Computer system for controlling values of operational parameters during an operation

A computer system for controlling the parameters used during molecule transfer operations. The computer system includes a processor coupled to a memory. The memory stores instructions that are executed by the processor. The computer system controls the value of parameters that affect the characteristics of pulses delivered by a pulse generating circuit to a solution. The pulse generating unit delivers individual pulses during cycles. The cumulative pulse delivered during a specified number of cycles is a pulse output. The magnitude of a pulse output is determined by the number of cycles corresponding to the pulse output and the characteristics of the individual pulses delivered during the cycles. A pulse group is a series of pulse outputs. The computer system controls a molecule transfer operation by causing the pulse generation circuit to deliver one or more pulse groups to the solution. A user selects the shape and number of pulse groups to be delivered for an operation. The computer alters the operational parameters used by the pulse generation circuit to cause the pulse generation circuit to deliver pulse groups having the selected shapes.

FIELD OF THE INVENTION 
This invention relates to computer systems and more particularly, to a 
computer control system for controlling the operative parameters in effect 
during molecule transfer operations. 
BACKGROUND OF THE INVENTION 
A high-voltage molecule transfer system based on the prior art is described 
in U.S. Pat. No. 4,663,292 issued to Wong et. al on May 5, 1987, the 
contents of which are incorporated herein by reference. The Wong system 
generates a high voltage discharge through a solution of cells and 
biological macromolecules to cause biological macromolecule transfers and 
cell fusions. 
The Wong system has numerous controls for setting certain characteristics 
of the high-voltage discharge. Specifically, an amplitude control sets the 
amplitude of the high-voltage discharge. A burst time control sets the 
duration or burst time of the high-voltage discharge output. A cycle 
number control sets the number of cycles in the high voltage discharge 
output. A pulse control sets the number of pulses within each burst of the 
high voltage discharge output. The duration of the individual pulses may 
also be adjusted. 
Once the values for these voltage discharge characteristics have been set, 
a master trigger switch is activated to produce a chain of continuously 
discharging high-voltage pulses into a solution or suspension of cells and 
biological macromolecules. These high-voltage pulses contain the 
characteristics of the parameters (amplitude, burst time, number of 
cycles, number of pulses and duration of individual pulses) which were 
selected. These parameters may be readjusted for each experiment but are 
fixed during a particular experiment. Moreover, there is no provision for 
automatically providing timed gaps between groups of pulses. 
It has been discovered that the results of molecule transfer operations may 
be affected by varying the characteristics of pulses generated during an 
experiment. However, current molecule transfer systems do not provide a 
mechanism to vary the pulses during an experiment in a consistent, 
repeatable manner. Therefore, it is desirable to provide a mechanism for 
consistently and predictably varying one or more characteristics of an 
output pulse during molecule transfer and cell fusion operations. 
SUMMARY OF THE INVENTION 
According to an embodiment of the invention, an apparatus for performing a 
molecule transfer operation is provided. The apparatus includes a computer 
system, an electronic pulse generating circuit, and an electronic pulse 
delivery device. 
The computer system includes a memory, a processor, and a bus. The memory 
contains a series of instructions for controlling a plurality of 
parameters. The processor executes the series of instructions. Execution 
of the series of instructions causes the processor to generate signals 
indicative of the plurality of parameters. The bus couples the processor 
to the memory. 
The electronic pulse generating circuit is coupled to the computer system. 
The electronic pulse generating circuit receives the signals indicative of 
the plurality of parameters from the computer system. The electronic pulse 
generating circuit generates pulses having characteristics based on the 
plurality of parameters. The electronic pulse delivery device delivers the 
pulses to a solution. 
According to one aspect of the invention, the electronic pulse generating 
circuit generates the pulses in cycles. The electronic pulse generating 
circuit generates a number of individual pulses in each cycle. The 
individual pulses have a pulse duration and a pulse amplitude. The series 
of instructions typically includes instructions for controlling the 
amplitude of the pulses, instructions for controlling the distance between 
the electronic pulse delivery device and the solution, instructions for 
controlling the duration of the cycles, instructions for setting the 
number of pulses, instructions for setting the pulse duration, and 
instructions for setting the pulse amplitude. 
According to one aspect of the invention, a computer controlled method for 
performing a molecule transfer operation is provided. According to the 
method, a first pulse output is delivered to a solution. The first pulse 
output has a first magnitude determined by a plurality of operational 
parameters. A computer system modifies at least one of the plurality of 
operational parameters after delivering the first pulse output and prior 
to delivering a second pulse output. The second pulse output is then 
delivered to the solution. The second pulse output has a second magnitude 
determined by the plurality of operational parameters. 
According to an aspect of the invention, the step of delivering the first 
pulse output may include the step of delivering a first series of 
individual pulses to the solution during a first series of cycles, where 
the first pulse output is the cumulative pulse of the first series of 
individual pulses. The step of delivering the second pulse output includes 
the step of delivering a second series of individual pulses to the 
solution during a second series of cycles, where the second pulse output 
is the cumulative pulse of the second series of individual pulses. 
According to another aspect of the invention, the plurality of operational 
parameters may include a parameter which determines a number of individual 
pulses applied to the solution during a given cycle, a parameter which 
determines a number of cycles executed during delivery of pulse outputs, 
an amplitude of individual pulses applied to the solution, a parameter 
which determines a duration of individual pulses applied to the solution, 
and/or a parameter which determines a duration of cycles executed during 
delivery of pulse outputs. 
According to another aspect of the invention, the steps of delivering the 
first pulse output and delivering the second pulse output are performed by 
a pulse delivery mechanism positioned at a distance from the solution. The 
pulse delivery mechanism may be, for example, an electrode. The plurality 
of operational parameters includes a parameter which determines the 
distance between the pulse delivery mechanism and the solution. Modifying 
the plurality of operational parameters causes the computer system to 
change the parameter which determines the distance between the pulse 
delivery mechanism and the solution. 
According to another aspect of the invention, the method includes 
delivering a first pulse group and a second pulse group to the solution, 
where the first pulse output is a last pulse output in the first pulse 
group and the second pulse output is a first pulse output in the second 
pulse group. Thus, the step of causing the computer system to modify at 
least one of the plurality of operational parameters includes causing the 
computer system to modify the operational parameters after delivering the 
first pulse group and before delivering the second pulse group. 
According to an alternative aspect of the invention, both the first pulse 
output and the second pulse output belong to a single pulse group. Thus, 
the step of causing the computer system to modify at least one of the 
plurality of operational parameters includes causing the computer system 
to modify the operational parameters during the delivery of the pulse 
group. 
According to yet another aspect of the present invention, a method for 
performing a molecule transfer operation is provided. The method includes 
receiving input from a user indicating a selected pulse group shape, and 
delivering a pulse group having the selected pulse group shape to a 
solution. The pulse group is delivered to the solution by repeatedly 
performing the steps of (1) causing a computer system to set one or more 
parameters of a plurality of parameters based on the selected pulse group 
shape and (2) delivering a pulse output to the solution, where the pulse 
output has a magnitude determined by the plurality of parameters.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
A method and apparatus for performing a computer-controlled high-voltage 
molecule transfer and cell fusion operation is described. In the following 
description, for the purposes of explanation, numerous specific details 
are set forth in order to provide a thorough understanding of the present 
invention. It will be apparent, however, to one skilled in the art that 
the present invention may be practiced without these specific details. In 
other instances, well-known structures and devices are shown in block 
diagram form in order to avoid unnecessarily obscuring the present 
invention. 
Referring to FIG. 1, it illustrates a computer system 100 which may be used 
to implement the preferred embodiment of the present invention. Computer 
system 100 comprises a bus or other communication means 101 for 
communicating information, and a processor 102 coupled with bus 101 for 
processing information. System 100 further comprises a random access 
memory (RAM) or other dynamic storage device 104 (referred to as main 
memory), coupled to bus 101 for storing information and instructions to be 
executed by processor 102. Main memory 104 also may be used for storing 
temporary variables or other intermediate information during execution of 
instructions by processor 102. Computer system 100 may also comprise a 
read only memory (ROM) and/or other static storage device 106 coupled to 
bus 101 for storing static information and instructions for processor 102. 
Data storage device 107 is coupled to bus 101 for storing information and 
instructions. 
Furthermore, a data storage device 107 such as a magnetic disk or optical 
disk and its corresponding disk drive can be coupled to computer system 
100. Computer system 100 can also be coupled via bus 101 to a display 
device 121, such as a cathode ray tube (CRT), for displaying information 
to a computer user. An alphanumeric input device 122, including 
alphanumeric and other keys, is typically coupled to bus 101 for 
communicating information and command selections to processor 102. Another 
type of user input device is cursor control 123, such as a mouse, a 
trackball, or cursor direction keys for communicating direction 
information and command selections to processor 102 and for controlling 
cursor movement on display 121. This input device typically has two 
degrees of freedom in two axes, a first axis (e.g., x) and a second axis 
(e.g., y), which allows the device to specify positions in a plane. 
Alternatively, other input devices such as a stylus or pen can be used to 
interact with the display. A displayed object on a computer screen can be 
selected by using a stylus or pen to touch the displayed object. The 
computer detects the selection by implementing a touch sensitive screen. 
Similarly, a light pen and a light sensitive screen can be used for 
selecting a displayed object. Such devices may thus detect selection 
position and the selection as a single operation instead of the "point and 
click," as in a system incorporating a mouse or trackball. Stylus and pen 
based input devices as well as touch and light sensitive screens are well 
known in the art. Such a system may also lack a keyboard such as 122 
wherein all interface is provided via the stylus as a writing instrument 
(like a pen) and the written text is interpreted using optical character 
recognition (OCR) techniques. Also, alternatively, the processor may be a 
portion of a semiconductor chip which is coupled to a memory on the chip 
by a bus also on the chip; in this embodiment, the memory stores the 
instructions for varying the pulses as described herein. An example of 
this implementation would use a microcontroller having on chip memory, 
with the microcontroller's processing ALU being the processor. 
In the currently preferred embodiment, the present invention is related to 
the use of computer system 100 to control parameters during, in one 
embodiment, high-voltage biological macromolecule transfer operations. 
Therefore, computer system 100 is one component of a larger high-voltage 
molecule transfer system 120. In one embodiment, a biological molecule or 
a nonbiological molecule may be transferred into a cell using the present 
invention. This operation is refereed to as a molecule transfer operation. 
Various techniques have been developed for transferring biological 
macromolecules such as genes into cells. Biological macromolecules are 
defined as those molecules which cannot be readily diffused through cell 
membranes, such as DNA, RNA, protein, etc. Some examples of gene transfers 
are described in G. Scangos and F. Ruddle, "Mechanisms and applications of 
DNA-mediated gene transfer in mammalian cells--a review," 14 Gene 1 
(1981); and W. Anderson, "Prospects for Human Gene Therapy," 226 Science 
401 (Oct. 26, 1984). 
One method of transferring genes into cells uses electric field pulses. 
This method is based on the theory that electric pulses above a certain 
threshold field strength would induce alternation of cell membranes, 
resulting in the delivery of molecules into the cells. Such a method is 
described in T. Wong and E. Neumann, "Electric Field Mediated Gene 
Transfer," 107 Biochemical and Biophysical Research Communications 584 
(Jul. 30, 1982). 
High-voltage molecule transfer system 120 includes, in addition to computer 
system 100, an electronic pulse generating circuit 140, a probe or 
electrode 142 and a suspension receptacle 146. According to one 
embodiment, receptacle 146 is a hollow member having at one end a metallic 
grounding member 152. The grounding member 152, which is another 
electrode, may be, for example, a steel spherical electrode. According to 
an alternative embodiment an electronic pulse delivery (EPD) reaction 
chamber may be used and has in one embodiment, two, generally flat, 
electrodes which are both insulated electrically from the solution 
containing the cells and the macromolecules. These insulated electrodes 
create an electric field according to the voltages applied to the 
electrodes; because they are insulated from the solution, very low current 
flows through the solution. An example of a reaction chamber having these 
insulated electrodes is shown in FIG. 1b, in which electrodes 905 and 903 
are disposed opposite each other with a dish 901 between the electrodes. 
The dish 901 contains the solution and cells and molecules which are to be 
transferred into the cells. This dish 901 in this embodiment of FIG. 1b 
replaces the receptacle 146 in the embodiment of FIG. 1a; similarly, the 
electrodes 903 and 905 of this embodiment replace the electrodes 142 and 
152 of the embodiment shown in FIG. 1a. Various EPD reaction chambers are 
disclosed in U.S. patent application Ser. No. 08/337,862, entitled "Method 
and Apparatus for Gene Therapy", filed by Xi Zhao on Nov. 14, 1994, the 
contents of which are incorporated herein by reference. 
Suspension receptacle 146 contains a solution or suspension 148 of cells 
and biological macromolecules such as DNA, RNA or proteins or smaller 
biological molecules or nombiological molecules. System 120 is operable 
without the necessity of immersing electrode 142 into suspension 148. 
Rather, electrode 142 is positioned above the top surface of suspension 
148 in a noncontact fashion, e.g., 0.1 to 10 millimeters above the top 
surface. Typically, the solution is a pH buffered solution which includes 
the cells which are to be transformed and the macromolecules (e.g. DNA or 
RNA) which will be introduced into the cells according to a method of the 
invention. The solution is typically at a suitable, physiological pH and 
salinity. 
Prior to performing a molecule transfer operation, receptacle 146 and 
grounding electrode 152 are preferably sterilized. Receptacle 146 is then 
filled with a suspension 148 of cells and, in one embodiment, biological 
macromolecules. Electrode 142 is positioned above the top surface of 
suspension 148 in a non-contact fashion. The distance between electrode 
142 and the top surface of suspension 148, a distance D,-has an effect on 
the outcome of the experiment. For example, a small distance would enable 
the discharge of a greater amount of energy which in mm varies the 
membrane permeability of cells, permitting macromolecule transfers or cell 
fusions. The experimenter may vary this distance D to control the amount 
of discharging energy in order to optimize the efficiency of the 
biological macromolecule transfer or cell fusion. Various details 
concerning the transfer process are described in U.S. Pat. No. 4,849,355. 
ELECTRONIC PULSE DELIVERY 
During a molecule transfer operation, electrode 142 delivers high-voltage 
electronic pulses to suspension 148. In the alternative embodiment of FIG. 
1b, the electrodes 903 and 905 deliver, through an electromagnetic field, 
pulses to the solution in the dish 901. It will be appreciated that 
reference in this description to electrode 142 is for purposes of 
illustration, and that electrodes 903 and 905, with dish 901, may also be 
used in accordance with the present invention. The pulses delivered by 
electrode 142 have certain characteristics that will be described 
hereafter with reference to FIG. 2. The characteristics of the pulses 
delivered during an operation affect the outcome of the operation. 
Referring to FIG. 2, it illustrates various characteristics of the pulses 
delivered by electrode 142 or electrodes 903 and 905 during an operation. 
Specifically, pulses are delivered in cycles (Cyn, Cyn+1, . . . Cyn+m). 
Within a given cycle, individual pulses (P) typically have a uniform 
amplitude (A) and a uniform duration or pulse time (Tp). The duty cycle of 
a pulse is typically fifty percent. Therefore, power is applied to 
electrode 142 during only one half of the pulse time. Alternatively, each 
pulse may have a varying amplitude or the pulses may be in the form of 
alternating current with the frequency being varied. In one embodiment of 
the present invention the amplitude may be up to 30 kilovolts and as few 
as about 100 volts. 
A cycle has two components, an action time (T.sub.A) in which pulses are 
generated, and a relaxation time (T.sub.R) in which no pulses are 
generated. The action time of a cycle equals the pulse time of the 
individual pulses multiplied by the number of individual pulses (Np) 
generated during the cycle. For example, given a pulse duration of 62.5 
microseconds, a cycle with 64 pulses will have an action time of 0.04 
seconds. The length of the relaxation time of a cycle is equal to the 
duration or burst time of the cycle (T.sub.B) minus the duration of the 
action time. 
PULSE OUTPUTS 
A pulse output is the cumulative pulses delivered during one or more 
cycles. According to the invention, several pulse outputs are provided in 
sequence with timed gaps between the pulse outputs. The magnitude, in a 
broad sense, of a pulse output is the sum of the pulses generated during 
the cycles of the pulse output. Therefore, the magnitude of a pulse output 
is a function of the number of cycles (Nc) in the pulse output, the number 
of pulses within each cycle, the amplitude of the individual pulses in 
each cycle, the pulse time of the individual pulses in each cycle, the 
burst time of a cycle, and the distance between the electrode 142 and the 
suspension 148 during the delivery of the pulse output (or the distance 
between electrode 905 and disk 901). This relationship between the 
magnitude of a pulse output and the various parameters in effect during 
the delivery of the pulse output is illustrated by the expression: 
PO=F(Nc, Np, A, Tp, T.sub.B, D) where 
PO is the magnitude of a pulse output, 
Nc is the number of cycles in the pulse output, 
Np is the number of pulses within each cycle, 
A is the amplitude of the individual pulses in each cycle, 
Tp is the pulse time of the individual pulses in each cycle, 
T.sub.B is the burst time of a cycle, and 
D is the distance between the electrode and the solution. 
When the value of any one of the parameters (Nc, Np, A, Tp, T.sub.B, D) 
changes, the magnitude of the pulse output changes. 
PULSE GROUPS 
A pulse group is a sequentially delivered series of pulse outputs. 
Typically a sequence of pulse groups will be delivered and typically, 
according to the invention, a timed delay (having no pulses) between the 
pulse groups will be provided. An example of this timed delay is shown in 
FIG. 3a as delay 305. The length of the delay between pulse groups may be 
varied also as a parameter. As explained above, the magnitude (PO) of each 
pulse output in a pulse group is determined by the operational parameters 
that are in effect at the time the pulse output is delivered. Thus, if the 
parameters (Nc, Np, A, Tp, T.sub.B, D) remain constant during the delivery 
of a pulse group, then all of the pulse outputs in the pulse group will 
have the same magnitude. If one or more of the parameters is altered 
between the delivery of one pulse output and the next, then the pulse 
outputs will have different magnitudes. 
The characteristics of a pulse group include the number of pulse outputs 
within the pulse group (PULSE-QUANTITY) and the shape of the pulse group 
(GROUP-SHAPE). As shall be illustrated hereafter, the shape of a pulse 
group is determined by variations in the magnitude of the pulse outputs 
within the pulse group. 
MOLECULE TRANSFER OPERATIONS 
Molecule transfer operations are performed by causing electrode 142 to 
deliver one or more pulse groups to the suspension 148 solution (or by 
causing electrodes 903 and 905 to deliver pulses of an electromagnetic 
field to the solution in the dish 901). FIG. 3a is a graph illustrating a 
molecule transfer operation that includes the delivery Of three pulse 
groups 302, 304 and 306. Each pulse group is represented by a plurality of 
lines. Each line, such as line 308, represents a pulse output or a cycle 
of pulses; that is, each line in FIG. 3a represents a cycle, such as cycle 
Cyn of FIG. 2, of pulses where there are Np pulses in a cycle. 
In the graph, the x-axis represents time. Thus, in the illustrated 
operation, pulse group 302 is delivered before pulse group 304, and the 
cycle of pulses (a pulse output) represented by line 307 is delivered 
before the cycle of pulses (a pulse output) represented by line 308. 
The y-axis of the graph represents magnitude. Consequently, the length of 
each line illustrates the magnitude of each of the pulses in the cycle of 
pulses represented by the line. For example, line 308 of pulse group 302 
represents a pulse with a magnitude of 30. In one embodiment, a voltage of 
up to 30 kilovolts may be applied to the electrodes, such as electrodes 
903 and 905. 
In the operation represented in FIG. 3a, all three of the pulse groups have 
identical characteristics. Specifically, the magnitude of each line of 
each pulse output in each of the three pulse groups 302, 304 and 306 is 
constant. Consequently, all of the lines within each of the pulse groups 
have the same length. Therefore, the GROUP-SHAPE of pulse groups 302, 304 
and 306 is identical. In addition, each of pulse groups 302, 304 and 306 
have the same number of pulse outputs. Therefore, the PULSE-QUANTITY of 
pulse groups 302, 304 and 306 is identical. 
In general, different reactions may be caused by changing the number of 
pulse groups (GROUP-QUANTITY) delivered during an operation and/or 
changing the characteristics (GROUP-SHAPE and PULSE-QUANTITY) of the pulse 
groups delivered during the operation. According to an embodiment of the 
present invention, a computer system is used to change the value of one or 
more operational parameters between the delivery of pulse groups or 
between the delivery of pulse outputs. As explained above, a change in the 
value of certain operational parameters will affect the magnitude of pulse 
outputs and the magnitude of the pulses during a cycle. Therefore, by 
changing the value of parameters between pulse groups or between pulse 
outputs in a single operation, operations that involve non-identical pulse 
groups or non-identical pulse outputs may be performed. 
FIG. 3b illustrates an operation which involves multiple pulse groups 350, 
352, 354 and 356. Within each of the pulse groups, the magnitude of pulse 
outputs (PO) remains constant. However, the magnitude of pulse outputs of 
each pulse group differs from the magnitude of pulse outputs of the other 
pulse groups. The operation illustrated in FIG. 3b may be performed, for 
example, by causing a computer to change the value of the parameter that 
controls the amplitude of the individual pulses of each pulse group or the 
number of pulses per cycle (Np) between the delivery of each of the pulse 
groups. 
PULSE OUTPUT MAGNITUDE VARIATION WITHIN A PULSE GROUP 
As explained above, FIG. 3b illustrates an operation in which the magnitude 
of pulse outputs changes between pulse groups, but remains constant within 
each pulse group. According to another aspect of the invention, a computer 
system is used to alter the value of operation parameters during the 
delivery of pulse groups, as well as between the delivery of pulse groups. 
Varying the value of operational parameters during the delivery of a pulse 
group results in pulse groups that contain pulse outputs of differing 
magnitudes. The pattern of variation in the magnitude of pulse outputs 
within a pulse group dictates the GROUP-SHAPE of the pulse group. FIGS. 4a 
to 4k illustrate various pulse groups that can be delivered by a molecule 
transfer system that uses a computer to modify parameter values during the 
delivery of pulse groups. 
FIG. 4a is a graph of three pulse groups 402, 404 and 406. As with FIGS. 3a 
and 3b, each pulse group 402, 404 and 406 is represented by an array of 
lines. Each line represents a pulse output. The length of each line 
represents the magnitude of that pulse output represented by the line. In 
contrast to the pulse groups 302, 304 and 306 shown in FIG. 3a, the length 
of the lines in pulse groups 402, 404 and 406 varies over time. 
Specifically, each of pulse groups 402, 404 and 406 represents a series of 
pulse outputs in which the magnitude of pulse outputs increases for each 
subsequent pulse output within a pulse group. This may be achieved by, for 
example, increasing the amplitude of pulses within each new cycle of 
pulses within a pulse group. For example, pulses in the 8th cycle of 
pulses may each be at 5 kilovolts (Kv) and pulses in the 9th cycle of 
pulses may each be at 6 kilovolts. Alternatively, the pulses in the 8th 
cycle of pulses may steadily increase from 5 Kv to 6 kv and pulses in the 
9th cycle may steadily increase from 6 Kv to 7 Kv. Due to the increasing 
magnitude of the pulse outputs, the outcome of an operation that delivers 
pulse groups 402, 404 and 406 may differ from the outcome of the 
operations represented in FIG. 3a and FIG. 3b. 
While pulse groups 402, 404 and 406 are similar in that they all represent 
pulse groups in which the magnitude of pulse outputs increases over time, 
they differ from each other in other aspects. For example, the magnitude 
of the pulse outputs in pulse group 404 increases at a faster rate than 
the magnitude of the pulse outputs in pulse group 402. Therefore, pulse 
group 402 has a slightly different GROUP-SHAPE than pulse group 402. In 
contrast, the magnitude of the pulse outputs in pulse group 404 increases 
at the same rate as it does for pulse group 406, but the number of pulse 
outputs in pulse group 406 is less than the number of pulse outputs of 
pulse group 404. Thus, pulse groups 404 and 406 have the same GROUP-SHAPE, 
but have different PULSE-QUANTITIES. Each of these differences may affect 
the outcome of an operation. Thus, an operation in which a pulse group 
with one shape is delivered may yield different results than an operation 
in which a pulse group with a different shape is delivered, even if the 
shape of the two pulse groups have some characteristics in common. 
FIGS. 4b through 4k illustrate pulse groups that have a variety of shapes. 
Specifically, FIG. 4b illustrates three pulse groups 410, 412 and 414 in 
which the magnitude of pulse outputs decreases at a constant rate. The 
rate at which the magnitude of pulse outputs decreases is less for pulse 
group 410 than it is for pulse groups 412 and 414. Pulse group 414 has the 
same rate at which the magnitude of pulse outputs decreases as pulse group 
412, but has a lower number of pulse outputs. 
FIG. 4c illustrates three pulse groups 418, 420 and 422 in which the 
magnitude of pulse outputs increases at a non-constant rate. Pulse group 
418 has a lower number of pulse outputs than pulse group 420 and a higher 
number of pulse outputs than pulse group 422. FIG. 4d illustrates three 
pulse groups 424, 426 and 428 in which the magnitude of pulse outputs 
decreases at a non-constant rate. Pulse group 424 has a lower number of 
pulse outputs than pulse group 426, and a higher number of pulse outputs 
than pulse group 428. 
FIG. 4e illustrates three pulse groups 430, 432 and 434 in which the 
magnitude of pulse outputs increases at a constant rate for the first half 
of pulse outputs in each pulse group, and decreases at a constant rate for 
the second half of pulse outputs in each pulse group. Pulse group 432 has 
higher rates of magnitude of pulse outputs increases and decreases than 
pulse groups 430 and 434. 
FIG. 4f illustrates three pulse groups 436, 438 and 440 in which the 
magnitude of pulse outputs increases at a non-constant rate for the first 
half of pulse outputs in each pulse group, and decreases at a non-constant 
rate for the second half of pulse outputs in each pulse group. 
FIG. 4g illustrates three pulse groups 442, 444 and 446 in which the 
magnitude of pulse outputs decreases at a non-constant rate for the first 
half of pulse outputs in each pulse group, and increases at a non-constant 
rate for the second half of pulse outputs in each pulse group. 
Similar to the pulse groups illustrated in FIG. 4f, the magnitude of pulse 
outputs of the three pulse groups 448, 450 and 452 of FIG. 4h increases at 
a non-constant rate for the first half of pulse outputs in each pulse 
group, and decreases at a non-constant rate for the second half of pulse 
outputs in each pulse group. The difference between the overall shape of 
pulse groups 436, 438 and 440 of FIG. 4f and pulse groups 448, 450 and 452 
of FIG. 4h is that the rate at which the magnitude of pulse outputs 
increases for the pulse groups of FIG. 4f increases over time, while the 
rate at which the magnitude of pulse outputs increases for the pulse 
groups of FIG. 4h decreases over time. Similarly, the rate at which the 
magnitude of pulse outputs decreases for the pulse groups of FIG. 4f 
decreases over time, while the rate at which the magnitude of pulse 
outputs decreases for the pulse groups of FIG. 4h increases over time. 
Pulse group 452 has a higher number of pulse outputs than pulse group 448. 
Similar to the pulse groups illustrated in FIG. 4f and 4h, the magnitude of 
pulse outputs of the three pulse groups 454, 456 and 458 of FIG. 4i 
increases at a non-constant rate for the first half of pulse outputs in 
each pulse group, and decreases at a non-constant rate for the second half 
of pulse outputs in each pulse group. However, the rate at which the 
magnitude of pulse outputs increases for the pulse groups in FIG. 4i 
decreases over time, and the rate at which the magnitude of pulse outputs 
decreases for the pulse groups in FIG. 4i also decreases over time. Pulse 
group 454 has a lower number of pulse outputs than pulse group 456. 
FIG. 4j illustrates a pulse group 460 in which the magnitude of pulse 
outputs of each pulse output is randomly determined. Note that there are 
no timed delays between groups of pulse outputs. 
FIG. 4k illustrates how pulse groups with differing GROUP-SHAPES may be 
combined in a sequence to perform a molecule transfer operation. 
Specifically, the cell transfer operation illustrated in FIG. 4j involves 
four pulse groups 462, 464, 466 and 468 of pulse outputs. Each pulse group 
has a fundamentally different shape. The magnitude of pulse outputs of 
pulse group 462 increases at a constant rate for the first half of pulse 
outputs, and decreases at a constant rate for the second half of pulse 
outputs. The magnitude of pulse outputs of pulse group 464 decreases at a 
non-constant rate for the first half of pulse outputs, and decreases at a 
non-constant rate for the second half of pulse outputs. The magnitude of 
pulse outputs of pulse group 466 increases at a non-constant rate for the 
first half of pulse groups, and decreases at a non-constant rate for the 
second half of pulse outputs. The magnitude of pulse outputs of pulse 
group 468 increases at a constant rate. 
AUTOMATED AMETER CONTROL 
As explained above, the delivery of pulse groups with different 
characteristics will yield different molecule transfer results. However, 
in prior art systems, the parameters that affect the characteristics of 
pulse groups were determined by human-operated controls. Due to the timing 
required for the delivery of pulse groups, it was virtually impossible to 
alter the pulse group characteristics with any degree of accuracy. 
Consequently, operations typically involved the delivery of one group of 
cycles of identically-shaped pulses with no timed delays between groups of 
pulses. 
To make possible the performance of accurate and repeatable operations that 
involve the delivery of non-identical pulse groups, the present invention 
controls the parameters that affect the magnitude of pulse outputs in 
pulse groups with a computer system. As was explained with reference to 
FIG. 1, the computer system 100 communicates with electronic pulse 
generating circuit 140 to cause electronic pulse generating circuit 140 to 
perform specified cell transfer operations having a specified number of 
pulse groups, where each pulse group has a specified shape and a specified 
number of pulse outputs. One embodiment of the system used to perform such 
operations shall now be described in greater detail with reference to FIG. 
5. 
Referring to FIG. 5, it illustrates bus connector 130 and electronic pulse 
generating circuit 140 in greater detail. Bus connector 130 is connected 
to bus 101 through which it receives control information from processor 
102. Such control information includes data indicating the values for 
parameters that affect the magnitude and timing of pulse outputs delivered 
by electronic pulse generating circuit 140. 
BUS CONNECTOR 
Bus connector generally includes an address selection circuit 502, a data 
buffer 504, a programmable pulse generator 506, and a digital-to-analog 
conversion circuit 508. Address selection circuit 502 allows a user to 
specify an address. Computer system 100 controls electronic pulse 
generating circuit 140 by sending control information to the selected 
address over bus 101. Data buffer 504 temporarily stores the control 
information received from computer system 100 over bus 101. 
Programmable pulse generator 506 generates a waveform control signal based 
on control information received over bus 101. The waveform control signal 
is used to control the waveform of the pulses generated by electronic 
pulse generating circuit 140. Such a pulse, having a duration Tp, is 
generated using an oscillating circuit and a programmable counter. In a 
preferred embodiment, Tp is fixed at 62.5 microseconds. This duration is 
merely exemplary, and the present invention is not limited to that value. 
The resultant waveform control signal that appears at the output of 
programmable pulse generator 506 is a signal that specifies the 
characteristics of parameters T.sub.B, Cy, Np, Tp, and the group number 
(e.g. group 302 versus group 304), the timed delay gap between groups 
(e.g. the time of delay for delay 305), and the group shape. These 
parameters were produced by programmable pulse generator 506 based on the 
control data received from computer system 100. 
Digital-m-analog conversion circuit 508 generates an analog voltage control 
signal based on control information received from computer system 100. The 
voltage control signal is sent to electronic pulse generating circuit 140 
and controls the voltage of the signal generated by electronic pulse 
generating circuit 140. In one embodiment, bus connector 130 may be 
implemented as shown in FIG. 6 where the illustrated computer chips may be 
the items identified in Table 1. 
TABLE 1 
______________________________________ 
Circuit 602 
Part number 74LS138 generally available from National 
Semiconductor Corporation. 
Circuit 604 
Part number 74LS138 generally available from National 
Semiconductor Corporation. 
Circuit 606 
Part number 74LS682 generally available from National 
Semiconductor Corporation. 
Circuit 608 
Part number 74LS245 generally available from National 
Semiconductor Corporation. 
Circuit 610 
Part number Intel 8253 generally available from Intel. 
Circuit 612 
Part number 74LS273 generally available from National 
Semiconductor Corporation. 
Circuit 614 
Part number 74LS373 generally available from National 
Semiconductor Corporation. 
Circuit 616 
Part number 74LS273 generally available from National 
Semiconductor Corporation. 
Circuit 618 
Part number 74LS273 generally available from National 
Semiconductor Corporation. 
Circuit 620 
Part number 74LS244 generally available from National 
Semiconductor Corporation. 
Circuit 622 
Part number 74LS244 generally available from National 
Semiconductor Corporation. 
Circuit 624 
Part number 74LS244 generally available from National 
Semiconductor Corporation. 
Circuit 626 
Part number 74LS93 generally available from National 
Semiconductor Corporation. 
Circuit 628 
Part number 74LS390 generally available from National 
Semiconductor Corporation. 
Circuit 630 
Part number 74LS390 generally available from National 
Semiconductor Corporation. 
Circuit 632 
Part number AD7545 generally available from National 
Semiconductor Corporation. 
______________________________________ 
Referring to FIG. 6, address selection circuit 502 includes a plurality of 
switches 654 for specifying an address. The control signals on bus 101 
that are sent to the address specified by switches 654 are latched into 
data buffer 504. Data buffer 504 includes a circuit 608 that latches the 
control signals into buffers provided by circuits 612, 614, 616, 618, 620, 
622 and 624. The control signals specify parameters for a waveform control 
signal generated by programmable pulse generator 506 and for a voltage 
control signal generated by digital-to-analog conversion circuit 508. 
Programmable pulse generator 506 includes an oscillator circuit 656 for 
generating an oscillating signal. Circuits 626, 628 and 630 transmit a 
clock signal at a predetermined frequency based on the oscillating signal. 
The clock signal is sent to a programmable counter 610 over line 658. The 
clock signal drives programmable counter 610 to generate a waveform 
control signal that is ultimately output on line 652. 
The characteristics of the waveform control signal generated by 
programmable counter 610 are determined by how programmable counter 610 is 
currently programmed. Programmable counter 610 is programmed by control 
signals from bus 101 that are sent through latch circuit 608 to the data 
inputs of programmable counter 610. These control signals are initiated by 
the processor 102 of computer system 100 while executing software designed 
to drive electric pulse generating circuit 140. 
Digital-to-analog conversion circuit 508 receives a control signal on the 
data inputs of circuit 632 and generates an analog signal on line 650 with 
a voltage that corresponds to the value of the digital control signal. As 
shall be explained hereafter, the voltage of the signal generated by 
digital-to-analog conversion circuit 508 ultimately determines the voltage 
(or amplitude A) of the pulses discharged by electrode 142 or electrodes 
903 and 905. Therefore, the signal produced by digital-to-analog 
conversion circuit 508 on line 650 is hereafter referred to as the voltage 
control signal. 
POWER SUPPLY UNIT 
Referring again to FIG. 5, electronic pulse generating circuit 140 includes 
a power supply unit 510 and a pulse generating unit 512. Power supply unit 
510 includes a voltage control signal amplifier 514, a lower voltage power 
supply 516 and a high voltage power supply 518. High voltage power supply 
518 converts a high voltage alternating current to a high voltage direct 
current. In an implemented embodiment of the invention, high voltage power 
supply 518 converts a standard 120 V alternating current to a 27 V direct 
current. Lower voltage power supply unit 516 receives the high voltage DC 
signal from the high voltage power supply 518 and converts the signal to 
produce signals with lower voltages. In an implemented embodiment, lower 
voltage power supply 516 produces a 5 V signal and a 12 V signal based on 
the 27 V signal produced by high voltage power supply 518. 
Voltage control signal amplifier 514 amplifies the voltage control signal 
generated by digital-to-analog conversion circuit 508. In one embodiment, 
the analog reference signal generated by digital-to-analog conversion 
circuit 508 fluctuates between zero and five volts. Voltage control signal 
amplifier 514 amplifies the analog reference signal to produce an 
amplified reference signal that fluctuates between zero and twenty-four 
volts. FIG. 7 illustrates one implementation of power supply unit 510. 
Referring now to FIG. 7, a transformer 704 transforms a standard 120 V AC 
signal to a lower voltage (e.g. 28 V) higher current signal which is 
applied to a bridge circuit 706. Bridge circuit 706 generates a DC signal 
from the lower voltage AC signal and transmits the DC signal through a 
regulator 710 to a line 708. In the illustrated embodiment, the resulting 
DC signal on line 708 is at 27 V. The 27 V DC signal on line 708 is passed 
through a voltage converter 700 (e.g. a resistor network) to produce a 12 
V DC signal on a line 712. The 12 V DC signal on line 712 is passed 
through a second voltage converter 702 to produce a 5 V DC signal on a 
line 714. 
The 27 V DC signal is also sent to voltage control signal amplifier 514 to 
provide the power to amplify the voltage control signal generated by 
digital-to-analog conversion circuit 508 over line 650. The voltage 
control signal generated by the digits-to-analog conversion circuit 508 
has a voltage level between zero and five volts. The circuits within 
voltage control signal amplifier 514 amplify the power of the voltage 
control signal so that the resulting high voltage control signal generated 
on line 716 has substantially the same characteristics as the voltage 
control signal on line 650, but has a higher current and a voltage level 
that falls between zero and twenty-seven volts. 
PULSE GENERATION UNIT 
Pulse generation unit 512 generates a high-voltage discharge output capable 
of enabling efficacious biological macromolecule or non-biological 
molecule transfers or cell fusions based on the waveform control signal 
and the voltage control signal. Pulse generation unit 512 includes a 
high-voltage output 520, a voltage stabilizer 522, a first level pulse 
transformer 524, an input driver 526 and a power voltage range selector 
528. Pulse generation unit 512 is shown in greater detail in FIG. 8. 
Referring to FIG. 8 which shows an embodiment of a pulse generation unit, 
pulse generation unit 512 comprises a first level pulse transformer 524 
for transforming the waveform control signal on line 652 from the 
generator 506 into an amplified waveform control signal. When the waveform 
control signal on line 652 goes low, current flows from line 712, through 
transformer 524 to ground through input driver 526 over line 830. 
When the waveform control signal on line 652 goes high, the circuit from 
line 712 to line 830 is broken. However, due to capacitor 832, current 
flows back through transformer 524 to line 712. Consequently, the signal 
applied to transformer 524 has a current that alternates based on the 
waveform control signal on line 652. 
An input driver protection circuit 838 is used to prevent the back 
electromagnetic force from damaging input driver 526 during the "turn off" 
period. The "turn off" period is defined as that portion of the pulse 
duration P which falls from amplitude A to zero. 
In one embodiment the waveform control signal on line 652 may specify the 
characteristics of parameters such as pulse duration Tp, number of pulses 
Np, duration of burst T.sub.B, and the cycle number Cy, the number of 
cycles in a pulse output Nc, the group number, the timed delay between 
groups, and the group shape. The amplified waveform control signal, at the 
output of first level pulse transformer 524, on line 844 (FIG. 8) is a 
pulse train representing the parameters T.sub.B, Cy, Np, Tp, Nc, and Group 
Quantity. 
Power and voltage range selector 528 determines the power level of the 
amplified waveform control signal. When switch 842 is open, the current 
flow between line 844 and ground through transformer 524 is limited by the 
resistance of resistor 842. When switch 840 is closed, the resistance 
between line 844 and ground is decreased based on the resistance of 
resistor 846. By affecting the amount of current that flows between line 
844 and ground, switch 840 affects the voltage level of the resulting 
amplified waveform control signal. 
High-voltage output 520 includes a modulation circuit 804 for modulating 
the relatively low-voltage, direct-current voltage control signal on line 
716. Modulation circuit 804, a conventional transistor in the preferred 
embodiment, employs the amplified waveform control signal as its base 
voltage. The output of modulation circuit 804, at its collector, is a 
modulating waveform control signal. The modulating waveform control signal 
continues to contain the characteristics of parameters T.sub.B, Cy, Np and 
Tp, Nc, and Group Quantity. It will be appreciated that Cy represents the 
PULSE-QUANTITY defined above. Modulation circuit 804, therefore, uses the 
modulating waveform control signal to modulate the direct-current voltage 
control signal into the modulated system voltage signal. The modulated 
system voltage signal, (appearing on lines 520a and 520b which are coupled 
to electrodes, such as electrodes 903 and 905) thus, contains (over time 
as a waveform) the characteristics of parameters A, T.sub.B, Np, Tp, Cy, 
Nc, Group Quantity and Group Shape. This signal will also reflect the 
timed delay between the pulse groups, as specified by the software on the 
computer. 
A snubber circuit 802 is provided to control the operation of modulation 
circuit 804. Snubber circuit 802, a capacitive-resistive network circuit 
in the preferred embodiment, is capable of generating a negative voltage 
potential to "turn off" modulation circuit 804 as fast as possible during 
the "turn off" periods of the pulses of first level pulse transformer 524. 
Three modulation protection circuits are also provided: modulation 
protection circuit 806, and snubber circuit 808. Snubber circuit 808 is a 
capacitive-inductive network circuit adapted to reduce the power 
dissipation of modulating modulation circuit 804 during the "turn off" 
period. Modulation protection circuit 806 is used to prevent the back 
electromagnetic force from damaging modulation circuit 804. 
Pulse generation unit 512 also comprises an output transformer circuit 810 
for transforming the modulated system voltage into the high-voltage 
discharge output. The high-voltage discharge output appearing at outputs 
520a and 520b and, containing the characteristics of parameters A, 
T.sub.B, Cy, Np, Tp, Nc and GROUP QUANTITY and GROUP SHAPE, is a chain of 
continuously discharging pulses. The voltage level of the pulses is 
determined by the 0-24 V voltage control signal on line 716 that is used 
to drive output transformer circuit 810. 
In the preferred embodiment, processor 102 executes an application that 
cause user interface controls to be generated on display 121. The user 
interface controls allow a user to specify an operation by selecting the 
number of pulse groups for the operation and the characteristics of each 
of the pulse groups and the timed delay gap between the pulse groups. As 
mentioned above, the characteristics of pulse groups include GROUP-SHAPE 
and PULSE-QUANTITY. In one embodiment, the controls allow a user to select 
the parameters that will be altered to vary the magnitude of pulse outputs 
during an operation. The selected parameters may include one or more of 
the parameters that affect pulse output magnitude (No, Np, A, Tp, T.sub.B 
and D). In addition, the GROUP SHAPE, GROUP-QUANTITY, the timed delay gap, 
and the number of cycles (Cy) in a pulse group (PULSE-QUANTITY) may be 
specified by the user in order to select a particular type of molecule 
transfer operation. In one embodiment, these various parameters for a 
particular molecule transfer may be stored on a computer readable medium 
(e.g. device 107). The user may from a visual display listing various 
different molecule transfers, select a particular one (the desired 
molecule transfer) and the computer can retrieve the parameters for the 
desired transfer and provide these parameters to the bus connector 130 
which then causes the electronic pulse generating circuit to deliver the 
selected pulses to the solution in the reactor chamber, such as the 
chamber which includes dish 901 and electrodes 903 and 905. 
Once the information entered by the user has been entered, the application 
determines the data that must be sent to bus connector 130 perform the 
specified operation. During the operation, the application sends the data 
over bus 101 to connector 130 to control the characteristics of the pulses 
being generated by electronic pulse generating circuit 140. The 
transmission of the parameter data is timed to deliver the number and type 
of pulse groups that were specified by the user. 
According to one embodiment of the invention, the distance D between the 
electrode 142 and the solution may also be selected as a parameter to vary 
during an operation. In this embodiment, electrode 142 (or electrode 905) 
is connected to a positioning mechanism (not shown) controlled by computer 
system 100. During an operation, the application transmits a control 
signal to the positioning mechanism to vary the distance D. The control 
signal is timed so as to deliver the pulse groups specified by the user. 
While specific embodiments of the present invention have been described, 
various modifications and substitutions will become apparent to one 
skilled in the art by this disclosure. Such modifications and 
substitutions are within the scope of the present invention, and are 
intended to be covered by the following claims.