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BACKGROUND OF THE INVENTION
The general field of the invention is polymer characterization.
Rapid, reliable, and inexpensive characterization of polymers, particularly
nucleic acids, has become increasingly important. One notable project,
known as the Human Genome Project, has as its goal sequencing the entire
human genome, over three billion nucleotides.
Typical current nucleic acid sequencing methods depend either on chemical
reactions that yield multiple length DNA strands cleaved at specific
bases, or on enzymatic reactions that yield multiple length DNA strands
terminated at specific bases. In each of these methods, the resulting DNA
strands of differing length are then separated from each other and
identified in strand length order. The chemical or enzymatic reactions, as
well as the technology for separating and identifying the different length
strands, usually involve tedious, repetitive work. A method that reduces
the time and effort required would represent a highly significant advance
in biotechnology.
SUMMARY OF THE INVENTION
We have discovered a method for rapid, easy characterization of individual
polymer molecules, for example polymer size or sequence determination.
Individual molecules in a population may be characterized in rapid
succession.
Stated generally, the invention features a method for evaluating a polymer
molecule which includes linearly connected (sequential) monomer residues.
Two separate pools of liquid-containing medium and an interface between
the pools are provided. The interface between the pools is capable of
interacting sequentially with the individual monomer residues of a single
polymer present in one of the pools. Interface-dependent measurements are
continued over time, as individual monomer residues of a single polymer
interact sequentially with the interface, yielding data suitable to infer
a monomer-dependent characteristic of the polymer. Several individual
polymers, e.g., in a heterogenous mixture, can be characterized or
evaluated in rapid succession, one polymer at a time, leading to
characterization of the polymers in the mixture.
The method is broadly useful for characterizing polymers that are strands
of monomers which, in general (if not entirely), are arranged in linear
strands. Any polymer whose monomer units interact with the interface can
be characterized. The method is particularly useful for characterizing
biological polymers such as deoxyribonucleic acids, ribonucleic acids,
polypeptides, and oligosaccharides, although other polymers may be
evaluated. In some embodiments, a polymer which carries one or more
charges (e.g., nucleic acids, polypeptides) will facilitate implementation
of the invention.
The monomer-dependent characterization achieved by the invention may
include identifying physical characteristics such as the number and
composition of monomers that make up each individual molecule, preferably
in sequential order from any starting point within the polymer or its
beginning or end. A heterogenous population of polymers may be
characterized, providing a distribution of characteristics (such as size)
within the population. Where the monomers within a given polymer molecule
are heterogenous, the method can be used to determine their sequence.
The pools of medium used in the invention may be any fluid that permits
adequate polymer mobility for interface interaction. Typically, the pools
will be liquids, usually aqueous solutions or other liquids or solutions
in which the polymers can be distributed.
The interface between the pools is designed to interact sequentially with
the monomers of one polymer molecule at a time. As described in greater
detail below, the useful portion of the interface may be a passage in or
through an otherwise impermeable barrier, or it may be an interface
between immiscible liquids. It is preferable that only one passage is
present or functional in the impermeable barrier.
The interface-dependent measurements made according to the invention can be
any measurement, e.g., physical or electrical, that varies with
polymer-interface interaction. For example, physical changes the monomers
cause as they interact sequentially with the interface may be measured.
Current changes resulting from the polymer's interference with ion flow at
the interface may be measured. The measurements may reflect the sequential
interaction of the monomers with the interface, so as to permit evaluation
of monomer-dependent characteristics of the polymer molecule (e.g., size
or mass of individual monomers or of the entire polymer, or the sequence
or identity of individual monomers which make up the polymer).
In one embodiment, the pools include electrically conductive medium which
can be of the same or different compositions. The pools with conducting
media are separated by an impermeable barrier containing an ion-permeable
passage, and measurements of the interface characteristics include
establishing an electrical potential between the two pools such that ionic
current can flow across the ion permeable passage. When the polymer
interacts sequentially with the interface at the ion permeable passage,
the ionic conductance of the passage will change (e.g., decrease or
increase) as each monomer interacts, thus indicating characteristics of
the monomers (e.g., size, identity) and/or the polymer as a whole (e.g.,
size).
In a different embodiment, the concentration of polymers in a solution can
be determined, using the conducting medium and ion-permeable passage
described above. As a voltage differential is applied across the pools,
the polymer molecules interact with the ion-permeable passage. The number
of interactions (conductance change events) per unit time is proportional
to the number of polymer molecules in the solution. This measurement is
preferably made under relatively low resolution recording conditions,
e.g., below the level of resolution of individual monomer/pore
interactions.
The conducting medium used can be any medium, preferably a solution, more
preferably an aqueous solution, which is able to carry electrical current.
Such solutions generally contain ions as the current conducting agents,
e.g., sodium, potassium, chloride, calcium, cesium, barium, sulfate,
phosphate. Conductance (g) across the pore or channel is determined by
measuring the flow of current across the pore or channel via the
conducting medium. A voltage difference can be imposed across the barrier
between the pools by conventional means, e.g., via a voltage source which
injects or administers current to at least one of the pools to establish a
potential difference. Alternatively, an electrochemical gradient may be
established by a difference in the ionic composition of the two pools,
either with different ions in each pool, or different concentrations of at
least one of the ions in the solutions or media of the pools. In this
embodiment of the invention, conductance changes are measured and are
indicative of monomer-dependent characteristics.
The term "ion permeable passages" used in this embodiment of the invention
includes ion channels, ion-permeable pores, and other ion-permeable
passages, and all are used herein to include any local site of transport
through an otherwise impermeable barrier. For example, the term includes
naturally occurring, recombinant, or mutant proteins which permit the
passage of ions under conditions where ions are present in the medium
contacting the channel or pore. Synthetic pores are also included in the
definition. Examples of such pores can include, but are not limited to,
chemical pores formed, e.g., by nystatin, ionophores (e.g., A23187;
Pressman et al., Ann. Rev. Biochem. 45:501, 1976), or mechanical
perforations of a membranous material. Proteinaceous ion channels can be
voltage-gated or voltage independent, including mechanically gated
channels (e.g., stretch-activated K.sup.+ channels), or recombinantly
engineered or mutated voltage dependent channels (e.g., Na.sup.+ or
K.sup.+ channels constructed as is known in the art).
Another preferred type of passage is a protein which includes a portion of
a bacteriophage receptor which is capable of binding all or part of a
bacteriophage ligand (either a natural or functional ligand) and
transporting bacteriophage DNA from one side of the interface to the
other. The polymer to be characterized includes a portion which acts as a
specific ligand for the bacteriophage receptor, so that it may be injected
across the barrier/interface from one pool to the other.
The protein channels or pores of the invention can include those translated
from one or more natural and/or recombinant DNA molecule(s) which includes
a first DNA which encodes a channel or pore forming protein and a second
DNA which encodes a monomer-interacting portion of a monomer polymerizing
agent (e.g., a nucleic acid polymerase). The expressed protein or proteins
are capable of non-covalent association or covalent linkage (any linkage
herein referred to as forming an "assemblage" of "heterologous units"),
and when so associated or linked, the polymerizing portion of the protein
structure is able to polymerize monomers from a template polymer, close
enough to the channel forming portion of the protein structure to
measurably affect ion conductance across the channel. Alternatively,
assemblages can be formed from unlike molecules, e.g., a chemical pore
linked to a protein polymerase, but these assemblages still fall under the
definition of a "heterologous" assemblage.
The invention also includes the recombinant fusion protein(s) translated
from the recombinant DNA molecule(s) described above, so that a fusion
protein is formed which includes a channel forming protein linked as
described above to a monomer-interacting portion of a nucleic acid
polymerase. Preferably, the nucleic acid polymerase portion of the
recombinant fusion protein is capable of catalyzing polymerization of
nucleotides. Preferably, the nucleic acid polymerase is a DNA or RNA
polymerase, more preferably T7 RNA polymerase.
The polymer being characterized may remain in its original pool, or it may
cross the passage. Either way, as a given polymer molecule moves in
relation to the passage, individual monomers interact sequentially with
the elements of the interface to induce a change in the conductance of the
passage. The passages can be traversed either by polymer transport through
the central opening of the passage so that the polymer passes from one of
the pools into the other, or by the polymer traversing across the opening
of the passage without crossing into the other pool. In the latter
situation, the polymer is close enough to the channel for its monomers to
interact with the passage and bring about the conductance changes which
are indicative of polymer characteristics. The polymer can be induced to
interact with or traverse the pore, e.g., as described below, by a
polymerase or other template-dependent polymer replicating catalyst linked
to the pore which draws the polymer across the surface of the pore as it
synthesizes a new polymer from the template polymer, or by a polymerase in
the opposite pool which pulls the polymer through the passage as it
synthesizes a new polymer from the template polymer. In such an
embodiment, the polymer replicating catalyst is physically linked to the
ion-permeable passage, and at least one of the conducting pools contains
monomers suitable to be catalytically linked in the presence of the
catalyst. A "polymer replicating catalyst," "polymerizing agent" or
"polymerizing catalyst" is an agent that can catalytically assemble
monomers into a polymer in a template dependent fashion--i.e., in a manner
that uses the polymer molecule originally provided as a template for
reproducing that molecule from a pool of suitable monomers. Such agents
include, but are not limited to, nucleotide polymerases of any type, e.g.,
DNA polymerases, RNA polymerases, tRNA and ribosomes.
The characteristics of the polymer can be identified by the amplitude or
duration of individual conductance changes across the passage. Such
changes can identify the monomers in sequence, as each monomer will have a
characteristic conductance change signature. For instance, the volume,
shape, or charges on each monomer will affect conductance in a
characteristic way. Likewise, the size of the entire polymer can be
determined by observing the length of time (duration) that
monomer-dependent conductance changes occur. Alternatively, the number of
monomers in a polymer (also a measure of size) can be determined as a
function of the number of monomer-dependent conductance changes for a
given polymer traversing a passage. The number of monomers may not
correspond exactly to the number of conductance changes, because there may
be more than one conductance level change as each monomer of the polymer
passes sequentially through the channel. However, there will be a
proportional relationship between the two values which can be determined
by preparing a standard with a polymer of known sequence.
The mixture of polymers used in the invention does not need to be
homogenous. Even when the mixture is heterogenous, only one molecule
interacts with a passage at a time, yielding a size distribution of
molecules in the mixture, and/or sequence data for multiple polymer
molecules in the mixture.
In preferred embodiments, the passage is a natural or recombinant bacterial
porin molecule. In other preferred embodiments, the passage is a natural
or recombinant voltage-sensitive or voltage gated ion channel, preferably
one which does not inactivate (whether naturally or through recombinant
engineering as is known in the art). "Voltage sensitive" or "gated"
indicates that the channel displays activation and/or inactivation
properties when exposed to a particular range of voltages. Preferred
channels for use in the invention include the .alpha.-hemolysin toxin from
S. aureus and maltoporin channels.
In an alternate embodiment of the invention, the pools of medium are not
necessarily conductive, but are of different compositions so that the
liquid of one pool is not miscible in the liquid of the other pool and the
interface is the immiscible interface between the pools. In order to
measure the characteristics of the polymer, a polymer molecule is drawn
through the interface of the liquids, resulting in an interaction between
each sequential monomer of the polymer and the interface. The sequence of
interactions as the monomers of the polymer are drawn through the
interface is measured, yielding information about the sequence of monomers
that characterize the polymer. The measurement of the interactions can be
by a detector that measures the deflection of the interface (caused by
each monomer passing through the interface) using reflected or refracted
light, or a sensitive gauge capable of measuring intermolecular forces.
Several methods are available for measurement of forces between
macromolecules and interfacial assemblies, including the surface forces
apparatus (Israelachvili, Intermolecular and Surface Forces, Academic
Press, New York, 1992), optical tweezers (Ashkin et al., Oppt. Lett.,
11:288, 1986; Kuo and Sheetz, Science, 260:232, 1993; Svoboda et al.,
Nature 365:721, 1993), and atomic force microscopy (Quate, F. Surf. Sci.
299:980, 1994; Mate et al., Phys. Rev. Lett. 59:1942, 1987; Frisbie et
al., Science 265:71, 1994; all hereby incorporated by reference).
The interactions between the interface and the monomers in the polymer are
suitable to identify the size of the polymer, e.g., by measuring the
length of time during which the polymer interacts with the interface as it
is drawn across the interface at a known rate, or by measuring some
feature of the interaction (such as deflection of the interface, as
described above) as each monomer of the polymer is sequentially drawn
across the interface. The interactions can also be sufficient to ascertain
the identity of individual monomers in the polymer.
This invention offers advantages particularly in nucleotide sequencing,
e.g., reduction in the number of sequencing steps, and increasing the
speed of sequencing and the length of molecule capable of being sequenced.
The speed of the method and the size of the polymers it can sequence are
particular advantages of the invention. The linear polymer may be very
large, and this advantage will be especially useful in reducing template
preparation time, sequencing errors and analysis time currently needed to
piece together small overlapping fragments of a large gene or stretch of
polymer.
Other features and advantages of the invention will be apparent from the
following description of the preferred embodiments thereof, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of an embodiment of DNA
characterization by the method of the invention. The unobstructed ionic
current (illustrated for the channel at the top of the diagram), is
reduced as a polymeric molecule begins its traversal through the pore
(illustrated for the channel at the bottom of the diagram). The monomeric
units of the polymer (drawn as different sized ovals on the strand)
interfere sequentially and differentially with the flow of ions through
the channel.
FIG. 2 is a schematic representation of an implementation of DNA sequencing
by the method of the invention. In this embodiment, the polymer is drawn
across the opening of the channel, but is not drawn through the channel.
The channel, e.g., a porin, is inserted in the phospholipid bilayer. A
polymerase domain is fused by its N-terminus to the C-terminus of one of
the porin monomers (the porin C-termini are on the periplasmic side of the
membrane in both Rhodobacter capsulatus and LamB porins). Fusions on the
other side of the membrane can also be made. Malto-oligosaccharides can
bind and block current from either side. The polymerase is shown just
prior to binding to the promoter. A non-glucosylated base is shown near a
pore opening, while a penta-glucosylated cytosine is shown 10 bp away. The
polymerase structure represented is that of DNA polymerase I (taken from
Ollis et al., 1985, Nature, 313:762-66), and the general porin model is
from Jap (1989, J. Mol. Biol., 205:407-19).
FIG. 3 is a schematic representation of DNA sequencing results by the
method of the invention. The schematic depicts, at very high resolution,
one of the longer transient blockages such as can be seen in FIG. 4. The
monomeric units of DNA (bases G, A, T, and C) interfere differentially
with the flow of ions through the pore, resulting in discrete conductance
levels that are characteristic of each base. The order of appearance of
the conductance levels sequentially identifies the monomers of the DNA.
FIG. 4 is a recording of the effect of polyadenylic acid (poly A) on the
conductance of a single .alpha.-hemolysin channel in a lipid bilayer
between two aqueous compartments containing 1 M NaCl, 10 mM Tris, pH 7.4.
Before addition of RNA, the conductance of the channel was around 850 pS.
The cis compartment, to which poly A is added, is -120 mV with respect to
the trans compartment. After adding poly A to the cis compartment, the
conductance of the .alpha.-hemolysin channel begins to exhibit transient
blockages (conductance decreases to about 100 pS) as individual poly A
molecules are drawn across the channel from the cis to the trans
compartment. When viewed at higher resolution (expanded time scale, at
top), the duration of each transient blockage is seen to vary between less
than 1 msec up to 10 msec. Arrows point to two of the longer duration
blockages. See FIGS. 5A and 5B for histograms of blockage duration.
FIGS. 5A and 5B are comparisons of blockage duration with purified RNA
fragments of 320nt (FIG. 5A) and 1100nt (FIG. 5B) lengths. The absolute
number of blockades plotted in the two histograms are not comparable
because they have not been normalized to take into account the different
lengths of time over which the data in the two graphs were collected.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As summarized above, we have determined a new method for rapidly analyzing
polymers such as DNA and RNA. We illustrate the invention with two primary
embodiments. In one embodiment, the method involves measurements of ionic
current modulation as the monomers (e.g., nucleotides) of a linear polymer
(e.g., nucleic acid molecule) pass through or across a channel in an
artificial membrane. During polymer passage through or across the channel,
ionic currents are reduced in a manner that reflects the properties of the
polymer (length, concentration of polymers in solution, etc.) and the
identities of the monomers. In the second embodiment, an immiscible
interface is created between two immiscible liquids, and, as above,
polymer passage through the interface results in monomer interactions with
the interface which are sufficient to identify characteristics of the
polymer and/or the identity of the monomers.
The description of the invention will be primarily concerned with
sequencing nucleic acids, but this is not intended to be limiting. It is
feasible to size and sequence polymers other than nucleic acids by the
method of the invention, including linear protein molecules which include
monomers of amino acids. Other linear arrays of monomers, including
chemicals (e.g., biochemicals such as polysaccharides), may also be
sequenced and characterized by size.
I. Polymer Analysis Using Conductance Changes Across An Interface
Sensitive single channel recording techniques (i.e., the patch clamp
technique) can be used in the invention, as a rapid, high-resolution
approach allowing differentiation of nucleotide bases of single DNA
molecules, and thus a fast and efficient DNA sequencing technique or a
method to determine polymer size or concentration (FIGS. 1 and 2). We will
describe methods to orient DNA to a pore molecule in two general
configurations (see FIGS. 1 and 2) and record conductance changes across
the pore (FIG. 3). One method is to use a pore molecule such as the
receptor for bacteriophage lambda (LamB) or .alpha.-hemolysin, and to
record the process of DNA injection or traversal through the channel pore
when that channel has been isolated on a membrane patch or inserted into a
synthetic lipid bilayer (FIG. 1). Another method is to fuse a DNA
polymerase molecule to a pore molecule and allow the polymerase to move
DNA over the pore's opening while recording the conductance across the
pore (FIG. 2). A third method is to use a polymerase on the trans side of
the membrane/pore divider to pull a single stranded nucleic acid through
the pore from the cis side (making it double stranded) while recording
conductance changes. A fourth method is to establish a voltage gradient
across a membrane containing a channel (e.g., .alpha.-hemolysin) through
which a single stranded or double stranded DNA is electrophoresed.
The apparatus used for this embodiment includes 1) an ion-conducting pore
or channel, perhaps modified to include a linked or fused polymerizing
agent, 2) the reagents necessary to construct and produce a linear polymer
to be characterized, or the polymerized molecule itself, and 3) an
amplifier and recording mechanism to detect changes in conductance of ions
across the pore as the polymer traverses its opening.
A variety of electronic devices are available which are sensitive enough to
perform the measurements used in the invention, and computer acquisition
rates and storage capabilities are adequate for the rapid pace of sequence
data accumulation.
A. Characteristics Identified by the Methods
1) Size/Length of Molecules The size or length of a polymer can be
determined by measuring its residence time in the pore or channel, e.g.,
by measuring duration of transient blockade of current. The relationship
between this time period and the length of the polymer can be described by
a reproducible mathematical function which depends on the experimental
condition used. The function is likely a linear function for a given type
of polymer (e.g., DNA, RNA, polypeptide), but if it is described by
another function (e.g., sigmoidal or exponential), accurate size estimates
may be made by first preparing a standard curve using known sizes of like
linear molecules.
2) Identity of Residues/Monomers
The chemical composition of individual monomers is sufficiently variant to
cause characteristic changes in channel conductance as each monomer
traverses the pore due to physical configuration, size/volume, charge,
interactions with the medium, etc. For example, our experimental data
suggest that poly A reduces conductance more than does poly U, indicating
a physical difference between purines and pyrimidines that forms the basis
of nucleotide identification in this invention.
The nucleotide bases of DNA will influence pore conductance during
traversal, but if the single channel recording techniques are not
sensitive enough to detect differences between normal bases in DNA, it is
practical to supplement the system's specificity by using modified bases.
The modifications should be asymmetrical (on only one strand of double
stranded template), to distinguish otherwise symmetrical base pairs.
Modified bases are readily available. These include: 1) methylated bases
(lambda can package and inject DNA with or without methylated A's and
C's), 2) highly modified bases found in the DNAs of several bacteriophage
(e.g. T4, SP15), many of which involve glycosylations coupled with other
changes (Warren, 1980, Ann. Rev. Microbiol., 34:137-58), and 3) the
modified nucleotide triphosphates that can be incorporated by DNA
polymerase (e.g. biotinylated, digoxigenated, and fluorescently tagged
triphosphates).
In order to identify the monomers, conditions should be appropriate to
avoid secondary structure in the polymer to be sequenced (e.g., nucleic
acids); if necessary, this can be achieved by using a recording solution
which is denaturing. Using single stranded DNA, single channel recordings
can be made in up to 40% formamide and at temperatures as high as
45.degree. C. using e.g., the .alpha.hemolysin toxin protein in a lipid
bilayer. These conditions are not intended to exclude use of any other
denaturing conditions. One skilled in the art of electrophysiology will
readily be able to determine suitable conditions by 1) observing
incorporation into the bilayer of functional channels or pores, and 2)
observing transient blockades of conductance uninterrupted by long-lived
blockades caused by polymers becoming stuck in the channel because of
secondary structure. Denaturing conditions are not always necessary for
the polymerase-based methods or for double stranded DNA methods of the
invention. They may not be necessary for single stranded methods either,
if the pore itself is able to cause denaturation, or if the secondary
structure does not interfere.
3) Concentration of Polymers in Solutions
Concentration of polymers can be rapidly and accurately assessed by using
relatively low resolution recording conditions and analyzing the number of
conductance blockade events in a given unit of time. This relationship
should be linear and proportional (the greater the concentration of
polymers, the more frequent the current blockage events), and a
standardized curve can be prepared using known concentrations of polymer.
B. Principles and Techniques
1) Recording Techniques
The conductance monitoring methods of the invention rely on an established
technique, single-channel recording, which detects the activity of
molecules that form channels in biological membranes. When a voltage
potential difference is established across a bilayer containing an open
pore molecule, a steady current of ions flows through the pore from one
side of the bilayer to the other. The nucleotide bases of a DNA molecule,
for example, passing through or over the opening of a channel protein,
disrupt the flow of ions through the pore in a predictable way.
Fluctuations in the pore's conductance caused by this interference can be
detected and recorded by conventional single-channel recording techniques.
Under appropriate conditions, with modified nucleotides if necessary, the
conductance of a pore can change to unique states in response to the
specific bases in DNA.
This flux of ions can be detected, and the magnitude of the current
describes the conductance state of the pore. Multiple conductance states
of a channel can be measured in a single recording as is well known in the
art. By recording the fluctuations in conductance of the maltoporin (LamB)
pore, for example, when DNA is passed through it by phage lambda injection
or over its opening by the action of a polymerase fused to the surface of
the LamB protein, we estimate that a sequencing rate of 100-1000
bases/sec/pore can be achieved.
The monitoring of single ion channel conductance is an inexpensive, viable
method that has been successful for the last two decades and is in very
wide spread current use. It directly connects movements of single ions or
channel proteins to digital computers via amplifiers and analog to digital
(A to D, A/D) converters. Single channel events taking place in the range
of a few microseconds can be detected and recorded (Hamill et al., 1981,
Pfluegers Arch. Eur. J. Physiol., 391:85-100). This level of time
resolution ranges from just sufficient to orders of magnitude greater than
the level we need, since the time frame for movement of nucleotide bases
relative to the pore for the sequencing method is in the range of
microseconds to milliseconds. The level of time resolution required
depends on the voltage gradient or the enzyme turnover number if the
polymer is moved by an enzyme. Other factors controlling the level of time
resolution include medium viscosity, temperature, etc.
The characteristics and conductance properties of any pore molecule that
can be purified can be studied in detail using art-known methods (Sigworth
et al., supra; Heinemann et al., 1988, Biophys. J., 54:757-64; Wonderlin
et al., 1990, Biophys. J., 58:289-97). These optimized methods are ideal
for our polymer sequencing application. For example, in the pipette
bilayer technique, an artificial bilayer containing at least one pore
protein is attached to the tip of a patch-clamp pipette by applying the
pipette to a preformed bilayer reconstituted with the purified pore
protein in advance. Due to the very narrow aperture diameter of the patch
pipette tip (2 microns), the background noise for this technique is
significantly reduced, and the limit for detectable current interruptions
is about 10 microseconds (Sigworth et al., supra; Heinemann et al., 1990,
Biophys. J., 57:499-514). Purified channel protein can be inserted in a
known orientation into preformed lipid bilayers by standard vesicle fusion
techniques (Schindler, 1980, FEBS Letters, 122:77-79), or any other means
known in the art, and high resolution recordings are made. The membrane
surface away from the pipette is easily accessible while recording. This
is important for the subsequent recordings that involve added DNA. The
pore can be introduced into the solution within the patch pipette rather
than into the bath solution.
An optimized planar lipid bilayer method has recently been introduced for
high resolution recordings in purified systems (Wonderlin et al., supra).
In this method, bilayers are formed over very small diameter apertures
(25-80 microns) in plastic. This technique has the advantage of allowing
access to both sides of the bilayer, and involves a slightly larger
bilayer target for reconstitution with the pore protein. This optimized
bilayer technique is an alternative to the pipette bilayer technique.
Instrumentation is needed which can apply a variable range of voltages from
about +400 mV to -400 mV across the channel/membrane, assuming that the
trans compartment is established to be 0 mV; a very low-noise amplifier
and current injector, analog to digital (A/D) converter, data acquisition
software, and electronic storage medium (e.g., computer disk, magnetic
tape). Equipment meeting these criteria is readily available, such as from
Axon Instruments, Foster City, Calif. (e.g., Axopatch 200A system; pclamp
6.0.2 software).
Preferred methods of large scale DNA sequencing involve translating from
base pairs to electronic signals as directly and as quickly as possible in
a way that is compatible with high levels of parallelism, miniaturization
and manufacture. The method should allow long stretches (even stretches
over 40 kbp) to be read so that errors associated with assembly and
repetitive sequence can be minimized. The method should also allow
automatic loading of (possibly non-redundant) fresh sequences.
2) Channels and Pores Useful in the Invention Any channel protein which has
the characteristics useful in the invention (e.g., minimum pore size
around 2 .ANG. , maximum around 9 nm; conducts current) may be employed.
Pore sizes across which polymers can be drawn may be quite small and do
not necessarily differ for different polymers. Pore sizes through which a
polymer is drawn will be e.g., approximately 0.5 -2.0 nm for single
stranded DNA; 1.0 -3.0 nm for double stranded DNA; and 1.0 - 4.0 nm for
polypeptides. These values are not absolute, however, and other pore sizes
might be equally functional for the polymer types mentioned above.
Examples of bacterial pore-forming proteins which can be used in the
invention include Gramicidin (e.g., Gramicidin A, B, C, D, or S, from
Bacillus brevis; available from Fluka, Ronkonkoma, N.Y.); Valinomycin
(from Streptomyces fulvissimus; available from Fluka), LamB (maltoporin),
OmpF, OmpC, or PhoE from Escherichia coli, Shigella, and other
Enterobacteriaceae, alpha-hemolysin (from S. aureus), Tsx, the F-pilus,
and mitochondrial porin (VDAC). This list is not intended to be limiting.
A modified voltage-gated channel can also be used in the invention, as long
as it does not inactivate quickly, e.g., in less than about 500 msec
(whether naturally or following modification to remove inactivation) and
has physical parameters suitable for e.g., polymerase attachment
(recombinant fusion proteins) or has a pore diameter suitable for polymer
passage. Methods to alter inactivation characteristics of voltage gated
channels are well known in the art (see e.g., Patton, et al., Proc. Natl.
Acad. Sci. USA, 89:10905-09 (1992); West, et al., Proc. Natl. Acad. Sci.
USA, 89:10910-14 (1992); Auld, et al., Proc. Natl. Acad. Sci. USA,
87:323-27 (1990); Lopez, et al., Neuron, 7:327-36 (1991); Hoshi, et al.,
Neuron, 7:547-56 (1991); Hoshi, et al., Science, 250:533-38 (1990), all
hereby incorporated by reference).
Appropriately sized physical or chemical pores may be induced in a
water-impermeable barrier (solid or membranous) up to a diameter of about
9 nm, which should be large enough to accommodate most polymers (either
through the pore or across its opening). Any methods and materials known
in the art may be used to form pores, including track etching and the use
of porous membrane templates which can be used to produce pores of the
desired material (e.g., scanning-tunneling microscope or atomic force
microscope related methods).
Chemical channels or pores can be formed in a lipid bilayer using chemicals
(or peptides) such as Nystatin, as is well known in the art of whole-cell
patch clamping ("perforated patch" technique); ionophores such as A23187
(Calcimycin), ETH 5234, ETH 157 (all chemicals available from Fluka,
Ronkonkoma, N.Y.; this list is not intended to be limiting), peptide
channels such as Alamethicin, etc.
To produce pores linked with polymerase, synthetic/recombinant DNA coding
for a fusion protein can be transcribed and translated, then inserted into
an artificial membrane in vitro. For example, the C-terminus of E. coli
DNA polymerase I (and by homology, T7 polymerase) is very close to the
surface of the major groove of the newly synthesized DNA. If the
C-terminus of a polymerase is fused to the N-terminus of a pore forming
protein such as colicin E1 and the colicin is inserted into an artificial
membrane, one opening of the colicin pore should face the DNA's major
groove and one should face the opposite side of the lipid bilayer. For
example, the colicin molecule can be modified to achieve a pH optimum
compatible with the polymerase as in Shiver et al. (J. Biol. Chem.,
262:14273-14281 1987, hereby incorporated by reference). Both pore and
polymerase domain can be modified to contain cysteine replacements at
points such that disulfide bridges form to stabilize a geometry that
forces the pore opening closer to the major groove surface and steadies
the polymer as it passes the pore opening. The loops of the pore domain at
this surface can be systematically modified to maximize sensitivity to
changes in the DNA sequence.
C. General Considerations for Conductance Based Measurements
1) Electrical/Channel Optimization
The conductance of a pore at any given time is determined by its resistance
to ions passing through the pore (pore resistance) and by the resistance
to ions entering or leaving the pore (access resistance). For a pore's
conductance to be altered in discrete steps, changes in one or both of
these resistance factors will occur by unit values. The base pairs of a
DNA molecule represent discrete units that are distinct from each other
along the phosphate backbone. As long as the orientation of DNA to the
pore remains relatively constant, and the membrane potential does not
change, as each base pair passes by (or through) the pore, it is likely to
interfere with a reproducible number of ions. Modifications made to the
individual bases would influence the magnitude of this effect.
To resolve stretches of repeating identical bases accurately, and to
minimize reading errors in general, it may be useful for the pore to
register a distinct (probably higher) level of conductance in between the
bases. This can take place naturally in the pore-polymerase system with
helix rotation during polymerization, or in the phage system between entry
of base pairs into the pore, or when the regions in between base pairs
pass by a rate limiting site for ion flux inside the pore. Modified bases
used to distinguish nucleotide identities may also contribute
significantly to this issue, because they should magnify the conductance
effect of the bases relative to the effect of regions in between the
bases. With single strand passage through a pore, charged phosphates may
punctuate the passage of each base by brief, higher conductance states.
Also, if the rate of movement is constant, then punctuation between bases
may not be required to resolve stretches of repeating identical bases.
Altered conductance states have been described for many channels, including
some LamB mutants (Dargent et al., 1988, supra). A mutant may be a
valuable alternative to a wild type channel protein if its fluctuation to
a given state is sensitive to nucleotide bases in DNA. Alternative systems
can also be developed from other channel proteins that are known to have
multiple single channel conductance states. Examples of these are the
alamethicin channel, which under certain conditions fluctuates through at
least 20 discrete states (Taylor et al., 1991, Biophys. J., 59:873-79),
and the OmpF porin, which shows gating of its individual monomers giving
rise to four discrete states (Lakey et al., 1989, Eur. J. Biochem.,
186:303-308).
Since channel events can be resolved in the microsecond range with the high
resolution recording techniques available, the limiting issue for
sensitivity with the techniques of our invention is the amplitude of the
current change between bases. Resolution limits for detectable current are
in the 0.2 pA range (1 pA=6.24.times.10.sup.6 ions/sec). Each base
affecting pore current by at least this magnitude is detected as a
separate base. It is the function of modified bases to affect current
amplitude for specific bases if the bases by themselves are poorly
distinguishable.
One skilled in the art will recognize that there are many possible
configurations of the sequencing method described herein. For instance,
lipid composition of the bilayer may include any combination of non-polar
(and polar) components which is compatible with pore or channel protein
incorporation. Any configuration of recording apparatus may be used (e.g.,
bilayer across aperture, micropipette patches, intra-vesicular recording)
so long as its limit of signal detection is below about 0.5 pA, or in a
range appropriate to detect monomeric signals of the polymer being
evaluated. If polymer | | |
