U.S. patent application number 15/610579 was filed with the patent office on 2017-11-23 for sensor arrays and nucleic acid sequencing applications.
The applicant listed for this patent is INTEL CORPORATION. Invention is credited to Xing SU, Kai WU.
Application Number | 20170335389 15/610579 |
Document ID | / |
Family ID | 43731144 |
Filed Date | 2017-11-23 |
United States Patent
Application |
20170335389 |
Kind Code |
A1 |
SU; Xing ; et al. |
November 23, 2017 |
SENSOR ARRAYS AND NUCLEIC ACID SEQUENCING APPLICATIONS
Abstract
Embodiments of the present invention provide devices methods for
sequencing DNA using arrays of reaction regions containing
electronic sensors to monitor changes in solutions contained in the
reaction regions. Test and fill reaction schemes are disclosed that
allow DNA to be sequenced. By sequencing DNA using parallel
reactions contained in large arrays, DNA can be rapidly
sequenced.
Inventors: |
SU; Xing; (Cupertino,
CA) ; WU; Kai; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INTEL CORPORATION |
Santa Clara |
CA |
US |
|
|
Family ID: |
43731144 |
Appl. No.: |
15/610579 |
Filed: |
May 31, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12950729 |
Nov 19, 2010 |
9695472 |
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15610579 |
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11226696 |
Sep 13, 2005 |
9040237 |
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12950729 |
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11073160 |
Mar 4, 2005 |
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11226696 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 2219/00722
20130101; B01L 3/5085 20130101; B01J 2219/00317 20130101; C12Q
1/6874 20130101; B01J 2219/00612 20130101; C12Q 1/6825 20130101;
B01J 2219/00511 20130101; C12Q 1/6874 20130101; B82Y 30/00
20130101; C12Q 2565/607 20130101; B01J 2219/00641 20130101; B01J
2219/00621 20130101; B01J 2219/00626 20130101; G01N 27/414
20130101; C12Q 1/6874 20130101; B01J 2219/00605 20130101; B01J
2219/0063 20130101; C12Q 2525/125 20130101; C12Q 2521/101 20130101;
C12Q 2533/101 20130101; C12Q 2565/301 20130101; C12Q 2521/319
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B82Y 30/00 20110101 B82Y030/00 |
Claims
1. A sensor comprising a plurality of reaction cavities for holding
reactants, the cavities having DNA molecules to be sequenced with
each of the cavities having statistically one DNA molecule to be
sequenced, each of the cavities having a surface for attachment of
the one DNA molecule to be sequenced and a coupled optical sensor
for detecting changes resulting from a chemical reaction in each of
the cavities, furthermore each of the cavities comprises a DNA
oligo, the DNA oligo comprising a first nuclease-resistant
nucleotide or a first nuclease-resistant nucleotide analog at the
3'-end of the DNA oligo, wherein the sensor is configured to
perform: repeated nucleotide addition and excision reactions
without cleavage of the first nuclease-resistant nucleotide or the
first nuclease-resistant nucleotide analog; identify the base of
the DNA molecule to be sequenced immediately upstream from the base
of the DNA molecule complementary to the first nuclease-resistant
nucleotide by monitoring increases in reaction; and attach a second
nuclease-resistant blocking nucleotide or a second
nuclease-resistant blocking nucleotide analog to the 3'-end of the
DNA oligo, the second nuclease-resistant blocking nucleotide or the
second nuclease-resistant blocking nucleotide analog being
complementary to the identified base,
2. The sensor of claim 1, wherein the sensor comprises a single
walled carbon nanotube that is capable of acting as field effect
transistor.
3. The sensor of claim 1, wherein the reactants comprise a
polymerase.
4. The sensor of claim 1, wherein the reactants comprises an
exonuclease.
5. The sensor of claim 1, wherein the first nuclease-resistant
blocking nucleoside comprises alpha-thiophosphate.
6. The sensor of claim 1, wherein the nucleotide is a naturally
occurring nucleotide or an analog thereof,
7. The sensor of claim 1, wherein the nucleotide is labeled,
8. The sensor of claim 1, further wherein the sensor is configured
to deblock the second nuclease-resistant blocking nucleotide or the
second nuclease-resistant blocking nucleotide analog.
9. The sensor of claim 1, wherein the reactants comprises only one
type of nucleobase selected from the group consisting of adenine,
cytosine, guanine, thymine and uracil.
10. The sensor of claim 3, wherein the polymerase is Klenow
(exo-).
11. The sensor of claim 4, wherein the exonuclease is exonuclease
III.
12. A sensor comprising a plurality of reaction cavities for
holding reactants, the cavities having DNA molecules to be
sequenced with each of the cavities having statistically one DNA
molecule to be sequenced, each of the cavities having a surface for
attachment of the one DNA molecule to be sequenced and a coupled
electronic sensor for detecting changes resulting from a chemical
reaction in each of the cavities, furthermore each of the cavities
comprises a DNA oligo, the DNA oligo comprising a first
nuclease-resistant nucleotide or a first nuclease-resistant
nucleotide analog at the 3'-end of the DNA oligo, wherein the
sensor is configured to perform: repeated nucleotide addition and
excision reactions without cleavage of the first nuclease-resistant
nucleotide or the first nuclease-resistant nucleotide analog;
identify the base of the DNA molecule to be sequenced immediately
upstream from the base of the DNA molecule complementary to the
first nuclease-resistant nucleotide by monitoring increases in
reaction; and attach a second nuclease-resistant blocking
nucleotide or a second nuclease-resistant blocking nucleotide
analog to the 3'-end of the DNA oligo, the second
nuclease-resistant blocking nucleotide or the second
nuclease-resistant blocking nucleotide analog being complementary
to the identified base,
13. The sensor of claim 12, wherein the sensor comprises a single
walled carbon nanotube that is capable of acting as field effect
transistor.
14. The sensor of claim 12, wherein the reactants comprise a
polymerase.
15. The sensor of claim 12, wherein the reactants comprises an
exonuclease.
16. The sensor of claim 12, wherein the first nuclease-resistant
blocking nucleoside comprises alpha-thiophosphate.
17. The sensor of claim 12, wherein the nucleotide is a naturally
occurring nucleotide or an analog thereof.
18. The sensor of claim 12, wherein the nucleotide is labeled.
19. The sensor of claim 12, further wherein the sensor is
configured to deblock the second nuclease-resistant blocking
nucleotide or the second nuclease-resistant blocking nucleotide
analog.
20. The sensor of claim 12, wherein the reactants comprises only
one type of nucleobase selected from the group consisting of
adenine, cytosine, guanine, thymine and uracil.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 12/950,729, filed Nov. 19, 2010, which is a
continuation-in-part of U.S. patent application Ser. No.
11/226,696, filed Sep. 13, 2005, which is a continuation-in-part of
U.S. patent application Ser. No. 11/073,160, filed Mar. 4, 2005,
the disclosures of which are incorporated herein by reference. An
additional related application is U.S. patent application Ser. No.
12/823,995, entitled "Nucleotides and Oligonucleotides for Nucleic
Acid Sequencing," filed Jun. 25, 2010 the disclosure of which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The embodiments of the present invention relate generally to
methods and devices for nucleic acid sequence detection.
Background Information
[0003] Genetic information in living organisms is contained in the
form of very long nucleic acid molecules such as deoxyribonucleic
acid (DNA) and ribonucleic acid (RNA). Naturally occuring DNA and
RNA molecules are typically composed of repeating chemical building
blocks called nucleotides which are in turn made up of a sugar
(deoxyribose or ribose, respectively), phosphoric acid, and one off
our bases, adenine (A), cytosine (C), guanine (G), and thymine (T)
or uracil (U). The human genome, for example, contains
approximately three billion nucleotides of DNA sequence and an
estimated 20,000 to 25,000 genes. DNA sequence information can be
used to determine multiple characteristics of an individual as well
as the presence of and or susceptibility to many common diseases,
such as cancer, cystic fibrosis, and sickle cell anemia.
Determination of the entire three billion nucleotide sequence of
the human genome has provided a foundation for identifying the
genetic basis of such diseases. An determination of the sequence of
the human genome required years to accomplish using traditional
technologies. The need for nucleic acid sequence information also
exists in research, environmental protection, food safety,
biodefense, and clinical applications, such as for example,
pathogen detection (the detection of the presence or absence of
pathogens or their genetic varients).
[0004] Thus, because DNA sequencing is an important technology for
applications in bioscience and personalized medicine, such as, for
example, the analysis of genetic information content for an
organism, tools that allow for faster and or more reliable sequence
determination are valuable. Applications such as, for example,
population-based biodiversity projects, disease detection,
prediction of effectiveness of drugs, and genotyping using
single-nucleotide polymorphisms, stimulate the need for simple and
robust methods for sequencing short lengths of nucleic acids (such
as, for example, those containing 1-20 bases). Sequencing methods
that provide increased accuracy and or robustness, decreased need
for analysis sample, and or high throughput are valuable analytical
and biomedical tools.
BRIEF DESCRIPTION OF THE FIGURES
[0005] The following drawings are included to further demonstrate
certain aspects of the disclosed embodiments of the invention.
[0006] FIG. 1 shows an outline for a general nucleic acid
sequencing strategy using test and fill (TAF) reactions and a
sensor array to detect the generation of reaction products.
[0007] FIG. 2 shows a general scheme for a test and fill sequencing
reaction.
[0008] FIG. 3 shows nucleotides that are useful in sequencing
reactions.
[0009] FIG. 4 shows a schematic of a method for achieving chemical
signal amplification in a test and fill sequencing reaction.
[0010] FIG. 5 demonstrates a method for parallel sequencing of
nucleic acids using the test and fill sequencing principle.
[0011] FIG. 6 is a schematic of device employing a field effect
transistor that can be used for analyzing a solution-based nucleic
acid sequencing reaction.
[0012] FIG. 7 is a schematic of an array of reaction sites
employing field effect transistors that can be used for analyzing
nucleic acid sequencing reactions.
[0013] FIG. 8 shows an alternate design for a sensor array that can
be used for analyzing nucleic acid sequencing reactions.
[0014] FIG. 9 is a schematic of a method for achieving chemical
signal amplification for optical detection of reaction products of
a nucleic acid sequencing reaction.
[0015] FIG. 10 shows a design for a microfluidic device that can be
used for optically detecting the reaction products of a nucleic
acid sequencing reaction.
[0016] FIG. 11 shows a design for a system that can be used for
sequencing nucleic acids.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Embodiments of the present invention provide devices and
methods for sequencing nucleic acids and nucleic acid detection. In
general, nucleic acids useful in the present invention include
polymers of deoxyribonucleotides (DNA) or ribonucleotides (RNA) and
analogs thereof that are linked together by a phosphodiester bond.
A polynucleotide can be a segment of a genome, a gene or a portion
thereof, a cDNA, or a synthetic polydeoxyribonucleic acid sequence.
A polynucleotide, including an oligonucleotide (for example, a
probe or a primer) can contain nucleoside or nucleotide analogs, or
a backbone bond other than a phosphodiester bond. In general, the
nucleotides comprising a polynucleotide are naturally occurring
deoxyribonucleotides, such as adenine, cytosine, guanine or thymine
linked to 2'-deoxyribose, or ribonucleotides such as adenine,
cytosine, guanine or uracil linked to ribose. However, a
polynucleotide or oligonucleotide also can contain nucleotide
analogs, including non-naturally occurring synthetic nucleotides or
modified naturally occurring nucleotides.
[0018] The covalent bond linking the nucleotides of a
polynucleotide generally is a phosphodiester bond. However, the
covalent bond also can be any of a number of other types of bonds,
including a thiodiester bond, a phosphorothioate bond, a
peptide-like amide bond or any other bond known to those in the art
as useful for linking nucleotides to produce synthetic
polynucleotides. The incorporation of non-naturally occurring
nucleotide analogs or bonds linking the nucleotides or analogs can
be particularly useful where the polynucleotide is to be exposed to
an environment that can contain nucleolytic activity, since the
modified polynucleotides can be less susceptible to
degradation.
[0019] Virtually any naturally occurring nucleic acid may be
sequenced including, for example, chromosomal, mitochondrial or
chloroplast DNA or ribosomal, transfer, heterogeneous nuclear or
messenger RNA. RNA can be converted into more stable cDNA through
the use of a reverse transcription enzyme (reverse transcriptase).
Additionally, non-naturally occuring nucleic acids that are
susceptible to enzymatic synthesis and degredation may be used in
embodiments of the present invention.
[0020] Methods for preparing and isolating various forms of nucleic
acids are known. See for example, Berger and Kimmel, eds., Guide to
Molecular Cloning Techniques, Academic Press, New York, N.Y.
(1987); and Sambrook, Fritsch and Maniatis, eds., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1989). However, embodiments of the
present invention are not limited to a particular method for the
preparation of nucleic acids.
[0021] Referring now to FIG. 1, an array of electronic sensors 100,
such as, for example, field-effect-transistor (FET) sensors, having
reaction sensor regions 110 (reaction regions) and immobilized DNA
molecules 120 is depicted. A primer molecule 125 is hybridized to
the immobilized DNA molecules 120. Sensors can also be impedance
meters, for example. One DNA molecule to be sequenced 120 is
immobilized per sensor region 110 in this example. Before
sequencing a sample of DNA, overlapped DNA fragments are
immobilized randomly on the array so that statistically one DNA
molecule occupies the reaction region 110 of a sensor. A sample of
DNA can be fragmented into smaller polymeric molecules using, for
example, restriction enzymes or mechanical forces (shearing). Test
and fill (TAF) reactions are performed and amplified chemical
products 130 are created in the reaction regions 110. Chemical
products 130 are amplified through repeated cycles of matching a
base (or nucleotide, used interchangeably here) in a DNA template
molecule to be sequenced with a testing base, filling the next
available position on the growing DNA strand with the matching
base, and then removing the newly added matching base. The
identified base position is then filled with a nuclease resistant
base, and the reaction is repeated to determine a matching base for
the next available position on the DNA strand to be sequenced. In
this example, the amplified chemical products 130 are detected
electronically and sequence data for the immobilized DNA molecules
is assembled. Amplified chemical products 130 in a reaction region
110, such as, for example, a gate or an extended gate of a FET,
change the ionic conditions or pH of the region and thus alter the
current flow between the source and the drain. In the case in which
sensors are electrodes, changes in chemical products change the
capacitance and or resistance of the sensor electrodes. Reaction
conditions and their corresponding positions and electronic signals
are recorded and analyzed with a computer and software.
[0022] Referring now to FIG. 2, an exemplary test and fill chemical
reaction is shown. In this reaction, a portion of a DNA molecule
205 is sequenced through testing with four nucleotides. In one
embodiment, regular dNTPs (deoxynucleoside triphosphates) are used
and a determination is made as to which one is incorporated into
the primed DNA molecule. In further embodiments, non-natural dNTPs
are used to determine sequence information through incorporation
and excision reactions. In FIG. 2, a DNA molecule to be sequenced
205 is primed with a primer 210 that is terminated with an
exonuclease resistant nucleotide which, in this example, is a
thymine (nuclease resistance being indicated in FIG. 2 with a " ").
The chemical products resulting from the incorporation of a
complementary dNTP deoxynucleoside triphosphate, e.g., dATP
(deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate),
dGTP (deoxyguanosine triphosphate), or dTTP (deoxythymidine
triphosphate), for example) or dNTP analog, complementary to a base
of the nucleic acid strand to be sequenced 205 are amplified
through the repeated addition and excision of the next
complementary nucleoside onto the priming sequence 210, In general,
a test reaction comprises a polymerase, an exonuclease, a
deoxynucleoside triphosphate (dATP (deoxyadenosine triphosphate),
dCTP (deoxycytidine triphosphate), dGMP (deoxyguanosine
triphosphate), dTMP (deoxythymidine triphosphate)) or a nucleoside
tetra- or pentaphosphate), and optionally a pyrophosphatase. A
nucleoside is incorporated into the primed growing DNA molecule
that is terminated with a nuclease resistant base through the
action of a polymerase enzyme. Typical useful polymerase enzymes
include DNA polymerases with or without 3' to 5' exonuclease
activities, such as for example, E. coli DNA polymerase I, Klenow
fragment of E. Coli DNA polymerase I, exo-minus Klenow fragment of
E. Coli DNA polymerase I (Klenow exo- or exonuclease free klenow
polymerase), Therminator DNA polymerase, reverse transcriptase, Taq
DNA polymerase, Vent DNA polymerase (all available from New England
Biolabs, Inc., Beverly, Mass.), T4 DNA polymerase, and Sequenase
(both available from USB, Cleveland, Ohio). Thus where there is a
cytosine on the strand to be sequenced, a guanine will be
incorporated, where there is a thymidine, an adenine will be
incoporated, and vice versa. If the nucleoside triphosphate is
incorporated into the growing strand in the test reaction, then
pyrophosphate (PPi) is released. The pyrophosphate can optionally
be degraded into two inorganic phosphates through ionic
dissociation caused by water and catalyzed by pyrophosphatase. In
an amplification reaction, an exonuclease is used to remove the
incorporated nucleoside monophosphate (NMP.sup.-2), allowing
another nucleoside triphosphate to be incorporated and a PPi to be
released. Repetition of these reactions provides linear
amplification of PPi and or inorganic phosphates. Thus, a positive
test reaction indicates that the base on the template DNA strand to
be sequenced immediately after the priming base (the 3' base) of
the primer strand is complementary to the test base introduced into
the reaction. To sequence the next base on the template, the first
identified base on the primer strand is filled or replaced with a
nuclease-resistant nucleotide that then becomes the priming base
for the test reaction. Nuclease-resistant nucleotides can be
ribonucleotides or other modified nucleotides. A variety of
polymerases are available that can incorporate ribonucleotides or
modified nucleotides into DNA, such as for example, the
commercially available Therminator DNA polymerases (available from
New England Biolabs, Inc., Beverly, Mass.) or genetically
engineered DNA polymerase. See also, for example, DeLucia, A. M.,
Grindley, N. D. F., Joyce, C. M., Nucleic Acids Research, 31:14,
4129-4137 (2003); and Gao, G., Orlova, M., Georgiadis, M. M.,
Hendrickson, W. A., Goff, S. P., Proceedings of the National
Academy of Sciences, 94, 407-411 (1997), Exemplary nuclease
resistant bases that can be incorporated into growing DNA strands
but that are resistant to digestion by exonucleases (such as the 3'
to 5' exonuclease active DNA polymerases or exonuclease I or III)
include alpha phosphorothioate nucleotides (available from Trilink
Biotechnologies, Inc., San Diego, Calif.). Additionally,
ribonucleotides can be incorporated into a growing DNA strand by
Therminator DNA polymerase or other genetically engineered or
mutated polymerases, but the ribonucleotide bases are resistant to
digestion by exonucleases, such as exonucleases I or exonuclease
III (available from New England Biolabs), Exemplary nucleases that
cannot digest these resistant bases include exonuclease I, nuclease
III, and 3' to 5' exonuclease active DNA polymerases.
[0023] In FIG. 2, to sequence the next base on the template, the
first identified base on the primer strand 210 is filled or
replaced with an identified nuclease-resistant nucleoside that then
becomes the priming base for the next test reaction after
deblocking. Optionally, the nucleoside is nuclease-resistant
blocking nucleoside (3' blocking is indicated with a ".degree." in
FIG. 2). In general, blocking nucleosides prevent further nucleic
acid synthesis by reversibly blocking the addition of a nucleic
acid to the end of the nucleic acid molecule. Nuclease-resistant
blocking nucleosides are, for example, ribonucleosides or other
modified nucleosides and are described more fully herein. A variety
of polymerases are available that can incorporate ribonucleotides
or modified nucleosides into DNA, such as for example, the
commercially available Therminator DNA polymerases (available from
New England Biolabs, Inc., Ipswitch, Mass.). See also, for example,
DeLucia, A. M., Grindley, N. D. F., Joyce, C. M., Nucleic Acids
Research, 31:14, 4129-4137 (2003); and Gao, G., Orlova, M.,
Georgiadis, M. M., Hendrickson, W. A., Goff, S. P., Proceedings of
the National Academy of Sciences, 94, 407-411 (1997). Exemplary
nuclease resistant bases include alpha-phosphorothioate nucleosides
having different chiralities, and exemplary nucleases that cannot
digest the specific chiral isomer of the phosphorothioate bond
include polymerase associated exonuclease such as the exonuclease
activity of T4 or T7 Polymerase (which can not digest S-chiral
conformation of the phosphorothioate bond) or a 3' to 5'
exonuclease with chiral specificity (such as Exonuclease III which
would not digest R-chiral conformation of phosphorothioate bond).
Some polymerase enzymes possess intrinsic exonuclease activity
therefore it is not always necessary to use two different enzymes
for the addition and excision reactions. Reactions in which no
significant amount of product is detected indicate that the test
reaction provided a nucleotide that was not complementary to the
next base of the nucleic acid to be sequenced. After addition of
the next known complementary nucleotide to the primer 210, the
primer 210 is deblocked through removal of the 3' blocking group
and the identity of the next complementary nucleotide is determined
by repeating the test reactions as described above.
[0024] Reversible terminators that have been modified at the 3'
position with, for example, 3'-azidomethyl or 3'-allyl, are cleaved
chemically to deblock the nucleotide, using for example, TCEP
(tricarboxylethylphosphine) for 3'-azidomethyl and aqueous Pd-based
catalyst to remove 3'-allyl group, and 3'O-nitrobenzyl blocking
groups are cleaved photochemically.
[0025] In an embodiment of the invention, the nuclease resistant
nucleoside is the molecule shown in FIG. 3, an alpha-thiophosphate
nucleoside. In FIG. 3, the base, B, is adenine (A), cytosine (C),
guanine (G), thymine (T), uracil (U), or an analog thereof, such
as, for example, a modified base, a hydrophobic base analog, such
as for example, 2,4-difluoro-5-toluene (Kool E. T., Acc. Chem.
Res., 35, 936-943 (2002)), universal base (3-nitropyrrole or
5-nitroindole) or isoguanine and isocytosine; n is a number between
and including 2 and 4, and Y is H, azidomethyl, allyl, acetyl, or
O-nitrobenzyl. In embodiments of the invention, the
alpha-thiophosphate nucleoside is the R or the S enantiomer of the
thiophosphate group. The polyphosphate nucleoside of FIG. 3 is
optionally a blocking nucleoside, and is not a blocking nucleotide
when Y is H. Optionally, the nucleotide of FIG. 3 is provided as an
acid or a neutral salt that is compatible with nucleic acid
synthesis reactions, such as for example, as a H.sup.+, Na.sup.+,
K.sup.+ triethylamine, or tributlyamine.
[0026] According to embodiments of the invention, the primer
molecule that is hybridized to the nucleic acid molecule to be
sequenced is terminated with a nucleoside as shown in FIG. 3 that
is either blocking or not blocking. Sequencing reactions involving
nucleoside polyphosphate incorporation and excision as described
herein are performed. The one or more enzymes used to add and
excise a next complementary nucleoside polyphosphate are not able
to digest the alpha-thiophosphate nucleoside that terminates the
primer molecule. In an embodiment, the alpha-thiophosphate
nucleoside that terminates the primer molecule is the R enantiomer
of the thiophosphate group. An exemplary enzyme that cannot digest
alpha-thiophosphate nucleosides is exonuclease III (Exo III). More
specifically, Exo III cannot digest the R enantiomer of the
thiophosphate group. In an embodiment of the invention, a
combination of Klenow (exo-) polymerase enzyme lacking the 3' to 5'
exonuclease activity) and Exo III (an exonuclease) are used in
sequencing reactions in which a nucleoside polyphosphate is
incorporated and excised repeatedly. An enzyme capable of
incorporating an alpha-thiolated nucleoside according to FIG. 3 is
Therminator II or III or .gamma. polymerase (ThII or Th III Pol. or
Th-.gamma. Pol.). After the identity of a base of the molecule to
be sequenced is determined, the position is filled with a
complementary alpha-thiolated nucleoside oligophosphate (tri-,
tetra-, or penta-phosphate).
[0027] Referring now to FIG. 4, a reaction that provides chemical
signal amplification for nucleotide incorporation into DNA is
shown. A test reaction for a single base position in a DNA molecule
can also be considered to be a chemical amplification reaction in
which many dNTP molecules are converted into pyrophosphate and
dNMP. In this reaction, a pyrophosphate can be further converted
into two phosphates by, for example, pyrophosphatase and
spontaneous hydrolysation. Thus, as can be seen in FIG. 3, after
incorporation of a nucleotide into a primed DNA molecule through,
for example, the action of a polymerase enzyme, the nucleotide can
then be deleted through, for example, the action of an exonuclease
enzyme. These reactions can be repeated many times for the same
incorporation event on a DNA molecule, resulting in the consumption
of many nucleotide triphosphates and the generation of many
molecules of pyrophosphate and/or phosphate. The generation of
pyrophosphate and/or phosphate can affect the local pH (by
providing a net proton (H.sup.+) increase) and the ionic strength
of the reaction solution. Since each dNTP has four ionizable groups
and its products have a total of eight ionizable groups, a net
increase of four ionizable groups and 4 protons (H.sup.+) is
realized through the incorporation-deletion reaction cycle. The
incorporation-deletion reaction cycle can be repeated many times as
long as a matched dNTP is present. For example, for ten rounds of
incorporation-deletion cycles, assuming a reaction cavity having
the dimensions of 100 nm in diameter and 100 nm in depth, the
change in ionizable groups would be equal to 85 .mu.m of monovalent
ions. The increase in acidity and/or ionic strength can be sensed
electronically, such as for example, with a reaction cavity coupled
to a FET sensor.
[0028] FIG. 5 shows how parallel sequencing of more than one DNA
molecule can be accomplished according to embodiments of the
present invention. In FIG. 4, four DNA molecules to be sequenced
are placed in four separate sensor cells. After each fill reaction
with a nuclease-resistant nucleotide, for example A (indicated in
FIG. 5 as A*), a test reaction is carried out with three other
regular nucleotides, such as for example, C, G, and T, separately
and sequentially (it is not necessary that the nucleotides be
tested in any particular order) to identify the next complementary
nucleotide (base) for each DNA molecule that is being sequenced,
The next filling nuclease resistant nucleotide should be the one
identified in the test reaction as the next complementary
nucleotide. These reactions are then repeated to sequence the DNA.
Thus, sequence information for each DNA molecule is obtained from
the results of positive test reactions for sequences without
repetitive bases. Sequence information for repeated nucleotides is
determined based on the amplitude of the measured chemical and/or
electronic signals. For sequences having repetitive bases, more
phosphates are generated per test reaction as compared to a single
base, therefore the quantity difference can be used to determine
the number of bases in a repetitive sequence. Alternatively, a
stronger exonuclease activity can be used so that the nucleotide
deletion rate is much higher than the nucleotide insertion
(extension)rate in the test reaction, and then a mixture of regular
dNTP and nuclease resistant nucleotides (NRN) is used for a fill
reaction with a polymerase and a strong exonuclease. Redundant
reactions can optionally be used to increase accuracy.
[0029] Referring now to FIG. 6, a sensor cell according to an
embodiment of the present invention is shown. In the cell the
amplified chemical signals from the TAF reactions can be converted
into an electronic signal by an electronic sensor. For example, the
sensor can be a P-type FET, a N-type FET, or a carbon nanotube
transistor. See, for example, Janicki, M., Daniel, M., Szermer, M.,
Napieralski, A., Microelectronics Journal, 35, 831-840 (2004) and
Rolka, D., Poghossian, A., Schoning, M., Sensors, 4, 84-94 (2004).
In one embodiment, each sensor has a nano-sized reaction cavity and
a semiconductor transistor that are separated by an insulating
layer. The insulating layer can, for example, be made from silicon
oxide, silicon nitride, aluminum nitride, and or silicon
oxynitride. The channel of the semiconductor transistor, for
example, can be comprised of a P- or N-type semiconductor, as is
well known in the art, such as for example, silicon or germanium
doped with boron, arsenic, phosphorous, or antimony. A solution in
the reaction cavity forms a gate and the components of the sensor
are typically placed on a substrate, The source electrode and the
drain electrode are typically comprised of conducting materials, as
are well know in the art of chip fabrication, such as for example,
gold, copper, silver, platinum, nickel, iron, tungsten, aluminum,
or titanium. The substrate can be comprised of, for example,
silicon, silica, quartz, germanium, or polysilicon. In a further
embodiment, the reaction cavity has dimensions of less than about
100 nm. The reaction cavity is used as part of the gate of the
transistor. DNA can be immobilized in the reaction cavity by
standard methods, such as for example, through biotin-avidin or
antibody-antigen binding. Biotin, avidin, antibodies, or antigens
can be attached, for example, to an insulating layer comprised of
silicon oxide through derivatization of the silica surface with,
for example, (3-aminopropyl)triethoxysilane to yield a surface that
presents an amine group for molecule attachment. The molecule can
be attached by using water-soluble carbodiimide coupling reagents,
such as EDC (1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide), which
couples carboxylic acid functional groups with amine groups. DNA
molecules bearing a corresponding coupling agent can then be
attached through the surface through, for example, a biotin-avidin
or antibody-antigen interaction. Additionally, acrydite-modified
DNA fragments can be attached to a surface modified with thiol
groups and amine-modified DNA fragments can be attached to epoxy or
aldehyde modified surfaces. In operation, variations in the
potential between the solution (the gate) in the reaction cavity
and the insulator surface modify the charge distribution in the
channel. Changes in the solution, such as changes in charge
distribution created by the linearly amplified PPi molecules and
protons, can be measured by changes in the conductivity or changes
in the capacitance across the channel,
[0030] In additional embodiments, the sensor can comprise a carbon
nanotube transistor. Carbon nanotube FET devices have been
described. See, for example, Star, A., Gabriel, J. P., Bradley, K.,
Gruner, G., Nano Letters, 3:4, 459-463 (2003) and Fritz, J, Cooper,
E. B., Gaudet, S., Sorger, Manalis, S. R., Proceedings of the
National Academy of Sciences, 99:22, 4984-4989 (2002). In general,
carbon nanotubes, such as for example, single-walled carbon
nanotubes (SWNTs), that are useful in a FET device, can be made
through the chemical vapor deposition of methane onto catalytic
iron nanoparticles, Metal evaporation through a mask can be used to
create the electrical contacts that form a source and a drain. DNA
can be attached to the carbon nanotube transistor, for example, by
coating the carbon nanotube with Tween-20.TM. or polyethylene
oxide, which readily adsorb to the surface of the nanotube,
activating the Tween-20.TM. or polyethylene oxide-containing
polymer with a water-soluble carbodiimide coupling reagent, such as
for example, 1,1-carbonyldiimidazole, for conjugation with coupling
agents such as biotin, avidin, antigens, or antibodies. DNA
molecules having a corresponding coupling agent can then be
attached through the surface through, for example, a biotin-avidin
or antibody-antigen interaction.
[0031] Referring now to FIG. 7, an array of electronic sensors is
shown. For simplicity, the array is shown having five rows and five
columns of sensors, however the invention is not so limited and
arrays can be built having a variety of dimensions and numbers of
sensor regions. For example, arrays of sensors could be
10.times.10, 100.times.100, 1,000.times.1,000,
10.sup.5.times.10.sup.5, and 10.sup.6.times.10.sup.6. In FIG. 7,
the sensors are depicted as FET sensors that are connected to a
source line 700 and a drain line 710. A reaction region 720 is
shown in FIG. 7 having circular dimensions, however embodiments of
the present invention are not so limited and other shapes and
dimensions are possible, such as for example, those having
rectangular or other multisided configurations are possible. The
reaction region 720 forms part of the transistor gate. The FET
sensors 730 can be monitored individually or as a group, The sensor
array allows many immobilized DNA molecules to be sequenced
simultaneously. The immobilized DNA molecules can either be a
sample to be sequenced or capture DNA probes of known sequence can
be first immobilized and then the sample to be sequenced can be
hybridized to the immobilized probes. The capture probes have a
sequence designed to hybridize to sections of the sample DNA.
Typically, DNA fragments to be immobilized are diluted so that
statistically each sensor has one DNA molecule immobilized.
Information from FET sensors showing ambiguous results can be
disregarded. Sequence information is assembled from the sensors
having a single DNA molecule immobilized. Chemical information,
such as for example a change in pH or in ionic concentration, from
each reaction cavity is sensed independently. Micro and
nano-structures on the array can be built to minimize diffusion.
For example, wells can be built over each sensor, the sensor array
can be placed upside down, well facing down, with the temperature
in the down side lower than the chip side, and a low melting point
gel (such as low melting point agarose) can be used to make the
reaction mixture. Standard silicon and semiconductor processing
methods allow a highly integrated sensor array to be made. For
example, a 1 cm.sup.2 silicon wafer chip can hold as many as
1.times.10.sup.8 sensors that are about 1 .mu.m.sup.2 and that
present a 0.1 .mu.m opening to the array surface. For example, we
calculate that only about 300 sensor arrays would be needed to
sequence the whole human genome in about an hour, assuming that
such an array having 1.times.10.sup.8 sensors is a 100 M-sensor
array, that 10% of the sensors on the array yield single molecule
sequencing information, that each immobilized DNA molecule provides
10 bases worth of sequencing information, that 90% of the sequences
are overlapped, and assuming conservatively that it takes about 6
minutes to determine sequence information for each base (one test
and fill cycle).
[0032] Referring now to FIG. 8, an additional design for a sensor
array having reaction cavities with downward facing openings is
shown. In this array design, a temperature gradient can be used to
minimize diffusion from the reaction cavity, The temperature
gradient is created, for example, by Peltier thermoelectric devices
placed above and below the sensor array. A temperature gradient is
maintained so that the temperature below the sensor array (T2) is
less than the temperature above the array (T1). Such a temperature
gradient reduces convection and thereby limits the diffusion of the
amplified reaction products out of the reaction cavity. A
temperature gradient, for example, may be created by setting T1 at
about 37.degree. C. and T2 to about 35.degree. C. or less (for
example, 30.degree. C. or 20.degree. C.). A DNA molecule to be
sequenced can be immobilized within the reaction cavity. A
substrate to house the sensor array and inlet channels and outlet
channels that are for supplying and removing reagents are
provided.
[0033] In further embodiments of the present invention, chemical
signal amplification can be used in conjunction with optical
detection in test and fill sequencing reactions, FIG. 9 provides a
reaction scheme whereby the results of a test reaction for DNA
sequencing are amplified and converted into a product that can be
optically detected. In the initial reaction depicted in FIG. 9,
after incorporation of a nucleotide into the DNA primer that
matches the base on the DNA to be sequenced through the action of a
polymerase enzyme, the nucleotide is then deleted through the
action of an exonuclease enzyme. These incorporation and deletion
reactions are repeated many times on the same DNA molecule
resulting in the consumption of many nucleotide triphosphates (NTP)
and the generation of many molecules of phosphate and or
pyrophosphate. Phosphate ions generated from test phosphate
amplification processes can be reacted, for example, with molybdate
ions to form phosphomolybdate. The abundance of phosphomolybdate
can be quantified optically by absorbance measurements at about 340
nm. Additionally, phosphomolybdate can be reduced and the resulting
molybdenum blue optically detected at about 600 to about 840 nm,
Useful reducing agents include, for example, aminonapthosulfonic
acid, ascorbic acid, methyl-p-aminophenol sulfate, and ferrous
sulfate. Alternatively, pyrophosphate molecules that are amplified
from a test reaction can be converted to ATP by ATP sulfurase, and
photons emitted by luciferase. See, for example, Ronaghi et al,
Science, 281, 363-365 (1998). In another embodiment, phosphate and
pyrophosphate ions form resorufin in presence of maltose, maltose
phosphorylase, glucose oxidase, horse radish peroxidase, and Amplex
Red reagent (10-acetyl-3,7-dihydroxyphenoxazine). The resorufin can
be detected fluorometrically or spectrophotometrically. See, for
example, The Handbook--A Guide to Fluorescent Probes and Labeling
Technologies Molecular Probes, Section 10.3, available from
Invitrogen.
[0034] FIG. 10 provides a schematic demonstrating a method by which
signals from amplified TAF reactions can be detected optically.
Amplified chemical signals from the TAF reactions are converted to
an optical signal which is then converted into an electrical signal
by an optical detector, for example, a photodiode, CMOS, or CCD
detector. In one embodiment, each sensor has a nano-sized reaction
cavity in the middle, a light emitting diode on one side, and a
photodiode detector on the opposite side. Ideally the cavity is
less than about 100 nm, however larger sizes are also useful, such
as for example, cavities that have dimensions that are less than
about 10 .mu.m in size. Optionally, a bandpass filter, which can be
for example, a dielectric filter, can be placed between a
photodiode/light emitting diode and the reaction cavity to tailor
the wavelength of light transmitted. DNA can be immobilized in the
reaction cavity by standard methods, such as streptavidin/biotin
binding method. The geometry of each reaction cavity (size, depth,
shape and orientation) can be optimized to minimize reaction time.
Porous silicon substrates can be used to hold more DNA molecules
and accordingly increase the sensitivity. Porous silicon substrates
can be used to hold DNA molecules within a smaller area.
Optionally, the whole sensor can be packaged in an optically opaque
material so that external light does not generate background
noise.
[0035] In FIG. 11, a system that can be used for sequencing is
shown. The system contains a fluidic system that regulates reagent
delivery and waste removal, a computer that collects and analyzes
sequence data, and an array reader that retrieves signals from the
array. The array reader can be either an FET sensor array reader or
an optical sensor array reader. Reagent delivery and array washing
can be controlled, for example with an electronic valve system.
Fluid delivery can be controlled by a computer that sends signals
to valve drives (not pictured) that control the electronic valve
system.
[0036] Array compositions may include at least a surface with a
plurality of discrete reaction cavities. The size of the array will
depend on the end use of the array. Arrays containing from about
two to many millions of different discrete reaction cavities can be
made. Generally, the array size will depend in part on the size of
the surface from which the array is made. Very high density, high
density, moderate density, low density, or very low density arrays
can be made. Some ranges for very high-density arrays are from
about 100,000,000 to about 1,000,000,000 cavities per array.
High-density arrays range from about 1,000,000 to about 100,000,000
cavities. Moderate density arrays range from about 10,000 to about
100,000 cavities. Low-density arrays are generally less than 10,000
cavities. Very low-density arrays are less than 1,000 cavities.
[0037] The reaction cavities can comprise a pattern or a regular
design or configuration or can be randomly distributed. A regular
pattern of cavities can be used such that the cavities can be
addressed in an X-Y coordinate plane. The surfaces within the
cavities can be modified to allow attachment of analytes in
individual cavities. In general, reaction cavities are a depression
or well in the surface of the substrate that is capable of
containing a liquid.
[0038] There are numerous suitable methods for patterning an array
of nanoscale features on a surface of a substrate. Examples of such
suitable methods include lithography methods such as, for example,
interferometric lithography (IL), immersion interferometric
lithography, electron beam lithography, scanning probe lithography,
nanoimprint, extreme ultraviolet lithography, and X-ray
lithography, and stamping, etching, microetching, and molding
techniques. The technique used will depend in part on the
composition and shape of the substrate. Generally, lithography is a
highly specialized printing process used to create detailed
patterns on a substrate, such as a silicon wafer. An image
containing a desired pattern is projected onto the wafer, which is
coated by a thin layer of photosensitive material called resist.
The bright parts of the image pattern cause chemical reactions
which, in turn, render the resist material soluble, and, thus,
dissolve away in a developer liquid, whereas the dark portions of
the image remain insoluble. After development, the resist forms a
stenciled pattern across the wafer surface, which accurately
matches the desired pattern. Finally, the pattern is permanently
transferred into the wafer surface, for example by a chemical
etchant, which etches those parts of the surface unprotected by the
resist.
[0039] In various embodiments of the invention, arrays may be
incorporated into a larger apparatus and/or system. In certain
embodiments, the substrate may be incorporated into a
micro-electro-mechanical system (MEMS). MEMS are integrated systems
comprising mechanical elements, sensors, actuators, and
electronics. All of those components may be manufactured by known
microfabrication techniques on a common chip, comprising a
silicon-based or equivalent substrate (See for example, Voldman et
al., Ann. Rev. Biomed. Eng., 1:401-425 (1999).) The sensor
components of MEMS may be used to measure mechanical, thermal,
biological, chemical, optical and/or magnetic phenomena. The
electronics may process the information from the sensors and
control actuator components such as pumps, valves, heaters,
coolers, and filters, thereby controlling the function of the
MEMS.
[0040] The electronic components of MEMS may be fabricated using
integrated circuit (IC) processes (for example, CMOS, Bipolar, or
BICMOS processes). The components may be patterned using
photolithographic and etching methods known for computer chip
manufacture. The micromechanical components may be fabricated using
compatible micromachining processes that selectively etch away
parts of the silicon wafer or add new structural layers to form the
mechanical and/or electromechanical components.
[0041] Basic techniques in chip manufacture include depositing thin
films of material on a substrate, applying a patterned mask on top
of the films by photolithographic imaging or other known
lithographic methods, and selectively etching the films. A thin
film may have a thickness in the range of a few nanometers to 100
micrometers. Deposition techniques of use may include chemical
procedures such as chemical vapor deposition (CVD),
electrodeposition, epitaxy and thermal oxidation and physical
procedures like physical vapor deposition (PVD) and casting.
[0042] In some embodiments of the invention, substrates may be
connected to various fluid filled compartments, such as
microfluidic channels, nanochannels, and or microchannels. These
and other components of the apparatus may be formed as a single
unit, for example in the form of a chip, such as semiconductor
chips and or microcapillary or microfluidic chips. Alternatively,
the substrates may be removed from a silicon wafer and attached to
other components of an apparatus. Any materials known for use in
such chips may be used in the disclosed apparatus, including
silicon, silicon dioxide, silicon nitride, polydimethyl siloxane
(PDMS), polymethylmethacrylate (PMMA), plastic, glass, and
quartz.
[0043] Techniques for batch fabrication of chips are well known in
the fields of computer chip manufacture and or microcapillary chip
manufacture. Such chips may be manufactured by any method known in
the art, such as by photolithography and etching, laser ablation,
injection molding, casting, molecular beam epitaxy, dip-pen
nanolithography, chemical vapor deposition (CVD) fabrication,
electron beam or focused ion beam technology or imprinting
techniques. Non-limiting examples include conventional molding with
a flowable, optically clear material such as plastic or glass;
photolithography and dry etching of silicon dioxide; electron beam
lithography using polymethylmethacrylate resist to pattern an
aluminum mask on a silicon dioxide substrate, followed by reactive
ion etching. Methods for manufacture of nanoelectromechanical
systems may be used for certain embodiments of the invention. See
for example, Craighead, Science, 290:1532-36, (2000), Various forms
of microfabricated chips are commercially available from, for
example, Caliper Technologies Inc. (Mountain View, Calif.) and
ACLARA BioSciences Inc. (Mountain View, Calif.).
EXAMPLE
[0044] Sensor Array Fabrication: A field effective transistor array
having 10.sup.8 sensors that are pH sensitive is fabricated. The
array is a Al--Si--SiO.sub.2--Ta.sub.2O.sub.5 structure, fabricated
from a p-Si wafer with specific receptivity of 1-10 Ohm/cm. A
double layer that consists of 65 nm SiO.sub.2 and 67 nm
Ta.sub.2O.sub.5 which is made by thermal oxidation of sputtered Ta
(Rolka, D., Poghossian, A., Schoning, M., Sensors, 4, 84-94
(2004)). Each sensor in the array is connected to an electronic
control board for signal amplification and processing. Wells of
1.times.1.times.1 .mu.m are constructed over each sensor using
SiO.sub.2 by standard photolithography techniques. The SiO.sub.2
surface is modified to present free aldehyde groups through an
aldehyde trimethoxysilane process (Lobert, P. E., Hagelsieb, L. M.,
Pampin, R., Bourgeois, D., Akheyar, A., Flandre, D., Remacle, J.,
Sensors & Actuators B, Section .mu.Tas, (2002)). Streptavidin
is used to coat the well surface surrounding each sensor by
sheriff's reaction. Multiple streptavidin molecules are attached to
the well wall surface and the uncoated surface is blocked by BSA
(bovine serum albumin) molecules. The density of streptavidin
molecules on the wall can be adjusted by varying the ratio of
streptavidin and BSA. Ideally, the distance between two
streptavidin molecules is greater than about 50 nm. The array
device is sandwiched between two peltier thermoelectrical coolers,
which can be programmed and controlled by a computer.
[0045] Fluidic control: the sensor array surface is enclosed in a
chamber made of plastic, with an inlet and outlet, The inlet is
connected to reagent reservoirs and the outlet is connected to a
waste chamber. Several reagent reservoirs are kept separate.
[0046] Reagents: major reagent solutions include: 1) Reaction
buffer (also used as washing solution): 50 mM NaCl, 10 mM Tris-HCl
(pH 7.9), 10 mM MgCl.sub.2, 1 mM dithiothreitol, 100 .mu.g/ml BSA;
2) Regular deoxyribonucleotides, dATP, dCTP, dGTP, dTTP, each kept
separately at 100 .mu.M in reaction buffer; 3)
alpha-phosphorothioate nucleotides, each kept separately at 50
.mu.M in reaction buffer; 4) enzyme mix solution: T4 DNA polymerase
at 0.01 unit/.mu.l (with DNA polymerase activity and strong 3' to
5' exonuclease activity in the same enzyme) and pyrophosphotase at
0.001 unit/.mu.l in reaction buffer,
[0047] DNA sample preparation: The sensor chip is a universal chip
for DNA sequence detection, depending on DNA sample used. In this
example, bacterial contamination in water is to be determined. A
sample of water to be tested (1 L) is concentrated using a 0.22
.mu.m filter. DNA is extracted from the sample with a DNA
purification kit (Qiagen), and digested to an average length of 100
bp by DNase I digestion. The 3' end of the DNA fragments are
modified by biotin labeled alpha-phosphorothioate
dideoxyribonucleotides in a terminal transferase reaction, in which
dATP and the biotin nucleotide is in a 30:1 ratio, The modified DNA
fragments will have a poly A tail, terminated with a nuclease
resistant biotin-labeled nucleotide.
[0048] DNA template immobilization: About 1 pg of the modified DNA
fragments (about 1.times.10.sup.8 copies) is added to the chip so
that the DNA fragments are captured to the sensor well (1
fragment/senor on average; the distance between 2 streptavidin
molecules are longer than the length of 100 bp DNA,) by
biotin-strepavidin binding. The capture double-stranded DNA
molecules are denatutred by washing the chip with an alkaline
solution (50 mM, NaOH). The chip is neutralized and nuclease
resistant oligo dT primers (a set of 3 each terminated with with A,
C, and G, respectively) are hybridized to the immobilized
single-stranded DNA molecules.
[0049] Sequence detection operation: After immobilizing DNA and
loading the reagents into the system (all reagents and chips are
set at 4.degree. C.), the sequence detection reaction can start. 1)
The chip is washed and primed with the reaction buffer; 2) one of
the 4 regular nucleotides (dCTP, for example) is mixed with the
enzyme solution in 1:1 volume; 3) the mixture is introduce to the
chip to replace the priming buffer (all at 4.degree. C.), some
mixing in the chip is necessary to ensure all sensors received the
reagent at the same concentration; 4) the chip concentration is
raised to 37.degree. C. and for 3 min.; 5) a signal is recorded
from each sensor; 6) the sensor is cleaned with reaction buffer; 7)
the tested base (dCTP) is filled with the same base
(alpha-phosphorothioate dCTP) for 2 min.; 8) the chip is cleaned
with reaction buffer; 9) the preceding steps are repeated for each
of the other bases; and 10) the whole reaction cycle (steps 1-9) is
repeated.
[0050] Data analysis: Recorded data from each sensor is analyzed.
Sensors with no information or unidentifiable information from each
step are ignored for further data analysis. The rest of the data is
analyzed as follows: the frequency of given sequences is
calculated, the sequence information is analyzed based on sequence
alignment based on fragments of similar frequency to generate
longer fragment information, and the assembled sequence is searched
against a database of know sequences.
* * * * *