U.S. patent application number 12/361690 was filed with the patent office on 2009-11-19 for method and compositions for detection and enumeration of genetic variations.
This patent application is currently assigned to The Johns Hopkins University. Invention is credited to Devin DRESSMAN, Kenneth Kinzler, Bert Volgelstein, Hai Yan.
Application Number | 20090286687 12/361690 |
Document ID | / |
Family ID | 34107717 |
Filed Date | 2009-11-19 |
United States Patent
Application |
20090286687 |
Kind Code |
A1 |
DRESSMAN; Devin ; et
al. |
November 19, 2009 |
Method and Compositions for Detection and Enumeration of Genetic
Variations
Abstract
Many areas of biomedical research depend on the analysis of
uncommon variations in individual genes or transcripts. Here we
describe a method that can quantify such variation at a scale and
ease heretofore unattainable. Each DNA molecule in a collection of
such molecules is converted into a single particle to which
thousands of copies of DNA identical in sequence to the original
are bound. This population of beads then corresponds to a
one-to-one representation of the starting DNA molecules. Variation
within the original population of DNA molecules can then be simply
assessed by counting fluorescently-labeled particles via flow
cytometry. Millions of individual DNA molecules can be assessed in
this fashion with standard laboratory equipment. Moreover, specific
variants can be isolated by flow sorting and employed for further
experimentation. This approach can be used for the identification
and quantification of rare mutations as well as to study variations
in gene sequences or transcripts in specific populations or
tissues.
Inventors: |
DRESSMAN; Devin; (Baltimore,
MD) ; Yan; Hai; (Chapel Hill, NC) ; Kinzler;
Kenneth; (Baltimore, MD) ; Volgelstein; Bert;
(Baltimore, MD) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
1100 13th STREET, N.W., SUITE 1200
WASHINGTON
DC
20005-4051
US
|
Assignee: |
The Johns Hopkins
University
Baltimore
MD
|
Family ID: |
34107717 |
Appl. No.: |
12/361690 |
Filed: |
January 29, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10562840 |
Jun 22, 2006 |
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PCT/US04/15587 |
Jun 9, 2004 |
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12361690 |
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60485301 |
Jul 5, 2003 |
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60525859 |
Dec 1, 2003 |
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Current U.S.
Class: |
506/2 |
Current CPC
Class: |
C12N 15/1075 20130101;
C12Q 1/6858 20130101; C12Q 1/6858 20130101; C07H 21/04 20130101;
C12Q 1/686 20130101; C12Q 2565/537 20130101; C12Q 2563/143
20130101; C12Q 2563/149 20130101 |
Class at
Publication: |
506/2 |
International
Class: |
C40B 20/00 20060101
C40B020/00 |
Goverment Interests
[0002] The invention disclosed herein was made using funds from the
National Institutes of Health grants CA 43460, CA 57345, and
CA62924. The United States government therefore retains certain
rights in the invention.
Claims
1. A method for sequencing nucleic acids comprising: (a)
fragmenting genomic nucleic acid molecules to generate a plurality
of fragmented nucleic acids; (b) delivering the fragmented nucleic
acids into aqueous microreactors in a water-in-oil emulsion such
that a plurality of aqueous microreactors comprise a single copy of
a fragmented nucleic acid, a single bead capable of hybridizing to
the fragmented nucleic acid, and amplification reaction solution
containing reagents necessary to perform nucleic acid
amplification; (c) amplifying the fragmented nucleic acids in the
microreactors to form amplified copies of said nucleic acids and
hybridizing the amplified copies to beads in the microreactors; (d)
delivering the beads to an array of reaction chambers, wherein a
plurality of the reaction chambers comprise no more than a single
nucleic acid bound bead; and (e) performing a sequencing reaction
simultaneously on a plurality of the reaction chambers.
2. The method of claim 1 wherein step (c) is accomplished using
polymerase chain reaction.
3. The method of claim 1 wherein the sequencing reaction is a
pyrophosphate-based sequencing reaction.
4. The method of claim 1 wherein the sequencing reaction comprises
the steps of: (a) annealing an effective amount of a sequencing
primer to the amplified copies of the nucleic acid and extending
the sequencing primer with a polymerase and a predetermined
nucleotide triphosphate to yield a sequencing product and, if the
predetermined nucleotide triphosphate is incorporated onto a 3' end
of said sequencing primer, a sequencing reaction byproduct; and (b)
identifying the sequencing reaction byproduct, thereby determining
the sequence of the nucleic acid in a plurality of the reaction
chambers.
Description
[0001] This application claims the benefit of application Ser. No.
60/485,301 filed Jul. 5, 2003 and 60/525,859, filed Dec. 1, 2003,
the contents of both of which are expressly incorporated
herein.
[0003] A portion of the disclosure of this patent document contains
material which is subject to copyright protection. The copyright
owner has no objection to the facsimile reproduction by anyone of
the patent document or the patent disclosure, as it appears in the
Patent and Trademark Office patent file or records, but otherwise
reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
[0004] The invention relates to the field of genetic analysis. In
particular, it relates to methods and compositions for analyzing
variations in individual genes or transcripts and separating
variants.
BACKGROUND OF THE INVENTION
[0005] The study of DNA sequence variation is essential for many
areas of research. The study of germ-line variations is essential
for assessing the role of inheritance in normal and abnormal
physiologic states (1). Other variations, developed somatically,
are responsible for neoplasia (2). The identification of such
mutations in urine, sputum, and stool can therefore be used for the
detection of presymptomatic cancers (3-5). Similarly, the detection
of somatic mutations in lymph nodes, blood, or bone marrow can
provide data about the stage of disease, prognosis, and
appropriateness of various therapies (5). Somatic mutations in
non-neoplastic cells also occur and appear to accumulate as humans
age or are exposed to environmental hazards (6). Such mutations
occur in only a small fraction of the cells in a tissue, thereby
complicating their analysis.
[0006] Central to the investigation of many of these issues is the
detection and quantification of sequence variants within a
population of DNA molecules. The number of molecules in each such
collection is finite and therefore countable. Consider, for
example, a collection of red and green balls. Counting these balls
is simple in principle but subject to basic probability theory. If
there is only one red ball for every 500 green balls, then it is
necessary to count several thousand balls to get an accurate
estimate of the proportion of red balls. If it is difficult to
count enough balls to make a reliable estimate, one can elute the
paint off all the balls and measure the color of the resultant
paint mix.
[0007] In analogous fashion, small numbers of DNA molecules that
vary by subtle changes (single base pair substitutions or small
deletions or insertions) can be directly counted by amplifying
individual DNA molecules (single molecule PCR) (7-12). Such digital
techniques have been shown to be extremely useful for measuring
variation in genes or their transcripts. But digital technologies
have so far been limited to counting tens to thousands of
molecules, either in the wells of microtiter plates, on microscope
slides, or after electrophoresis of individual PCR products. Analog
techniques, analogous to the elution of paint from the balls
described above, are generally easier to implement and can assess
millions of molecules simultaneously (13). However, their accuracy
and sensitivity is limited by instrumental and experimental noise.
There is a continuing need in the art for methods which are
accurate and sensitive for measuring variation in genes or their
transcripts.
BRIEF SUMMARY OF THE INVENTION
[0008] In a first embodiment of the invention a composition is
provided. The composition comprises a plurality of beads. Each of
the plurality of beads comprises a plurality of bound
polynucleotides. The polynucleotides in the composition are
heterogeneous; however, on at least 1% of said beads the plurality
of bound polynucleotides is homogeneous.
[0009] In a second embodiment of the invention a liquid composition
is provided. The liquid composition comprises a plurality of
microemulsions forming aqueous compartments. At least a portion of
said aqueous compartments comprise a bead, a polynucleotide
template, and oligonucleotide primers for amplifying the template.
At least a portion of the oligonucleotide primers is bound to the
bead.
[0010] A third embodiment of the invention provides a method for
analyzing nucleotide sequence variations. Microemulsions comprising
one or more species of analyte DNA molecules are formed. The
analyte DNA molecules in the microemulsions are amplified in the
presence of reagent beads which are bound to a plurality of
molecules of a primer for amplifying the analyte DNA molecules.
Product beads are formed that are bound to a plurality of copies of
a single species of analyte DNA molecule. The product beads are
separated from analyte DNA molecules which are not bound to product
beads. A sequence feature of the single species of analyte DNA
molecule that is bound to the product beads is determined.
[0011] A fourth embodiment of the invention is a probe for use in
hybridization to a polynucleotide that is bound to a solid support.
The probe comprises an oligonucleotide with a stem-loop structure.
At one of the 5' or 3' ends there is a photoluminescent dye. The
oligonucleotide does not comprise a quenching agent at the opposite
5' or 3' end.
[0012] A fifth embodiment of the invention is a pair of molecular
probes. The first and second probes each comprise an
oligonucleotide with a stem-loop structure having a first
photoluminescent dye at one of the 5' or 3' ends, and not
comprising a quenching agent at the opposite 5' or 3' end. The
first oligonucleotide hybridizes to a wild-type selected genetic
sequence better than to a mutant selected genetic sequence and the
second oligonucleotide hybridizes to the mutant selected genetic
sequence better than to the wild-type selected genetic sequence.
The first and the second photoluminescent dyes are distinct.
[0013] In a sixth embodiment of the invention a method is provided
for isolating nucleotide sequence variants. Microemulsions
comprising one or more species of analyte DNA molecules are formed.
Analyte DNA molecules in the microemulsions are amplified in the
presence of reagent beads. The reagent beads are bound to a
plurality of molecules of a primer for amplifying the analyte DNA
molecules. Product beads are formed which are bound to a plurality
of copies of one species of analyte DNA molecule. The product beads
are separated from analyte DNA molecules which are not bound to
product beads. The product beads which are bound to a plurality of
copies of a first species of analyte DNA molecule are isolated from
product beads which are bound to a plurality of copies of a second
species of analyte DNA molecule.
[0014] These and other embodiments of the invention, which will be
apparent from the entire description of the invention, provide the
art with the ability to quantify genetic variations at a scale and
ease heretofore unattainable.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a schematic drawing of the BEAMing method. Step
1--Magnetic beads covalently coated with streptavidin are bound to
biotinylated oligonucleotides ("oligos"). Step 2--An aqueous mix
containing all the necessary components for PCR plus primer-bound
beads and template DNA are stirred together with an oil/detergent
mix to create microemulsions. The aqueous compartments (white
circles in the gray oil layer) contain an average of <1 template
molecule and <1 bead. Red and green templates represent two
template molecules whose sequences differ by one or many
nucleotides. Step 3--The microemulsions are temperature cycled as
in a conventional PCR. If a DNA template and a bead are present
together in a single aqueous compartment, the bead bound
oligonucleotides act as primers for amplification. The straight red
and green lines connected to the beads represent extension products
from the two different kinds of templates. Step 4--The emulsions
are broken and the beads are purified with a magnet. Step 5--After
denaturation, the beads are incubated with oligonucleotides that
can distinguish between the sequences of the different kinds of
templates. Fluorescently-labeled antibodies are then used to label
the bound hybridization probes. This renders the beads containing
PCR product as red or green upon appropriate laser excitation. Step
6--Flow cytometry is used to count the red and green beads.
[0016] FIG. 2 is a photograph of a typical microemulsion.
Microemulsions were made as described infra with the exception that
the aqueous compartments contained cascade blue-labeled dCTP and
the beads were pre-labeled with R-phycoerythrin (red) or Alexa 488
(green). One microliter of microemulsion was deposited in 1
microliter of oil on a microscope slide prior to photography. Of
the seven aqueous compartments visible in this picture, two contain
beads. Note the heterogeneous size of the aqueous compartments
(beads are 1.05 microns in diameter).
[0017] FIG. 3A to FIG. 3D show density plots of flow cytometric
data obtained from BEAMing. The locus queried in this experiment
was MID42 and PCR products generated from genomic DNA were used as
templates in the microemulsions. (FIG. 3A) Forward scatter (FSC)
and side scatter (SSC) of all beads show that .about.80% of the
total beads are singlets, with most of the remaining beads
aggregated as doublets. The "noise" is instrumental and is observed
with blank samples containing no beads. The instrument output was
gated so that only singlets were analyzed for fluorescence
analysis. The patterns observed from an individual homozygous for
the L allele (FIG. 3C), homozygous for the S allele (FIG. 3B), and
heterozygous for L and S (FIG. 3D) are shown. The regions
containing beads hybridizing to the L and S allele probes are
labeled green and red, respectively. The region containing beads
that did not hybridize to any probe is black and the region
containing beads that hybridized to both probes is blue. The blue
beads arose from aqueous compartments in which both types of
template molecules were present. The proportion of singlet beads
that hybridized to at least one of the probes was 2.9%, 4.3%, and
20.3% in (FIG. 3B) to (FIG. 3D), respectively. The FSC and SSC
plots in (FIG. 3A) represent the same beads analyzed in (FIG.
3D).
[0018] FIG. 4A to FIG. 4D show density plots of BEAMing using
genomic DNA or RT-PCR products as templates. The data in (FIG. 4A)
and (FIG. 4B) were generated by including 10 and 1 ug of human
genomic DNA, respectively, in the microemulsions, querying the
MID42 locus. The data in (FIG. 4C) and (FIG. 4D) were generated
using emulsions that contained .about.50 picograms of PCR products
synthesized from cDNA of lymphoblastoid cells, querying the
calpain-10 locus. The green and red regions correspond to the L and
S alleles for MID42 and to the A and G alleles for calpain-10. The
number of beads in the outlined regions containing red or green
beads is shown in each case. The proportion of singlet beads that
hybridized to at least one of the probes was 1.2%, 0.6%, 6.8% and
4.2% in (FIG. 4A) to (FIG. 4D), respectively. The outlined regions
used for counting in (FIG. 4A) and (FIG. 4B) were identical, as
were those used for (FIG. 4C) and (FIG. 4D). Beads that did not
hybridize to any probe were gated out and therefore not evident in
the graphs, while the region containing beads that hybridized to
both probes is labeled blue.
[0019] FIG. 5A to FIG. 5C show detection and validation of variants
present in a minor fraction of the DNA population. (FIG. 5A)
Mixtures of PCR products containing 0% to 4% L alleles of MID42
were used for BEAMing. Flow cytometry such as that shown in FIG. 3
was used to determine the fraction of singlet beads that were red
(y-axis). The proportion of singlet beads that hybridized to at
least one of the probes varied from 3.2% to 4.3%. (FIG. 5B and FIG.
5C) Beads were sorted with the FACS Vantage SE instrument and
individual red or green beads were used as templates for
conventional PCR employing the forward and reverse primers listed
in FIG. 8. Red beads generated only the S allele sequence (FIG. 5B;
SEQ ID NO: 1) while green beads generated only the L allele
sequence (FIG. 5C; SEQ ID NO: 2).
[0020] FIG. 6A to 6B demonstrate the use of agar in the aqueous
phase of the microemulsions. Emulsion bubbles that were formed by
including 1.5% agarose in the aqueous compartment are shown. FIG.
6A shows the bubbles that have fluorescents in them. FIG. 6B shows
a darkfield image of the bubbles with one of the bubbles containing
a bead in it. After breaking the emulsions, the droplets containing
magnetic beads can be recovered by centrifugation and size
fractionated through filtration or flow sorting.
[0021] FIG. 7 shows denaturing electrophoresis of two FAM-labeled
oligonucleotides, 50 and 20 bases in length, which had been
hybridized to a 100 bp product on beads. The beads were embedded in
an acrylamide gel in an oval shaped configuration and an electric
field was applied The labeled oligonucleotides migrated off the
beads and migrated a distance proportional to their size.
[0022] FIG. 8 shows oligonucleotides used.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The inventors describe herein a digital technology, called
BEAMing, that has the power to assess millions of molecules and can
be generally applied to the study of genetic variation. The
technology involves conversion of single DNA molecules to single
beads each containing thousands of copies of the sequence of the
original DNA molecule. The number of variant DNA molecules in the
population can then be assessed, for example, by staining the beads
with fluorescent probes and counting them using flow cytometry.
Beads representing specific variants can be optionally recovered
through flow sorting and used for subsequent confirmation and
experimentation.
[0024] Beads according to the present invention are also known as
microspheres or microparticles. Particle sizes can vary between
about 0.1 and 10 microns in diameter. Typically beads are made of a
polymeric material, such as polystyrene, although nonpolymeric
materials such as silica can also be used. Other materials which
can be used include styrene copolymers, methyl methacrylate,
functionalized polystyrene, glass, silicon, and carboxylate.
Optionally the particles are superparamagnetic, which facilitates
their purification after being used in reactions.
[0025] Beads can be modified by covalent or non-covalent
interactions with other materials, either to alter gross surface
properties, such as hydrophobicity or hydrophilicity, or to attach
molecules that impart binding specificity. Such molecules include
without limitation, antibodies, ligands, members of a
specific-binding protein pair, receptors, nucleic acids.
Specific-binding protein pairs include avidin-biotin,
streptavidin-biotin, and Factor VII-Tissue Factor.
[0026] Beads, after being prepared according to the present
invention as product beads, have more than one copy of the same
nucleic acid molecule bound to them. Preferably each bead is bound
to at least 10, 50, 100, 500, or 1000 molecules of the same nucleic
acid sequence. In some circumstances some of the product beads are
bound to more than one type of nucleic acid molecule. These product
beads are generally less useful in the analysis of ratios of
genetic sequences in a population of genetic sequences. Such
product beads can be readily discriminated and so will not distort
the analysis.
[0027] A population of product beads will often comprise two or
more types of nucleic acids. Such a population is heterogeneous
with respect to the nucleic acids. Desirably, a substantial
proportion of the product beads comprise only one type of nucleic
acid per bead. A substantial proportion can be for example, at
least 1%, at least 5%, at least 10%, or at least 50%. A product
bead with only one type of nucleic acid per bead is termed
homogeneous. Homogeneous beads with only one type of nucleic acid
per bead include those with nucleic acids containing errors due to
errors in polymerase chain reaction. A product bead with two types
of nucleic acid per bead is termed heterogeneous. Although not
wishing to be bound by any particular theory, heterogeneous product
beads are thought to result from aqueous compartments which have
more than two molecules of template of non-identical sequence. A
population of product beads can be heterogeneous as a population
but contain individual product beads that are homogeneous
[0028] Individual product beads preferably comprise more than one
copy of template analyte molecule. Each bead may comprise at least
10, at least 50, at least 100, at least 500, or at least 1000
copies of template analyte. If the bead is homogeneous, each of
those copies will be identical.
[0029] Populations of product beads can be maintained in a liquid
suspension. Alternatively they can be sedimented and dried or
frozen. The latter alternatives may be beneficial for storage
stability.
[0030] Analysis of populations of product beads can be useful for
distinguishing between many kinds of genetic variants.
Polynucleotides can be distinguished which differ by as little as a
single nucleotide polymorphism (SNP), by the presence or absence of
a mutation, by the presence or absence of an insertion or deletion,
by the presence or absence of a non-single nucleotide polymorphism.
Thus populations of product beads may be heterogeneous with regard
to these genetic variations.
[0031] One very convenient way for distinguishing genetic variants,
i.e., determining a sequence feature of the analyte, is by
differentially labeling the variants with fluorescent dyes. Such
labeling can be accomplished by hybridization of a fluorescently
labeled oligonucleotide probe to one species of polynucleotide.
Alternatively, a fluorescently labeled antibody can be used to
specifically attach to one oligonucleotide probe that hybridizes to
a particular genetic variant. Such antibody binding can be, for
example, mediated by a protein or polypeptide which is attached to
an oligonucleotide hybridization probe. Of course, other means of
labeling polynucleotides as are known in the art can be used
without limitation. Another means of labeling different
polynucleotide species is by primer extension. Primers can be
extended using labeled deoxyribonucleotides, such as fluorescently
labeled deoxyribonucleotides.
[0032] Populations of product beads can be used as templates.
Template analyte molecules on the product beads can be analyzed to
assess DNA sequence variations by hybridization, primer-extension
methods, mass spectroscopy, and other methods commonly used in the
art. Template analyte molecules on product beads can be employed
for solid phase sequencing. In one solid phase sequencing
technique, product beads are arrayed by placing them on slides
spotted with complementary oligonucleotides. In another solid phase
sequencing technique, product beads are placed into individual
wells. In still another solid phase sequencing technique product
beads are incorporated into acrylamide matrices (with or without
subsequent polony formation). Sequencing reactions can be performed
with any solid phase sequencing method, such as those using
unlabeled nucleotide precursors (e.g., pyrosequencing, as described
in Ronaghi et al. Anal. Biochem. 267: 65-71, 1999) or labeled
nucleotides (e.g., photocleavable reagents described by Mitra et
al. Anal. Biochem. 320:55-65, 2003). Product beads can thus be used
for and facilitate multiple parallel sequencing. Product beads can
also be used in sequencing employing Type IIS restriction
endonucleases. Product beads can also be used to provide templates
for conventional dideoxynucleotide sequencing. To obtain useful
data upon sequence analysis, a homogeneous template population is
desirable. To provide a homogenous template population, product
beads can be diluted, separated, or otherwise isolated so that each
sequencing reaction contains a single product bead. Alternatively,
product beads can be sorted to provide populations of beads with a
single species of template.
[0033] Oligonucleotide primers can be bound to beads by any means
known in the art. They can be bound covalently or non-covalently.
They can be bound via an intermediary, such as via a
protein-protein interaction, such as an antibody-antigen
interaction or a biotin-avidin interaction. Other specific binding
pairs as are known in the art can be used as well. To achieve
optimum amplification, primers bound to the bead may be longer than
necessary in a homogeneous, liquid phase reaction. Oligonucleotide
primers may be at least 12, at least 15, at least 18, at least 25,
at least 35, or at least 45 nucleotides in length. The length of
the oligonucleotide primers which are bound to the beads need not
be identical to that of the primers that are in the liquid phase.
Primers can be used in any type of amplification reaction known in
the art, including without limitation, polymerase chain reaction,
isothermal amplification, rolling circle amplification,
self-sustaining sequence replication (3SR), nucleic acid
sequence-based amplification (NASBA), transcription-mediated
amplification (TMA), strand-displacement amplification (SDA), and
ligase chain reaction (LCR).
[0034] Microemulsions are made by stirring or agitation of oil,
aqueous phase, and detergent. The microemulsions form small aqueous
compartments which have an average diameter of 0.5 to 50 microns.
The compartments may be from 1 to 10 microns, inclusive, from 11 to
100 microns, inclusive, or about 5 microns, on average. All such
compartments need not comprise a bead. Desirably, at least one in
10,000 of said aqueous compartments comprise a bead. Typically from
1/100 to 1/1 or from 1/50 to 1/1 of said aqueous compartments
comprise a bead. In order to maximize the proportion of beads which
are homogeneous with respect to oligonucleotide, it is desirable
that on average, each aqueous compartment contains less than 1
template molecule. Aqueous compartments will also desirably contain
whatever reagents and enzymes are necessary to carry out
amplification. For example, for polymerase chain reaction (PCR) the
compartments will desirably contain a DNA polymerase and
deoxyribonucleotides. For rolling circle amplification a DNA
polymerase and a generic DNA circle may be present.
[0035] Emulsions can be "broken" or disrupted by any means known in
the art. One particularly simple way to break the emulsions is to
add more detergent. Detergents which can be used include, but are
not limited to Triton X100, Laureth 4, Nonidet.
[0036] Sample DNA for amplification and analysis according to the
present invention can be genomic DNA, cDNA, PCR products of genomic
DNA, or PCR products of cDNA, for example. Samples can be derived
from a single individual, for example, from a body sample such as
urine, blood, sputum, stool, tissue or saliva. Samples can also be
derived from a population of individuals. The individuals can be
humans, but can be any organism, plant or animal, eukaryotic or
prokaryotic, viral or non-viral.
[0037] Any type of probe can be used for specific hybridization to
the amplified polynucleotides which are bound to the beads.
Fluorescently labeled probes are useful because their analysis can
be automated and can achieve high throughput. Fluorescence
activated cell sorting (FACS) permits both the analysis and the
isolation of different populations of beads. One type of
fluorescently labeled probe that can be used is a modified
molecular beacon probe. These probes have stem-loop structures and
an attached fluorescent moiety on the probe, typically on one end
of the probe, sometimes attached through a linker. Unlike standard
molecular beacon probes, modified molecular beacon probes do not
have a quenching moiety. The modified molecular beacon probe can
have the fluorescent moiety attached on either end of the probe, 5'
or 3'. One such probe will hybridize better to a wild-type sequence
than to a mutant. Another such probe will hybridize better to a
mutant sequence than to the wild type. Still other probes will
preferably hybridize to one polymorphic variant over another.
[0038] The method of the present invention provides a reliable and
sensitive assay for measuring variations in genes and transcripts.
It requires no instrumentation other than machines that are widely
available. There are several other advantages of this approach.
First, the sensitivity can be increased to meet the particular
specifications of an assay simply by analyzing more beads. Such
sensitivity is limited only by the error rate of the polymerases
used for amplification. Second, the data obtained can be used not
only to demonstrate that a variant is present in a particular
population of DNA molecules, but also quantifies the fraction of
variant DNA molecules in that population (FIG. 5A). Such
quantification is not possible with techniques that destroy or
ignore the wild type molecules as part of the assay, such as those
that use allele specific priming or endonuclease digestion during
PCR. Third, the beads containing variant alleles can easily be
purified through flow sorting. Such recovery is difficult with
digital techniques that count molecules deposited on microscope
slides. And finally, the method is automatable.
[0039] Several modifications of the basic principles described here
can be envisioned that will further simplify the technology or
widen its applications. For example, microemulsions were made by
stirring water/oil/detergent mixes. The sizes of the resultant
aqueous compartments were somewhat heterogeneous, as illustrated in
FIG. 2. A relatively large number of beads containing PCR products
of both alleles are obtained from large compartments because they
are more likely to contain >1 template molecule than smaller
compartments. Though this is not a problem for the analysis of
uncommon variants, it does pose a problem when the variant to be
analyzed is present in a substantial fraction of the DNA molecules.
For example, it is easy to distinguish a population containing 2%
of allele A and 98% of allele B from one that contains 0% of allele
A (FIG. 5A). But it is more difficult to distinguish a population
that contains 48% of allele A and 52% of allele B from a population
that contains 50% of allele A; the large number of heterozygote
beads formed in the latter analysis diffuse the boundaries of the
pure red and green channels. This limit to accuracy can be overcome
through the preparation of more uniformly sized aqueous
compartments. Sonication or pressure-driven emulsifiers can make
more uniform compartments.
[0040] Though flow cytometry requires only seconds to minutes per
sample, multiple parallel analyses could facilitate throughput.
Novel particle counting designs may prove useful for this purpose.
Another way to increase throughput would be to physically separate
the beads that contained PCR products prior to flow cytometry. This
could be accomplished with proteins such as antibodies or
streptavidin that bind to modified nucleotides incorporated into
the PCR product during amplification.
[0041] The methods of the invention can be applied to genes or
transcripts of any organism or population of organisms. These
include without limitation: humans, rodents, ungulates, mammals,
primates, cows, goats, pigs, rats, mice, yeast, poultry, fish,
shellfish, digs, cats, zebrafish, worms, algae. It can also be used
to quantify epigenetic alterations, such as methylation, if DNA is
first treated with bisulfite to convert methylated cytosine
residues to thymidine. Beads generated from random fragments of
whole genomes (24), rather than from individual genes as described
above, could be used to identify gene segments that bind to
specific DNA-binding proteins (25). And if the product beads are
used in compartmentalized in vitro transcription-translation
reactions, variant proteins can be bound to beads containing the
corresponding variant DNA sequences (23). This could allow facile
flow cytometric evaluation of rare mutations using antibodies that
distinguished between wild type and mutant gene products (26).
[0042] While the invention has been described with respect to
specific examples including presently preferred modes of carrying
out the invention, those skilled in the art will appreciate that
there are numerous variations and permutations of the above
described systems and techniques that fall within the spirit and
scope of the invention as set forth in the appended claims.
EXAMPLES
Example 1
Materials and Methods
[0043] Step 1--Coupling oligonucleotides to beads.
Superparamagnetic beads of 1.05+/-0. 1 um in diameter, covalently
bound to streptavidin, were purchased from Dynal Biotech, Inc.
(650.01, Lake Success, N.Y.). Beads were washed once with
1.times.PCR buffer (53286, Invitrogen, Carlsbad, Calif.) then
suspended in Bind and Wash Buffer (BWB) (5 mM Tris-HCl, 0.5 mM
EDTA, 1.0 M NaCl, pH 7.5). Beads were incubated in BWB for 30 min
at room temperature in the presence of 10 uM oligonucleotides (FIG.
8). These oligonucleotides were modified with a dual biotin group
at the 5' end with the biotin groups separated by a six-carbon
linker (IDT, Coralville, Iowa). After binding, the beads were
washed 3 times with 1.times.PCR buffer to thoroughly remove unbound
oligonucleotides.
[0044] Step 2--Preparing microemulsions. Microemulsions for PCR
were prepared by slight modifications of previously described
methods (14) (15). The oil phase was composed of 4.5% Span 80
(S6760, Sigma, St. Louis, Mo.), 0.40% Tween 80 (Sigma S-8074), and
0.05% Triton X-100 (Sigma T-9284) in mineral oil (Sigma M-3516).
The oil phase was freshly prepared each day. The aqueous phase
consisted of 67 mM Tris-HCl (pH 8.8), 16.6 mM NH4SO4, 6.7 mM MgCl2,
10 mM .beta.-mercaptoethanol, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM
dTTP, 0.05 uM forward primer, 25 uM reverse primer, 45 units
Platinum Taq (Invitrogen 10966-034), various amounts of template
DNA (see results), and .about.108 oligonucleotide-coupled beads in
a total volume of 300 ul. The forward primer was an oligonucleotide
whose sequence was identical to the 3' 20-22 nt of that described
in step 1 and was not modified with biotin.
[0045] Water-in-oil microemulsions were prepared by drop wise
addition of 200 microliters of the aqueous phase to 400 microliters
of the oil phase previously placed in a 2 ml round bottom cryogenic
vial (430661, Corning, Corning, N.Y.). The drop wise addition was
performed over .about. one minute while the mixture was being
stirred at 1400 RPM with a magnetic microstir bar (58948-353, VWR,
Plainfield, N.J.) on a VWR model 565 Magnetic Stirrer. After the
addition of the aqueous phase, the mixture continued to be stirred
for a total time of 30 minutes. Two emulsions were made at once by
placing two tubes in a rack placed at the center of the magnetic
stirrer.
[0046] Step 3--PCR cycling. The emulsions were aliquotted into five
wells of a 96 well PCR plate, each containing 100 ul. PCR was
carried out under the following cycling conditions: 94.degree. C.
for 2 minutes; 40 cycles of 94.degree. C. for 15 seconds,
57.degree. C. for 30 seconds, 70.degree. C. for 30 seconds. The PCR
products analyzed in this study ranged from 189 to 239 bp.
[0047] Step 4--Magnetic capture of beads. After PCR cycling, the
microemulsion from five wells of a PCR plate were pooled and broken
by the addition 800 microliters of NX buffer (100 mM NaCl
containing 1% Triton X-100, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) in a
1.5 ml tube (Corning 430909). After vortexing for .about.20 sec.
the beads were pelleted by centrifugation in a microcentrifuge at
8000 rpm (5000 g) for 90 seconds. The top oil phase and all but
.about.300 microliters of the aqueous phase was removed from the
tube and 600 microliters of NX buffer was added. After vortexing
for 20 sec. and centrifugation for 90 sec., the top oil phase and
all but .about.300 microliters of the aqueous phase was removed.
The addition of 600 microliters NX buffer, vortexing, and
centrifugation was repeated once more and the top oil portion and
all but .about.300 microliters of the aqueous phase was removed.
The tube was then placed on a magnet (Dynal MPC-S) and the rest of
the supernatant was carefully pipetted off. The beads were washed
an additional 3 times with 1.times.PCR buffer using magnetic
separation rather than centrifugation and finally resuspended in
100 microliters of 1.times.PCR buffer.
[0048] Step 5--Sequence differentiation. Two oligonucleotide probes
were used for each reaction. One was 5'-labeled with
6-carboxyfluorescein (6-FAM) and was specific for one allele while
the second was 5'-labeled with biotin and was specific for the
other allele. Probes were synthesized by IDT. The 30 microliters
hybridization reactions contained 10 uM of each probe and 5-25
million beads in 1.times.PCR buffer. Reactions were performed in
PCR plates on a thermal cycler by heating to 94.degree. C. for 30
seconds then cooling to 75.degree. C. at a rate of 0.5.degree. C.
per second, cooling to 45.degree. C. at 0.2.degree. C. per second,
and finally cooled to 30.degree. C. at 1.degree. C. per second. All
subsequent steps were performed at room temperature. The reactions
were transferred to a 96 well Costar plate (Corning 3797) and
placed on a 96 well magnet. Beads were collected magnetically by
exposing them to the magnet for 2 minutes. The supernatant was
removed and the beads washed 3 times with 1.times.PCR buffer by
pipetting them and collecting for two minutes. They were finally
resuspended in 100 microliters B-PCR buffer (1 mg/mL BSA in
1.times.PCR buffer). The beads were then incubated for 10 minutes
in a total volume of 100 microliters B-PCR buffer containing 3 ug
of Alexa-488 rabbit anti-fluorescein antibody (Molecular Probes
A-11090, Eugene, Oreg.) and 3 ug of Nutravidin labeled with
R-phycoerythrin (Molecular Probes A-2660) in B-PCR buffer. The
beads were washed three times and resuspended in B-PCR buffer as
described above. They were then incubated for ten minutes in a
total volume of 100 microliters B-PCR buffer containing 6 ug of
Alexa 488-conjugated chicken anti-rabbit antibody (Molecular Probes
A-21441) and 3 ug of biotinylated goat anti-avidin antibody
(BA-0300, Vector Laboratories, Burlingame, Calif.). The beads were
washed three times and resuspended in B-PCR buffer as described
above. They were then incubated for ten minutes in a total volume
of 100 microliters B-PCR buffer containing 3 ug of an Alexa
488-conjugated goat anti-chicken antibody (Molecular Probes
A-11039) and 3 micrograms of R-phycoerythrin-labeled streptavidin
(Molecular Probes S-866). This solution was then washed an
additional 3 times with 1.times.PCR buffer and resuspended in 20
microliters of 1.times.PCR buffer.
[0049] Step 6--Flow Cytometry. The bead suspension was diluted to a
concentration of .about.106-107 beads per ml in 10 mM Tris-HCl, 1
mM EDTA (351-010-131, Quality Biological, Inc., Gaithersburg, Md.)
and analyzed using a LSR instrument (BD Biosciences, Franklin
Lakes, N.J.). The instrument was set up for standard two-color
analysis using an argon laser and optical filters that
distinguished between the two fluorescent dyes. No spectral
deconvolution was required as the major bead populations were
well-separated. In some cases, scanning was performed with FACScan
or FACSCalibur instruments (BD Biosciences), yielding equivalent
results. Sorting was carried out with a FACS Vantage SE instrument
(BD Biosciences).
[0050] Template preparation and sequence analyses. Human genomic
DNA was purified with DNeasy (69504, Qiagen, Valencia, Calif.). RNA
was purified with Quickprep (27-9255-01, Amersham Biosciences,
Piscataway, N.J.). Reverse transcription of RNA was performed using
Superscript II reverse transcriptase (Invitrogen 18064014)
according to the manufacturer's instructions. PCR using genomic DNA
or reverse transcripts as templates was performed as described (7).
PCR products to be used as templates for BEAMing or for sequencing
were purified with QIAquick (Qiagen 28104). Sequencing reactions
were performed using Big Dye v3.0 reagents (Applied Biosystems,
Foster City, Calif.) and analyzed by capillary electrophoresis
(Spectrumedix 9600, State College, Pa.).
Example 2
Results
[0051] Step 1--Coupling oligonucleotides to beads. We used
streptavidin-beads because of the simplicity of coupling
biotinylated oligonucleotides to them. Oligonucleotides with just a
single 5' biotin group were found to dissociate from the beads
during temperature cycling, while oligonucleotides labeled with
dual biotin groups at their 5' end (separated by a six-carbon
linker) were stable to cycling. As determined by fluoroscopic
measurements of oligonucleotides doubly labeled with 6-FAM and
biotin, .about.105 oligonucleotide molecules were bound to each
bead. We found that short oligonucleotides (20 bases) did not work
as well for priming as longer ones (41 bp), perhaps because of
steric hindrance at the bead surface. It is likely that amino-,
sulfhydryl-, or carboxyl-modified oligonucleotides covalently
coupled to beads modified with corresponding reactive groups could
also function as bead-bound primers for BEAMing.
[0052] Step 2--Preparing microemulsions. The size of the individual
aqueous compartments ranged from less than 1 micron to >10
microns in diameter (FIG. 2). We estimated that an emulsion
comprising 200 microliters of aqueous solution and 400 microliters
of oil would contain .about.3.times.10.sup.9 compartments with an
average diameter of 5 microns. Approximately 10.sup.8 beads were
included in each emulsion, so that only one in .about.30
compartments contained a bead. The optimal amount of template was
experimentally determined to be .about.5.times.10.sup.8 molecules,
so that one in .about.six compartments contained a template
molecule.
[0053] Step 3--PCR cycling. PCR priming by oligonucleotides coupled
to beads was found to be very inefficient compared to the priming
by the same oligonucleotides when free in solution. For this
reason, a small amount of non-biotinylated forward primer identical
in sequence to the biotinylated oligonucleotide coupled to the
beads was included in the reactions. This facilitated the first few
rounds of amplification of the single template within each aqueous
compartment. In the absence of additional primer, no detectable
amplification on the beads was generated. Conversely, if too much
additional primer was included, no amplification on the beads
occurred because of competition with the primers in solution. An
excess of the reverse primer was included in the aqueous
compartment to maximize the probability that bead-bound
oligonucleotides extended by polymerase would serve as templates
for further amplification cycles.
[0054] Step 4--Magnetic capture of beads. There are several ways to
break water-in-oil emulsions, including extraction with organics
(14). We found that simply adding non-ionic detergents produced
phase separations without any detectable modification of the beads
or DNA molecules bound to them. By measuring the amount of DNA that
could be released from the beads following restriction endonuclease
digestion, we estimate that >10,000 extended PCR products were
present, on average, per bead.
[0055] Step 5--Sequence differentiation. Most fluorescence-based
methods for distinguishing alleles in homogeneous or two-phase
assays can be used to assess allelic variation captured on beads.
These methods include single nucleotide extension, allele specific
priming, or hybridization. We generally employed hybridization of
fluorescein-conjugated or biotin-conjugated oligonucleotides for
discrimination. As shown in FIG. 1 and FIG. 8, these
oligonucleotides had a stem-loop structure, with the middle of the
loop containing the variant nucleotide(s). This design was based on
studies of Molecular Beacons wherein a stem-loop structure was
shown to markedly improve allelic discrimination (16). The
oligonucleotides we used differed from Molecular Beacons in that
there was no need for a quenching group. Such quenching is required
for homogeneous assays when unhybridized oligonucleotides cannot be
removed from the reactions prior to assay but is not necessary for
solid phase assays such as those employed with beads.
[0056] Step 6--Flow Cytometry. Optimum results in flow cytometry
depend on high fluorescent signals on the beads. We generally
enhanced the fluorescence emanating from the hybridization probes
with secondary reagents. For example, Alexa 488--labeled antibodies
were used to enhance the signals emanating from fluorescein-coupled
oligonucleotide probes. Similarly, R-phycoerythrin-labeled
streptavidin was used to generate a signal from biotin-labeled
oligonucleotide probes. Flow cytometers equipped with two or three
lasers and appropriate filters have the capacity to distinguish
multi-allelic loci and to perform multiplex analysis of several
genes simultaneously. The newest generation of flow cytometers can
also analyze >70,000 events per second. In addition to the
analytical power of flow cytometry, FACS instruments can separate
specific populations of beads for further analysis.
Example 3
Characteristics of Microemulsions
[0057] Pilot experiments demonstrated that simply stirring the
water-oil mixtures described in Materials and Methods produced very
stable microemulsions of a size compatible with that of the beads.
In the experiment shown in FIG. 2, the aqueous compartment
contained a blue dye and 1 micron magnetic beads that were labeled
by binding to an oligonucleotide that was biotinylated at its 5'
end and labeled with fluorescein at its' 3' end. The appearance of
emulsions immediately after their formation is shown in FIG. 2. As
expected, this appearance was unchanged after temperature cycling
during PCR (15). Most aqueous compartments contained no beads, as
expected from the figures provided in the previous section. Those
compartments that did contain beads generally contained only one,
though a fraction contained more, as expected from a Poisson
distribution and non-uniform aqueous compartment sizes.
"Heterozygous" beads containing PCR products representing both
alleles are produced when two or more DNA template molecules are
contained within a single aqueous compartment. Such heterozygotes
can compromise the accuracy of the analyses under some
circumstances (see Discussion).
Example 4
Detection of Homozygotes and Heterozygotes
[0058] FIG. 3 shows typical results obtained with human DNA
samples. The MID42 marker used in this experiment was chosen from a
collection of diallelic short insertion/deletion polymorphisms
assembled by Weber and colleagues (17). These alleles are
particularly simple to distinguish with hybridization probes
because the two alleles at each locus differ by .about.4 bases. The
probe for the longer (L) allele was labeled with fluorescein
(green) and the probe for the shorter (S) allele labeled with
R-phycoerythrin (red).
[0059] FIG. 3A shows a plot of the side scatter vs. forward scatter
of beads following BEAMing. In general, >75% of beads were
dispersed as single particles, with the remainder aggregated in
groups of two or more. Subsequent flow cytometric analysis was
confined to the singlet beads, gated as outlined in FIG. 3A.
[0060] FIGS. 3B-D show density plots of gated beads generated with
various templates. In FIG. 3B, a template from an individual
homozygous for the L allele was included in the emulsion. Two
populations of beads were apparent. 98% of the beads contained no
PCR product (black) and the remaining 2% fluoresced in the FL1
channel (colored green in FIG. 3). FIG. 3C represents the analysis
of an individual homozygous for the S allele. Two populations of
beads were again apparent, but this time the labeled population
fluoresced in the FL2 channel (colored red in FIG. 3). FIG. 3D
presents density plots from the analysis of an individual
heterozygous at the MID42 locus. Four populations of beads are
evident: the black region represents beads without any PCR product,
the red region represents beads containing PCR products from the L
allele, the green region represents beads containing PCR products
from the S allele, and the blue region represents beads containing
PCR products from both alleles. Beads containing PCR products from
both alleles were derived from aqueous compartments which contained
more than one template molecule. The number of blue beads increased
in a non-linear fashion as more template molecules were added. At
the extreme, when all aqueous compartments are saturated, virtually
all beads will register as blue. Operationally, we found that the
bead populations were most distinct when the number of beads
containing any PCR product was <10% of the total beads
analyzed.
Example 5
PCR Products, Genomic DNA or cDNA as Templates
[0061] The results shown in FIG. 3 were generated using PCR
products made from human genomic DNA samples. As the ratio of the
beads representing L alleles to those representing S alleles was
1.0 in this experiment, it was clear that the initial PCR did not
preferentially amplify either allele. The use of PCR products
rather than genomic DNA permitted large numbers of alleles to be
amplified from even small quantities of starting DNA. In general,
10 to 100 picograms of PCR products of size 200 bp were found to be
optimal for BEAMing, producing PCR-mediated extension of primers on
.about.1 to 10% labeled beads.
[0062] In some situations it might be useful to use genomic DNA
rather than PCR products as templates for BEAMing. The data in
FIGS. 4A and B show flow cytometric data from an experiment wherein
10 ug or 1 ug of human genomic DNA was used as template for BEAMing
at the MID42 locus. Patterns very similar to those shown in FIG. 3
were observed, though fewer beads were labeled than when PCR
products were used as templates.
[0063] BEAMing could also be used to analyze variations in
expression from the two alleles of a heterozygous individual.
Heritable variations in the expression from individual alleles of
the same gene have been shown to occur often in humans (18) and
mice (19) and can have significant phenotypic effects (20). The
results shown in FIGS. 4C and D show that PCR products made from
reverse-transcribed mRNA can be used for BEAMing. In this case,
calpain-10 transcripts differing by a single nucleotide
polymorphism (SNP) were analyzed. For SNPs like these, probes that
incorporated an extra mismatched nucleotide adjacent to the
polymorphic nucleotide (see FIG. 8) can enhance the distinction
between alleles (21) (22). The results from two independent
emulsions made with aliquots of the same RT-PCR product are shown
to illustrate reproducibility. Though the number of beads that
functioned as templates in BEAMing varied up to 3-fold among
experiments with identical templates, the proportion of beads
representing the two alleles was reproducible (775 A allele beads
to 690 G allele beads in FIGS. 4C and 1380 A allele beads to 1227 G
allele beads in FIG. 4D) respectively).
Example 6
Analysis of Minor Variants in a DNA Population
[0064] The analysis of uncommon variations is ideally suited for
analysis via BEAMing because of the large number of molecules that
can be independently analyzed while retaining a high
signal-to-noise ratio. FIG. 5A shows representative data from
templates representing 1%, 2%, 3%, and 4% of the L allele of MID42.
The linearity of these measurements, with a correlation coefficient
of 0.99, demonstrates the utility of this approach for such
applications. We also applied this analysis to the detection of
KRAS and could easily observe 0.1% mutants when spiked into a
population of wt molecules (data not shown).
[0065] The rare beads representing the mutant alleles could not
only be quantified but could also be purified for subsequent
analysis. As a demonstration, samples of the beads enumerated in
FIG. 5A were additionally assessed using a flow cytometer equipped
with sorting capabilities. Beads were sorted and individual beads
used as templates for conventional PCR using the same primers
employed for BEAMing. As each bead contains thousands of bound
template molecules, single beads were expected to generate robust
PCR products (23) and this was experimentally confirmed. These PCR
products were then subjected to sequencing. As shown in FIGS. 5B
and C, green and red beads generated PCR products exclusively of
the L and S types, respectively.
Example 7
Electrophoresis of Oligonucleotides Hyrbridized to Beads
[0066] A 100 bp product was amplified on beads as described in
Example 1, steps 1 through 4. Two FAM-labeled oligonucleotides (50
and 20 bases in length) were annealed to the 100 bp product on the
beads. The beads were then embedded in an acrylamide gel (using
conventional Tris-Borate-EDTA electrophoresis buffer) in an oval
shaped configuration. An electric field (250 V) was applied under
denaturing conditions for 3 minutes. The labeled oligonucleotides
migrated off the beads and migrated a distance related to their
sizes. See FIG. 7. There was little diffusion, as evidenced by the
retention of the oval shape of the beads.
Example 8
Sequencing of Templates Immobilized to Beads
[0067] Sanger-type (dideoxynucleotide) sequencing is performed
using as templates oligonucleotides which have been amplified on
beads, as described in Example 1. Individual beads are subjected to
primer extension conditions in the presence of dideoxynucleotide
inhibitors. The beads are then subjected to electrophoresis under
denaturing conditions to separate the dideoxynucleotide-terminated,
primer extended oligonucleotides on the basis of length. A sequence
is compiled based on the length of the primer extended
oligonucleotides.
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Sequence CWU 1
1
17120DNAHomo sapiens 1ctttgtaact aactgtttaa 20216DNAHomo sapiens
2ctttgtaact gtttaa 16340DNAArtificial Sequenceprobe 3tactatgtat
ttatagttaa gacctctatg aatgaatgta 40426DNAArtificial Sequenceprobe
4cgttaagacc tctatgaatg aatgta 26522DNAArtificial Sequenceprobe
5gaaaggtaag tacagggaaa gg 22640DNAArtificial Sequenceprobe
6cacgcagatt gaattaaaca gttagttaca aagacacgtg 40736DNAArtificial
Sequenceprobe 7cacgcagatt gaattaaaca gttacaaaga cacgtg
36847DNAArtificial Sequenceprobe 8aggtcccaga gggtggaagg agccaggacg
cacccccact gctgctg 47919DNAArtificial Sequenceprobe 9aggtcccaga
gggtggaag 191020DNAArtificial Sequenceprobe 10ttgcgatggt cactgtgaag
201126DNAArtificial Sequenceprobe 11cacggtaggt gcttgcaggc agcgtg
261226DNAArtificial Sequenceprobe 12cacggtaggt gcccgcaggc agcgtg
261341DNAArtificial Sequenceprobe 13ttcgtccaca aaatgattct
gaattagctg tatcgtcaag g 411421DNAArtificial Sequenceprobe
14agaatggtcc tgcaccagta a 211526DNAArtificial Sequenceprobe
15catgttctaa tatagtcaca ttttca 261624DNAArtificial Sequenceprobe
16cacgggagct ggtggcgtag cgtg 241726DNAArtificial Sequenceprobe
17ccacgggagc tgatggcgta gcgtgg 26
* * * * *