Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1998-09-15
2003-10-14
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S283100, C435S007100, C204S450000, C204S600000, C204S166000, C204S298070, C204S166000, C209S155000, C209S156000, C422S051000, C422S051000, C436S527000, C436S164000, C436S149000
Reexamination Certificate
active
06632652
ABSTRACT:
BACKGROUND
1. The Field of the Invention
The invention relates to apparatus and methods for fractionating microstructures such as free cells, viruses, macromolecules, or minute particles. More particularly, the present invention relates to apparatus and methods for sorting such microstructures in suspension in a fluid medium, and if desired, for simultaneously viewing individual of those microstructures during the process.
2. Background Art
The sizing, separation, and study of microstructures such as free cells, viruses, macromolecules, and minute particles, are important tools in molecular biology. For example, this fractionation process when applied to DNA molecules is useful in the study of genes and ultimately in planning and the implementation of genetic engineering processes. The fractionation of larger microstructures, such as mammalian cells, promises to afford cell biologists new insight into the functioning of these basic building blocks of living creatures. One method for estimating the size of small DNA molecules is the process of gel electrophoresis.
In gel electrophoresis an agarose gel is spread in a thin layer and allowed to harden into a firm composition. The composition comprises a fine network of fibers retaining therewithin a liquid medium, such as water. The fibers of the agarose gel cross and interact with each other to form a lattice of pores through which molecules smaller than the pores may migrate in the liquid retained in the composition. The size of the pores in the lattice is determined generally by the concentration of the gel used.
Slots are cast in one end of the gel after the gel is hardened, and DNA molecules are placed into the slots. A weak electric field of typically 1-10 volts per centimeter is then generated in the gel by placing the positive pole of an electric power source in one end of the gel and the negative pole of the power source in the opposite end.
In a free solution, the mobility of a DNA molecule is independent of the length of the molecule or of the size of the applied electric field. In a hindered environment, however, aside from the structure of the hindered environment, the mobility of a molecule becomes a function of the length of the molecule and the intensity of the electric field.
The gels used in gel electrophoresis is just such a hindered environment. Molecules are hindered in their migration through the liquid medium in the gel by the lattice structure formed of the fibers in the gel. The molecules nevertheless when inducted by the electric field, move through the gel by migrating through the pores of the lattice structure. Smaller molecules are able to pass through the pores more easily and thus more quickly than are larger molecules. Thus, smaller molecules advance a greater distance through the gel composition in a given amount of time than do larger molecules. The smaller molecules thereby become separated from the larger molecules in the process. In this manner DNA fractionation occurs.
The process has several inherent limitations, however. For example, the pore size in the lattice of gels cannot be accurately measured or depicted. Therefore, the lengths of the molecules migrating through the lattice cannot be accurately measured. It has also been found that DNA molecules larger than 20 megabasepairs in length cannot be accurately fractionated in gels. Apparently, the pore size in the lattice of such materials cannot be increased to permit the fractionation of larger molecules, much less even larger particles, viruses, or free cells.
Further, the lattice structure formed when a gel hardens is not predictable. It is not possible to predict the configuration into which the lattice structure will form or how the pores therein will be positioned, sized, or shaped. The resulting lattice structure is different each time the process is carried out. Therefore, controls and the critical scientific criteria of repeatability cannot be established.
Gel electrophoresis experiments cannot be exactly duplicated in order to predictably repeat previous data. Even if the exact lattice structures formed in one experiment were determinable, the structure could still not be reproduced. Each experiment is different, and the scientific method is seriously slowed.
Also, the lattice structure of a gel is limited to whatever the gel will naturally produce. The general size of the pores can be dictated to a degree by varying the concentration of the gel, but the positioning of the pores and the overall lattice structure cannot be determined or designed. Distinctive lattice structures tailored to specific purposes cannot be created in a gel.
Further, because the lattice structure arrived at depends upon the conditions under which hardening of the gel occurs, the lattice structure even in a single composition need not be uniform throughout.
Another shortcoming of gel electrophoresis is caused by the fact that a gel can only be disposed in a layer that is relatively thick compared to the pores in its lattice structure, or correspondingly to the size of the DNA molecules to be fractionated. Thus, the DNA molecules pass through a gel in several superimposed and intertwined layers. Individual DNA molecules cannot be observed separately from the entire group. Even the most thinly spread gel is too thick to allow an individual DNA molecule moving through the gel to be spatially tracked or isolated from the group of DNA molecules.
The diffusion of long polymers in complex environments where the mobility of the polymer is greatly perturbed is both a challenging statistical physics problem and a problem of great importance in the biological sciences. The length fractionation of charged polymers, such as DNA in gels, is a primary tool of molecular biology. One of the main stumbling blocks to understanding quantitatively the physical principles behind the length-dependent mobility of long polymers in complex environments has, however, been the ill-characterized nature of the hindering environment, the gel.
A known sorting apparatus
20
is illustrated in FIG.
1
. Sorting apparatus
20
has utility in fractionating and optionally for simultaneously viewing microstructures, such as free cells, macromolecules, and minute particles in a fluid medium, and doing so as desired in essentially a single layer. Sorting apparatus
20
is comprised of a substrate
22
having a shallow receptacle
24
located on a side
26
thereof. In the embodiment shown, receptacle
24
is recessed in side
26
of substrate
22
, although other structures for producing a recess such as receptacle
24
would be workable in the context of the present invention.
Receptacle
24
includes a floor
28
shown to better advantage in
FIG. 2
as being bounded by a pair of upstanding opposing side walls
30
,
31
and a first end
32
and a second end
34
. The height of side walls
30
,
31
define a depth of receptacle
24
. The depth of receptacle
24
is commensurate with the size of the microstructures to be sorted in sorting apparatus
20
. The depth of receptacle
24
is specifically tailored to cause those microstructures in a fluid medium in receptacle
24
to form essentially a single layer. Thus, when the microstructures are caused to migrate in the fluid medium through receptacle
24
, the microstructures do so in essentially the single layer. The migration of the microstructures occurs in a migration direction indicated by arrow M defined relative to sorting apparatus
20
.
Substrate
22
may be comprised of any type material which can be photolithographically processed. Silicon is preferred, however other materials, such as quartz and sapphire can also be used.
In accordance with one aspect of a known sorting apparatus, such as sorting apparatus
20
, capping means are provided for covering receptacle
24
intermediate first end
32
and second end
34
thereof and for affording visual observation of the migration of the microstructures within receptacle
24
. As shown by way of example in
FIG. 1
, a coverslip
36
extends across receptacle
24
in substrate
22
from on
Austin Robert H.
Carlson Robert H.
Miles & Stockbridge P.C.
Princeton University
Taylor Janell E.
LandOfFree
Reversibly sealable microstructure sorting devices does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Reversibly sealable microstructure sorting devices, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Reversibly sealable microstructure sorting devices will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3146614