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Description  |
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TECHNICAL FIELD
The present invention pertains generally to the field of apparatus and
methods for analyzing the structure of nucleic acid molecules, including
DNA (Deoxyribose Nucleic Acid) and RNA (Ribonucleic Acid) molecules and
more particularly to determining the nucleotide sequence of such
molecules.
BACKGROUND ART
A chemical method of sequencing DNA molecules is disclosed in Maxam, Allan
M. and Walter Gilbert, "A New Method for Sequencing DNA," Proc. Natl.
Acad. Sci. USA, Vol. 74, No. 2, pp. 560-564, February, 1977, the entire
contents of which are incorporated herein by reference. The Maxam/Gilbert
method provides for the terminal labeling of several identical DNA strands
with radioactive tracers and then breaking the strands at each base into
fragments using chemical agents. The relative lengths of the labeled
fragments identify the position of the associated base in the strand. For
example, the shortest fragment is comprised of a single base, such base
being the first base in the sequence. The longest fragment terminates in a
base which is the last base of the sequence. The relative lengths of the
framents are resolved by electrophoresis thereby enabling the sequence to
be ascertained.
Further details of the Maxam/Gilbert sequencing method will be described in
connection with FIGS. 1A through 1G. Single-stranded DNA is comprised of
four bases, A (Adenine), G (Guanine), C (Cytosine) and T (Thymine). These
bases are arranged in the strand to form a sequence. The first step of the
sequencing process is to isolate a large number of strands having
identical DNA sequences. By way of example, FIG. 1A schematically depicts,
in very simplified terms, several DNA strands having the identical
sequence CAAGAGATAC. In actual practice, a large number of strands will be
isolated. Next, each strand is terminally labeled with a radioactive
tracer such as P32, as shown schematically in FIG. 1B.
Once the strands have been labeled, the strands are separated into four
groups. Each group is then chemically treated to cleave the base-to-base
bonds in a particular way. A first group is placed in a first vial,
labeled for convenience as Vial A. Vial A contains chemicals, well known
in the art, which, in simplified terms, cause the strands in the vial to
cleave the bond at the right of one of the A bases. Thus, the exemplary
sequence shown in FIGS. 1A and 1B will produce, with equal probability,
the following five different labeled fragments depicted in FIG. 1C: P32CA;
P32CAA; P32CAAGA; P32CAAGAGA; and P32CAAGAGATA. Fragments without a P32
tracer are also present in the vial, but will not be detected in the
electrophoresis process.
The second group of identically labeled strands is placed in a second vial,
labeled Vial C, which contains chemicals, well known in the art, which
cause the strands in the vial to break the sequence at the right of one of
the C bases. The following two labeled fragments will be produced, as
shown in FIG. 1D: P32C; and P32CAAGAGATAC.
The third and fourth groups of strands are placed in third and fourth
vials, labeled Vials G and T, respectively. Vials G and T contain
chemicals, which cause the strands in the vials to break the bonds to the
right of G and T bases, respectively. FIG. 1F depicts the following two
Vial G labeled fragments which result: P32CAAG and P32CAAGAC. FIG. 1F
shows the single Vial T labeled fragment which is produced: P32 CAAGAGAT.
The tagged P32 fragments are then separated by size using conventional
electrophoresis. A separate gel track, typically one meter in length is
provided for each of the four groups of fragments. Each track has a square
well at the top for the initial placement of the DNA fragments. A uniform
voltage is applied across the length of the gel, causing the fragments to
travel along the gel track with a velocity approximately according to the
following equation:
V=(K) (V) (-log (N)+A) (1)
where K and A are positive constants, V is the applied voltage and N is the
number of bases contained in the fragment.
It can be seen from equation (1) that the velocity of the fragments is a
non-linear function of the applied voltage, with the smaller fragments
traveling at the higher velocities.
After an elapsed time interval, the fragments will be distributed in
subgroups along the length of each of the parallel gel tracks in
accordance with the length of the fragments in the subgroup. The gel is
then removed and exposed to photographic paper to form an autoradiograph.
The paper is then developed, thereby producing a visual image which shows
the relative position of the subgroups of fragments along the length of
the gel.
FIG. 1G illustrates a developed exposure which was made for the FIG. 1A DNA
sequence in accordance with the previously-described autoradiography
procedure. The tagged fragments were initially positioned at the top of
the exposure, with the Vial A track being positioned along the left edge
of the exposure, followed by the Vial C, G and T tracks.
The subgroup comprised of the smallest fragments will be comprised of the
single base adjacent the P32 label of the original strand as shown in FIG.
1D. This subgroup of fragments will have traveled the greatest distance,
as indicated by equation (1). Since the smallest fragment came from Vial
C, the first base of the sequence is a C base. The next smallest subgroup
of fragments will be comprised of fragments having two bases, including
first base C followed by a second base. As can be seen from FIG. 1G, the
next smallest (fastest) fragment came from Vial A, therefor the second
base of the sequence is an A. This process is continued for each of the
remaining eight bases. The final sequence is P32CAAGAGATAC which
corresponds to the FIG. 1A sequence.
It was previously assumed that vials G and T originally contained fragments
which terminated in G at T bases, respectively. In actual practice and in
accordance with conventional chemical processes, vial G will probably
contain fragments which terminate in both G and A fragments and vial T
will contain fragments which terminate in both T and C fragments. In that
event, fragments terminating in T bases can be uniquely identified by
observing the presence of fragments at a particular position on the
exposure along the vial T track and the absence of fragments at the
corresponding position along the C vial track. The position of fragments
terminating in G bases can be uniquely determined in a similar manner.
Conventional gel electrophoresis utilizing autoradiography is quite time
consuming and very labor intensive. The electrophoresis gel must be
carefully prepared so as to provide a uniform structure through which the
fragments pass. Any nonuniformity may result in sequencing errors. A gel
approximately 1 meter long is capable of determining roughly 100 bases of
a sequence. For a DNA sequence of 1000 bases, the procedure requires 5-10
gel runs lasting 8 to 16 hours. After each gel is run, the gel must be
separated from its supporting glass plate and exposed for 8 to 48 hours
with photographic paper. After the DNA sequence is obtained, it is
typically manually entered into a computer for further analysis. The
entire procedure, which must be performed by a skilled technician,
requires at least a week of applied time and an elapsed time of several
weeks. In addition, evaluation of the autoradiation photographs and entry
of the sequence into a computer is susceptible to human error.
Some attempts have been made to overcome the above-noted limitation of the
Maxam/Gilbert sequencing procedure. For example, it is believed the
automated imaging techniques have been utilized to analyze the
autoradiation photographs. Despite such advances, the principal
shortcomings of the Maxam/Gilbert procedure remain.
The present apparatus and method for DNA and RNA sequencing overcomes the
limitations of the Maxam/Gilbert procedure. The time required to obtain a
DNA sequence is greatly reduced. Moreover, the procedure can be carried
out by persons having limited training. In addition, overall accuracy is
improved inasmuch as it is not necessary to interpret a photograph and
manually enter data into a computer. These and other advantages of the
subject invention will become apparent to those having average skill in
the art upon reading the following Best Mode For Carrying Out The
Invention.
DISCLOSURE OF THE INVENTION
Apparatus and method for analyzing the structure of a nucleic acid
molecule, such as a DNA or RNA molecule, is disclosed. Several copies of
the molecule to be analyzed are prepared and preferably labeled with a
radioactive tracer. The copies are then separated into four groups and
each group is chemically cleaved to produce labeled fragments which
terminate with predetermined bases using conventional and well-known
chemistry. In the case of DNA and RNA molecules, there are preferably four
separate groups of fragment.
A separate electrophoresis gel channel is provided for each of the groups
of labeled fragment. The gel channels are typically contained in separate
elongated glass tubes. A voltage is applied across each of the gel
channels and the fragment are introduced into the channels. The fragments
propogate along the length of the channel in accordance with well-known
principles of electrophoresis with the smaller fragments moving at greater
velocities.
A detector is located at a predetermined position adjacent the gel
channels, downstream from the point where the fragments were introduced.
In the event radioactive tracers are used, the detector is a radiation
detector. Light detectors would be used in the event the tracers are
fluorescent. Once the fragments have reached the detectors, the fragments
will have become separated into subgroups in accordance with the length of
the fragments because of the velocity differences. When the subgroups pass
a detector, the detector outputs a signal. Data corresponding to the
signal are preferably then stored in a memory for further processing. The
order in which the respective subgroups of labeled fragments are detected
may then be analyzed to determine the base sequence of the molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1G schematically illustrate the previously-noted Maxam/Gilbert
method of sequencing DNA.
FIG. 2 is a schematic diagram of the four electrophoresis columns and
associated apparatus of the subject invention.
FIG. 3 is a simplified block diagram of one of the four identical radiation
detectors of the subject invention.
FIG. 4 is a simplified block diagram of the data processing apparatus of
the subject invention.
FIGS. 5A-5C are flow charts generally illustrating the operation of the
control microprocessor of the FIG. 4 data processing apparatus.
FIG. 6 is a graphical illustration of the transfer characteristics of the
digital location filter of the subject invention.
FIG. 7 is a graphical illustration of the transfer characteristics of the
digital rate filter of the subject invention.
FIG. 8 is a flow chart generally illustrating the operation of the
microcomputer of the FIG. 4 data processing apparatus.
BEST MODE FOR CARRYING OUT THE INVENTION
Referring again to the drawings, the electrophoresis components of the
subject invention depicted in FIG. 2 include four glass tubes, each
generally designated by the numberal 10. Tubes 10 are preferably
maintained in a vertical position by a suitable support structure (not
shown).
Tubes 10 each include a relatively long, wide section 12, a relatively
short, narrow section 16 and a tapered transition section 14 intermediate
sections 12 and 16. Wide section 12 is typically on the order of 50 cm in
length with the narrow sections and transition sections typically being on
the order of 5 cm and 1 cm, respectively. The interior cross-sectional
area of narrow section 16 is preferably approximately one-tenth that of
wide section 12.
Tubes 10 are filled with a gel 20 such as used in conventional
electrophoresis processes. A polyacrylamide gel has been found suitable
for the present application. A separate reservoir 18 is positioned above
each tube 10 and is in communication with the interior of the tubes.
Reservoirs 18 contain a electrically conductive liquid buffer 22 such as
tris-acetate, tris-phosphate or tris-borate. The lower ends of each of
tubes 10 is disposed in an elongated reservoir 24 which is filled with a
liquid buffer 26 similar to buffer 22.
A separate power supply 32 is provided for each tube 10. Supplies 32 are
conventional power supplies having an adjustable output voltage which may
be varied between approximately 0 and 5,000 voltage in response to control
signals supplied to line 34, such line being coupled to the control input
of the associated supply. The power supplies are capable of supplying
current in excess of 200 ma.
The positive output of power supplies 32 are each connected to separate
electrodes 23 which are disposed in the liquid buffer 22 of reservoirs 18.
The negative output of the supplies are connected to a single electrode 25
disposed in lower reservoir 24. The conductive buffer solutions serve to
electrically couple the outputs of the power supplies to the upper and
lower surfaces of gel 20 contained in the tubes.
As will be subsequently described in greater detail, DNA fragments, or
fragment from other varieties of nucleic acid such as RNA, having
radioactive labels, propagate down the length of tubes 10 through gel 20
by electrophoresis. Radiation detectors 28 are provided for each tube 12
for detecting the passage of the labeled fragments. Each time a beta
emission is detected in the region of the detector, a digital output pulse
is provided on output line 30.
Referring now to FIG. 3, detail of the construction of an exemplary
radiation detector 28 may be seen. Detectors 28 include a conventional
plastic phosphor scintillation crystal 31 which extends around the narrow
section 16 of each of tubes 10. Crystal 31 is in the general form of a
cube having a central aperture through which tube 10 extends. When a
labeled fragment in gel 20 passes through the aperture, the emitted beta
particles strike crystal 31 causing the crystal to scintillate. The
probability of detecting such randomly produced beta particles is
increased by increasing the dimension of the crystal along the length of
the tube. However, the increased height of the crystal reduces the
capability of the detector to resolve the position of the fragment when
the emission is detected. A compromise crystal height of approximately 0.5
cm has been found to be optimum for the present application.
Detectors 28 each further include a conventional photo-multiplier 33 which
is optically-coupled to crystal 31. Multiplier 33 detects the light bursts
produced within crystal 31 when the crystal scintillates and produces a
low level electrical output signal in response thereto. The signal is
amplified by a low-noise amplifier 36 having a gain of approximately 100
to 300. Amplifier 36 also preferably includes conventional filtering
circuitry which integrates the multiplier output signal for approximately
1000 nanoseconds and then differentiates for 100 nanoseconds.
The output of amplifier 36 is coupled to a level detector 38. Detector 38
includes a monostable or single shot circuit which produces a single
digital pulse on output line 30 when the output of amplifier 36 exceeds a
predetermined threshold level.
Because of the noise inherent in photo-multiplier 33 and amplifier 36, it
is possible that an output pulse will be produced on line 30 even in the
absence of a beta emission. Accordingly, an optional second
photo-multiplier circuit 37 may be provided which is also optically
coupled to crystal 31. In that event, level detector 38 is deleted. The
output of the second photo-multiplier 37 is coupled to a second low noise
amplifier 40, similar to amplifier 36 on line 44. The outputs of both
amplifiers 36 and 40 are connected to a conventional summing amplifier 42
which sums the two signals together and produces an output which is
coupled to a level detector 46, similar to detector 38. The threshold
level of detector 46 is selected such that a simultaneous output from both
amplifier 36 or amplifier 40 is required to produce a digital output pulse
on line 30. Thus, background noise, which is likely to cause one, but not
both, of amplifier 36 and 40 to produce an output, will not cause a pulse
to be produced on line 30'.
Details of the signal processing aspect of the subject apparatus are shown
in the block diagram of FIG. 4. A control microprocessor, represented by
block 48, is coupled to a main bus 50. A microprocessor manufactured by
Intel having the designation 8085 has been found suitable for the present
application although there are many other devices which could also be
used. Microprocessor 48 is under program control by a program stored in a
memory 52 also coupled to the bus. Memory 52 may be a Read Only Memory
(ROM) or an Erasable Programmable Read Only Memory (EPROM), both of which
are non-volatile.
The outputs of the four radiation detectors 28 (FIG. 2) on lines 30 are
coupled to the Set inputs of separate latch circuits 56. The outputs of
the latch circuits are connected to separate input ports 62 which
interface with bus 50. The Reset inputs of latches 56 are connected to a
common output port 54 which is also coupled to bus 50. Four output ports
64 are further coupled to bus 50 and provide separate control signals on
lines 34 for independently controlling the magnitude of the output voltage
of power supplies 32 (FIG. 2).
The outputs of latches 56 are further connected to the inputs of a
four-input OR gate 58. The output of gate 58 is connected to the 6.5
interrupt input of the Intel 8085 processor 48 by way of line 60. An
interrupt timer circuit, represented by block 70, produces a 7.5 interrupt
signal one line 72 for processor 48 by way of line 72.
Control processor 48 is further in communication with a microcomputer 68
through an interface circuit 66. As will be subsequently described in
greater detail, control microprocessor 48 is generally dedicated to
collecting data provided by the four radiation detectors with
microcomputer 68 serving to process and record such data.
Having described the overall construction of the subject sequencing
apparatus, operation of the apparatus will now be given. The sequencing of
DNA will be used as an example, it being understood that the subject
invention can be used to analyze the structure of other varieties of
nucleic acids, including RNA. DNA fragments are first prepared, as
previously set forth in connection with the discussion associated with
FIGS. 1A-1G. It should be noted that the chemical process for cleaving the
DNA strand is not actually as selective as previously described. For
example, although vial A will primarily contain fragments terminating with
A bases, there will also be a significant number of fragments terminating
in the other three bases. In addition, as will be subsequently explained,
it may be desirable to add a small quantity of labeled fragments
terminating with each of the four bases to each vial.
The contents of each of the four vials are then introduced into the top
layer of gel in respective ones of the four tubes 10. Because of the
potential produced across the length of the column of gel 20, the
fragments will propogate towards the bottom of the column in accordance
with the well known principles of electrophoresis. As previously
described, the rate of propogation is a function of the size of the
fragments, with the subgroups of smaller fragments moving at a greater
velocity than the subgroups of the larger fragments.
As the fragments propogate along the length of the tubes, the fragments
become separated into subgroups in accordance with the length of the
fragments. Since there are ten different fragment lengths (ten bases) in
the example of FIGS. 1A-1F, there will be ten separated subgroups of
fragments in each tube 10. The tube containing the contents of vial C, for
examples, will have two large subgroups of fragments (P32C and
P32CAAGAGATAC) and eight small subgroups. The number of fragments in each
subgroup and the P32 radioactive label are selected such that the large
subgroups produce intense radiation bands which emit approximately 200
beta particles per minute. Such emissions result in a detector output of
200 counts per minute (cpm). The small subgroups are selected to produce
weak radiation bands which generate approximately 50 cpm. It may be
necessary to add labeled fragments terminating in each of the ten bases to
each vial in order to enhance the weak radiation bands.
An alternative method of synchronization is to utilize chemical reactions
which produce the following four groups of fragments terminating in the
following bases: A+C+G; A+C+T; A+G+T and C+T+G. The four groups can be run
in separate columns. There are always three bands in each column to be
aligned and the fourth band can be aligned by analysis. The fragment
groups may be prepared by mixing the results of two or more known chemical
reactions in the event such groups cannot be directly produced.
It is important that the radiation bands be separated from one another a
sufficient distance for the detectors 28 to resolve the different bands. A
band separation of 1.0 cm has been found to be suitable when a detector
having a crystal height of 1.0 cm is used. The desired separation can be
achieved utilizing glass tube of relatively great length having a constant
cross-section where the intensity of the electric field is uniform
throughout the length of the gel column. However, it is preferable to use
tubes as depicted in FIG. 2 having a relatively wide upper section 12 and
a relatively narrow lower section 16 with an intermediate tapered section.
The intensity of the electric field in narrow section 16 is greater then
that of the wide section by approximately a factor of ten since the ratio
of the cross-sectional area of the wide section is ten times that of the
narrow section. Accordingly, the velocity of the fragments substantially
increase when the fragments pass from the wide section to the narrow
section, thereby increasing the subgroup separation a sufficient amount so
that the radiation bands may be resolved by the detectors. In addition,
the reduced cross-sectional area increases the probability of detecting a
beta particle emission since there is a smaller amount of gel which the
particles must penetrate before entering the crystal.
It is desirable to maximize the velocity of the fragments through the gel
in order to reduce the time required to complete the sequencing process.
However, if the velocity is too great, the probability that a radiation
band will not be detected as it passes through the detectors 28 is
increased. If the potential applied across the gel is adjusted to achieve
the desired velocity for the short fragments, the velocity of the larger
fragments will be substantially reduced. It is preferable to periodically
increase the potential during the sequencing process so as to maintain a
constant fragment velocity. A constant fragment velocity of approximately
1.0 cm per second through narrow section 16 has been found to be
acceptable.
The desired constant velocity is maintained by independently controlling
each of the four adjustable power supplies 32. In addition, the adjustable
power supplies permit the fragment subgroups in each tube to be
synchronized with one another. Synchronization is achieved when an intense
radiation band is detected in one column at approximately the same time
weak bands are detected in the other three columns. If the bands do not
occur substantially simultaneously, the potential is across the gels are
adjusted, as required, to resynchronize the bands.
The first step in carrying out the sequencing procedure is to first
introduce the fragments from the four vials into respective ones of the
glass tubes. The operator then enters initial setup information into
microcomputer 68 using a conventional keyboard (not depicted) as indicated
by block 90 of the FIG. 8 flow chart which illustrates the operation of
microcomputer 68. Such information includes, for example, the approximate
number of bases in the strand to be sequenced. Also, information regarding
the chemistry used in producing the fragments in the four vials may be
entered. In the example given, the chemistry for cleaving the DNA strands
generally produces fragments terminating in one of four bases. However,
other chemical procedures can be used to produce different fragment
groupings. By way of example, another chemical cleaving process commonly
used in conventional DNA sequencing produces a first group of fragments
terminating primarily in A bases and secondarily in G bases. A second
separate group of fragments is produced terminating primarily in G bases
and secondarily in A bases, with a third separate group of fragments being
produced which terminates in C bases and a fourth separate group of
fragments being produced terminating in both C and T bases. It can be seen
that sufficient information is provided in the four groups to uniquely
determine the type of base. By way of example, if a radiation band is
detected in a gel column containing the fourth group of fragments, but not
in the column containing the third group, it is known that the detected
fragments terminate in base T.
Once the setup procedure has been completed, a Start command is forwarded
from microcomputer 68 to control microprocessor 48 through interface 66,
as indicated by block 92 of the FIG. 8 flow chart. Microcomputer 68 is
also capable of issuing a Stop command and a Voltage Adjust Command, these
being the three primary microcomputer commands.
Referring now to the FIG. 5A flow chart which illustrates the operation of
control microprocessor 48, as represented by element 112 the
microprocessor periodically determines whether microcomputer 68 has issued
one of the three primary commands by way of interface 66. As indicated by
elements 114 and 118 of the flow chart, a determination is made as to
whether the command was either a Stop or a Start. Since a Start command
had issued, the microprocessor will then initialize the system for taking
data as indicated by block 120. The sequence will then return to element
112 at which time a determination will be made as to whether another
command from the microcomputer has issued. Since no command will have been
given, the program will proceed to element 124 where it is determined that
data from the detectors are in the process of being recorded.
Data from the detectors are accumulated over a time period dT, with the
period typically being on the order of 10 seconds. When a beta emission is
detected by one of the detectors, a digital pulse is produced on the
associated output line 30. The emission may be the result of background
radiation, an intense radiation band caused by the passage of a large
subgroup of labeled fragments past the detector or a weak radiation band
caused by the passage of a small subgroup of labeled fragments.
A digital pulse from any of the detectors causes the associated latch 56 to
set. The latch output causes the output of OR gate 58 to go high thereby
producing a 6.5 interrupt for control microprocessor 48. In addition, the
latch output is forwarded to the associated input port 62.
Referring now to FIG. 5B flow chart, the sequence for processing the 6.5
interrupts is depicted. When an interrupt is detected, the interrupt is
processed and additional interrupts are disabled. Interrupt processing
typically requires only approximately 100 microseconds, therefore, it is
unlikely that a significant number of beta emissions will be missed during
the processing.
As indicated by element 80 and block 82, when the interrupt is detected,
the status of each of the latches 56 is read through input ports 62.
Microprocessor 48 has internal memory locations associated with each of
the four detectors which accumulate the number of output pulses produced
by each detector during a time period dT. Depending upon which latch was
set, the identity of the particular detector which produced the output
pulse is then determined as indicated by block 86. The digital value
stored in memory location associated with the detector is incremented, as
represented by block 86, and interrupts are then enabled as indicated by
block 88.
Returning again to FIG. 5A flow chart, data continues to be taken
throughout the remainder of time period dT, as indicated by elements 124
and 126. Once the time period has elapsed, the count totals from the four
memory locations are loaded into buffer registers (not depicted), as shown
by block 130 and the interrupts are enabled, as represented by block 132.
Next, a command is sent to microcomputer 68 through interface 66
indicating that the four groups or channels of data are about to be
forwarded to the microcomputer. The four channels of data are then
forwarded to the microcomputer as represented by block 134. The program
then returns to element 112 and the sequence is repeated every dT time
period.
Control microprocessor 48 is implemented to provide an internal clock for
measuring time interval dT. Interrupt timer 70 (FIG. 4), which produces an
output signal approximately once every one millisecond, is coupled to the
7.5 interrupt input of microprocessor 48. As shown in the 7.5 interrupt
processing sequence of the FIG. 5C flowchart, when the microprocessor
detects a 7.5 interrupt, further interrupts are disabled. Next, as
indicated by element 74 and and block 76, the internal microprocessor
clock is incremented followed by the reenabling of interrupts as
represented by block 78.
Returning to FIG. 8, once the four channels of data have been received by
microcomputer 68 from the control microprocessor, the data associated with
each of the detectors are filtered as indicated by block 96. The purpose
of filtering is to detect the presence of intense and weak radiation bands
and to distinguish such bands from background radiation. Filtering also
serves to distinguish intense bands from weak bands.
Filtering is carried out utilizing digital filtering techniques. A location
filter is first used to filter the input data so as to provide a
relatively smooth curve having a peak which correspond to the time in
which a possible radiation band was detected. The input data are also
filtered by a rate filter to determine the radiation rate at the time the
band was detected. The radiation rate is used to distinguish between
radiation bands and background radiation and between intense and weak
radiation bands.
The digital filters are implemented utilizing look-up tables which are
stored in the microcomputer memory. Each table contains twenty different
filter values or constants which define the filter transfer
characteristics. The preferred twenty values stored for the location
filter, in normalized form, are plotted on the graph of FIG. 6, with the
points on the graph being connected together to form a transfer
characteristic curve 108. FIG. 7 shows the values stored in the rate
filter look-up table, which are connected together to form curve 108.
Filtering is accomplished by multiplying the twenty stored filter values
together with data taken for twenty consecutive time periods dT. The
multiplication is repeated each time a new set of data is forwarded to the
computer. By way of example, the data forwarded after the first time
interval are multiplied by the filter value plotted on the FIG. 6 graph at
dT=10, with the subsequent nineteen sets of data being simultaneously
multiplied with the value plotted at dT=9 through dT=-10. During the next
time interval, the input data are shifted one location so that the data of
the second time interval will be multiplied with the filter value of dT=10
and the most recently inputted data will be multiplied with the filter
value plotted at dT=-10.
The peak of the filter output data from the location filter corresponds to
the time in which a potential radiation band passed through the associated
detector. The inputted data are also processed by the rate filter in a
similar manner to determine the radiation rate at the time the potential
band passed the detector, as determined from the output of the location
filter.
Microcomputer 68 is provided with an internal clock so that the relative
times that the four channels of data are received from control
microprocessor 48 may be determined. Referring back to the FIG. 8 flow
chart, once the four channels of data for a particular time interval dT
have been filtered, the internal clock is incremented, as indicated by
block 98. The filtered data may then be displayed, if desired, on a CRT
display (not shown) as represented by block 100.
If the radiation rate at the time the potential radiation band was detected
is below a predetermined value, it is assumed that the radiation was
caused by background radiation rather than a radiation band. In the
present example, where intense and weak radiation bands nominally produce
50 cpm and 200 cpm, respectively, any radiation rate less than 20 cpm is
treated as background radiation. In that event, the microcomputer program
will loop back to block 94, to read further incoming data, as indicated by
element 102. If the radiation rate is greater than 20 cpm, but less than
100 cpm, it is assumed that a weak band was detected. If the measured rate
is greater than 100 cpm, it is assumed that an intense radiation band was
detected.
If a radiation band has been detected, the program will proceed to block
104, at which time a determination is made as to which base was detected.
In the present example, assuming that the system is synchronized, one of
the detectors will detect an intense band while the remaining three
detectors will detect weak radiation bands. Since the chemistry is such
that each tube primarily contains fragments terminating in one of the four
bases, the base associated with the column which produced the intense band
will be the base at that point in the sequence.
Synchronization is desirable because, among other things, the processing of
the data is simplified. For example, when the system is synchronized, it
is not necessary to record the actual times that radiation bands are
detected since the base sequence of the molecule will be identical to the
order in which the bands are detected. It would be possible to determine
the base sequence if the system were not synchronized, provided the
absolute time in which each of the bands is detected is recorded. Such
recordation would be performed automatically by microcomputer 68.
As previously noted, one of the purposes of the weak radiation bands is to
facilitate synchronization. If synchronization is not desired, the weak
bands may be ignored. If synchronization is to be achieved, an appropriate
Adjust Voltage command is sent to control mircoprocessor 48 to maintain
synchronization, as indicated by block 106.
The Adjust Voltage command includes data for independently controlling each
of the power supplies. Referring to the FIG. 5A flow chart, when control
microprocessor 48 detects that a microcomputer command has been sent, a
determination is made that the command is neither a Stop nor a Start
command, as indicated by elements 114 and 118. Accordingly, the program
will advance to block 122 where the command will be decoded as an Adjust
Voltage command. The decoded data for each power supply will then be
forwarded to the respective supply by way of output ports 64. By way of
example, if a weak band in one column is lagging the one intense band and
two weak bands of the other three columns, the power supply associated
with the lagging column will be increased slightly to increase the
velocity of the fragment. The magnitude of the increase in voltage will be
selected in accordance with well known principles of feedback control
system design.
The Voltage Adjust commands are also used to maintain the velocity of the
radiation bands at the desired 1.0 cm per second past the detectors. The
velocity may be determined by measuring the time intervals between the
detection of radiation bands and is be corrected by issuing appropriate
Voltage Adjust commands, also in accordance with principles of feedback
control system design.
The above-described sequencing process continues during subsequent time
intervals dT until the last subgroup of labeled fragments has been
detected. If no radiation bands are detected for a predetermined time
period, it is assumed that the sequence has been completed. Microcomputer
68 then issues a Stop command to control microprocessor 48 which, as
indicated by element 114 and block 116 of the FIG. 5A flow chart, cause
the system to be reset. The sequence data are preferably recorded, as it
is processed, in some form of non-volatile memory storage medium such as
floppy-disks or the like. The data can then be further analyzed without
the necessity of manually entering the data into the computer.
Thus, a novel apparatus and process for sequencing nucleic acids have been
disclosed. Although a preferred embodiment has been described in some
detail, it is to be understood that various changes can be made by those
skilled in the art without departing from the spirit and scope of the
subject invention as defined by the appended claims.
* * * * *
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