently suited for increasing the thermal conductivity of
plastics. The powders may be deposited in such a way that they do not
directly lie on the surface.
The low flow rate necessary for this invention can be achieved in a
constantly circulating capillary system, whereby zones of different
temperatures are passed through, in such a way that the flow of the
reaction solution in the zone of reconstruction temperature branches off
into a multitude of parallel capillaries, in which the flow rate in each
of these parallel capillaries is reduced as shown in FIG. 4.
The reaction solution can also be moved on intermittently by pressure
pulses. After each filling of the capillary system for the reconstruction
of the DNA double strand, at the corresponding temperature, the flow of
the reaction solution stops, the incorporation reactions run down and only
then (after about 2 seconds) is the reaction solution pumped on. It is
therefore advantageous to keep each of the volumes at equal amounts for
the chamber systems for melting, attachment of primers, and
reconstruction, so that the reaction solution is always pressed on by
exactly this amount of volume. A pulsed process occurs which, however,
makes it imperative for the dwell times of the reaction solution to be
equal in the three temperature zones.
It is however also possible, in particular, to select a capillary system
large enough so that the entire quantity of reaction solution to be
processed can be held in it and then very quickly passed through the
temperature phases one after another using fast heating and cooling
elements with the solution at rest.
Such a type of capillary system may easily be aligned in one plane, as
shown in FIGS. 2a and 2b. The capillaries arranged in a plane are enclosed
in a thin membrane, on the surface of which there are heating elements,
also in a planar structure. Thus for example, 200 nanoliters of reaction
solution in 16 parallel capillaries with cross sections of 60.times.100
micrometers and 2 millimeters length can be located on a surface of about
2.times.1.6 millimeters. These capillaries are located in a silicon
membrane of 300 micrometers maximum thickness. Through the thin membrane
and through the bridges between the capillaries, heat can be applied or
discharged very efficiently. On the top and bottom of the membrane, there
are resistance grids planarly imbedded or otherwise attached, which take
care of the heating capacity. With less than two watts heating capacity,
the temperature of this type of thin silicon membrane with a surface of
3.times.3 mm.sup.2 can be raised by about 100.degree. C. per second, an
increase from 45.degree. C. to about 72.degree. C. can therefore be
achieved in 0.3 seconds. The temperature can itself be determined in the
known fashion via the thermal coefficient from the resistance of the
heating element. Control of the heating capacity with a slight overshoot
leads to quick adjustment of the equilibrium in the reaction solution.
Via gaseous, liquid or solid movable cooling means, which can be brought
into planar contact with the membrane the membrane can be cooled very
quickly. An arrangement with a solid cooling element is depicted in FIG.
2b. In the simplest case, the cooling means may be at room temperature, or
at a lower temperature for acceleration. Since the temperature for primer
attachment need not be exactly adjusted, a simple time control is
sufficient for the contact time. In more critical cases, the change in
resistance for the heating elements may be exploited as a control of the
contact time. The cooling means, moved for example electromechanically or
pneumatically, may be a part of the microsystem arrangement, or they may
also be brought in contact with the membrane through external movement
devices.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a cycle of an optimal thermal profile, unobtainable previously
without this invention, for fast DNA amplification by PCR. The three
thermal levels of the cycles are run through in only 5 seconds. A DNA
amplification with 30 thermal cycles therefore takes only 21/2 minutes.
FIG. 2 shows a microfabricated membrane for DNA amplification with the
reaction solution at rest. FIG. 2a shows the capillary structure with
inlet channel (1), flow distributor (2) for uniform filling of the
parallel capillaries, parallel capillaries (3), flow collector (4) and
outlet channel (5). FIG. 2b shows a cross section through the membrane (6)
with the parallel capillaries, the heating elements (7,8) and the movable
solid cooling elements (9,10).
FIG. 3 shows the principle of an (unfavorable) capillary arrangement in
which the reaction solution in the capillary flows through three places of
varying temperature per cycle. The upper edge (15) of this structure is in
contact with a heater which keeps the edge at about 100.degree. C., while
the lower edge (16) is kept at about 50.degree. C. through cooling. After
flowing through the melting region (11) at about 95.degree. C., the
reaction solution flows to the opposite edge and is cooled in a primer
attachment region (12) to about 55.degree. C. Then it flows to a
reconstruction region (13) in which it is heated to about 72.degree. C.
This area has a somewhat longer flow-through path to achieve a somewhat
longer time for the reconstruction phase. From there the reaction solution
flows into the next melting region (14) which belongs to the next
temperature cycle. FIG. 3 shows an unfavorable arrangement for this
capillary structure since the flow rate is equal for all thermal levels.
FIG. 4 shows a more favorable embodiment of a capillary arrangement for
constant flow. In the reconstruction region (23) the capillary branches
off into a number of parallel capillaries with equal cross sections, which
greatly reduces the flow rate here. Otherwise this arrangement is equal in
all parts to the arrangement in FIG. 3.
PARTICULARLY FAVORABLE EMBODIMENTS
It seems expedient to generate a capillary structure in a silicon chip by
microfabrication techniques with stationary thermal distribution as shown
and described in FIG. 3, and to have the reaction solution flow through it
at a constant rate. It however appears that the PCR reaction at capillary
diameters below about 400 micrometers are considerably disturbed by the
necessarily high flow rate in the capillaries. However this capillary
diameter is still much too great for the heating rates required here. On
the other hand, in order to maintain the polymerase work at the usual low
error rate of 10.sup.-4, a flow rate is necessary that is so low that no
substantial reduction in total time is achieved.
One embodiment greatly improved by the idea of the invention is therefore
provided by a capillary structure on a chip as shown in FIG. 4. Here the
capillary branches off without constrictions in the reconstruction region.
In this way, a reduction in flow speed for PCR amplification may be
achieved. It is an advantage of this arrangement that, due to the
continuous operation in this structure, alternating quantities of reaction
solution may be subjected to PCR amplification, although the time
advantage disappears.
This chip structure also has disadvantages, however. It is relatively long
and narrow (about 4.times.60 millimeters), unusual for a microfabricated
chip and very fragile, and it is additionally subject to strong thermal
stress. These disadvantages may be partially balanced out by a circular or
loop-shaped arrangement with central heating, or by a convoluted
arrangement with capillary levels lying on top of one another, which leads
to a reduction in the overall structure. A further disadvantage is the
fixation of the number of PCR cycles, strictly prescribed by the number of
structure repetitions in the microfabricated chip. Another disadvantage is
the relatively long duration of the overall process including emptying
after the work has already been completed for the front of the reaction
solution passing through.
It is therefore advantageous to fill a larger volume pattern with very fine
capillaries only once, to allow the PCR reactions in the reaction solution
at rest to run through temporal thermal cycles and then empty the
structure again once.
In principle, this type of operation may be performed in a single, multiply
convoluted, continuous capillary, however the process of filling and
emptying is then relatively long. Filling and emptying times are not
insignificant. For example, a capillary with a cross section of
100.times.60 micrometers, which should hold about 250 nanoliters, is
already over 40 millimeters long and requires 40 seconds already for these
processes at a filling and emptying rate of 2 millimeters per second. If
still other processing steps are included, the filling and emptying times
become prohibitively long.
A particularly favorable embodiment is therefore shown in FIGS. 2a and 2b.
This is a number of parallel capillaries (3) which lie in the central
level of a thin, microfabricated membrane (6). Two distributor systems
(2,4) at the start and end of the parallel capillaries, which guarantee
equal flow resistances for all inlet and outlet ways of the parallel
capillaries, ensure a strictly cophasal filling. This capillary structure
is filled at the beginning of PCR amplification, afterwards the reaction
solution is at rest. The heating elements (7, 8) on the surface of the
membrane can heat up the membrane and, with it, the reaction solution in a
very brief time. Thus 2 watts of heating capacity suffice in order to
generate a temperature increase of more than 100.degree. C. per second.
The increases from the primer attachment temperature (55.degree. C.) to
reconstruction temperature (72.degree. C.) and then to melting temperature
(95.degree. C.) may be passed through in about 1/4 second each. If the
heaters are operated, for example, by a high frequency alternating
current, the thermal coefficients may then be used in the known fashion to
measure the temperature in the heater and thus control the heating
process.
The membrane is cooled in this embodiment via two gold or silver-plated
elements made of copper (9, 10), which are pressed against the membrane by
an electromechanically or pneumatically generated movement, producing a
large area thermal contact. A mechanical forced coupling of the opposing
movements of both cooling elements can protect the membrane from damage.
The cooling outlets are provided with cooling vanes cooled using ambient
air. For strong cooling, a simple air or water cooling system may also be
considered. An air system is especially advantagous because the air may
serve as an thermal isolator as soon as the air flow stops. The thermal
discharge of the thin membrane then takes place in less than half a
second.
If the parallel capillaries are filled, at the beginning of the PCR
process, with a very few DNA double strangs only, it may happen that only
one or two capillaries contain amplifyable DNA. In this case, the complete
reaction solution may be drawn back after some initial PCR cycles, mixed,
and returned into the capillary system to have a better distribution among
the capillaries.
After completing the PCR amplification, the capillary structure is emptied
by washing liquid forced from behind. The DNA solution is cleaned by
well-known means and transferred to analysis. The capillary structure is
washed out sufficiently well and is once again available for the next PCR
amplification.
This capillary structure in a microfabricated membrane does not allow any
change in volume of the process reaction solution. Since for this type of
analysis firm amounts of DNA are required, this is not a serious
disadvantage. In contrast to this, this structure allows alternating
numbers of replication cycles. In this way DNA amplification may be
adapted in an advantageous manner to the amount of DNA in the original
materials. If the DNA from only a few cells (about 100) is available, 32
cycles may be run, for example, or if on the other hand, the DNA is from
several thousand or even tens of thousands of cells, 24 cycles may
suffice. Therefore, this type of temporal variation of temperature is more
flexible than the above described variations of reaction solution flowing
through areas of differing temperature.
The initial cycles may, in this type of device, also run more slowly in
order to ease the hybridization, and if enough short DNA segments are
generated, the rate can be increased. It should be mentioned, however,
that the number of DNA sets at the beginning should not be much below 100
DNA sets, because all of the parallel capillaries must be filled with an
appropriate number of DNA sets to be effective amplifiers.
Analysis of amplified DNA segments may for example proceed mass
spectrometrically through ionization using matrix-assisted laser
desorption (MALDI) in a time-of-flight mass spectrometer (TOF). To do
this, the DNA is applied together with suitable matrix substances onto a
sample support The MALDI sample supports are then introduced in a known
manner into the ion source of the mass spectrometer and the individual DNA
sample substances are automatically measured for the molecular weights of
the DNA substance in an equally known fashion. Electrospray ionization
with ion trap mass spectrometers, using well-known nanospray methods,
constitutes an alternative method of analysis.
All of the above described capillary systems require deactivation of the
inner capillary surfaces so that the polymerase work is not disturbed.
Experiments have shown that bare silicon surfaces inactivate the
polymerase immediately.
The inner capillary surfaces must therefore be coated with deactivating
layers. Very good coating methods for deactivation are known from
capillary gas chromatography. The glass or quartz glass capillaries used
there also have very active surfaces, in this case active in adsorbing
substances. The activity essentially proceeds from free OH groups. Such
free OH groups are also responsible for the disturbance of the polymerase.
For capillary gas chromatography, various coating substances have been
developed. Since these substances form the liquid phase of this type of
distribution chromatography (which is often called
GLC=gas-liquid-chromatography instead of just GC), the coating substances
are simply called "phases" here. There are polar and nonpolar phases,
hydrophilic and hydrophobic. For well over 20 years, so-called "chemically
bonded phases" have established themselves in which long, thread-shaped
molecules are bonded chemically covalently on the surface, side-by-side
like seaweed. These phases are thermally stable up to several hundred
degrees Celsius and long-lasting.
Due to the parallel arrangement of the phase molecules, any desired
arrangement can be custom-tailored here. Thus a superficially hydrophobic
layer may be made hydrophilic on the inside. The thickness can be adapted
to the requirements. Silicon rubber phases are primarily used standard
phases in gas chromatography, however they are less favorable for PCR
reactions, while on the other hand waxy phases are better, for example
Carbowax.
In the future, coatings with biomaterials such as proteins, lipid proteins
or glycoproteins will play a greater role as coating materials. It is
already possible to bind such molecules covalently onto the surfaces of
metals. It can be expected that these biomaterial coatings will be even
more favorable for deactivation of the surfaces for polymerase work.
However, it is also possible to generate the capillary system of polymer
plastics using microfabrication methods and tools. Microprinting processes
exist which proceed from a silicone structure as a matrix. Using known
microwelding or microadhesion techniques, the production of thin membranes
with imbedded capillaries is also possible. The finished membranes may be
printed with a resistance network; such resistance networks can be created
by applying metal layers and then etching. Plastics may be filled with
metallic powders to improve the thermal conductivity, such as with silver
nanopowder.
The methods and structures described may of course be varied in many ways.
It is simple for a specialist, following the indicated invention ideas, to
develop further capillary structures and other operating methods.
Thus it is possible, for example, to replicate and finally to analyze RNA
in the above described fashion as DNA after a first duplication step using
"inverse transcriptase", which reconverts the RNA back into a DIA
complementary sequence. This process, too, may be performed in a unified,
microfabricated apparatus. Extensive changes or derivations of DNA toward
the goal of achieving more easily analyzable output products for analysis
may also be performed in instruments especially adapted for this, produced
using microfabrication technologies.
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