U.S. patent application number 10/075579 was filed with the patent office on 2002-08-29 for primer extension detection methods on active electronic microarrays.
This patent application is currently assigned to Nanogen, Inc.. Invention is credited to Heller, Michael J., Kahl, Brenda F., Weidenhammer, Elaine M., Xu, Xiao.
Application Number | 20020119484 10/075579 |
Document ID | / |
Family ID | 24853035 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119484 |
Kind Code |
A1 |
Weidenhammer, Elaine M. ; et
al. |
August 29, 2002 |
Primer extension detection methods on active electronic
microarrays
Abstract
The present invention presents methods for gene expression
monitoring that utilize microelectronic arrays to drive the
transport and hybridization of nucleic acids. Procedures are
described for generating mRNA expression samples for use in these
methods from populations of cells, tissues, or other biological
source materials, that may differ in their physiological and/or
pathological state. Provided in the invention are methods for
generating a reusable nucleic acid transcript library from mRNA in
a sample of biological material. In order to improve gene
expression monitoring on the microelectronic arrays, the
transcripts are amplified to produce sample nucleic acid amplicons
of a defined length. Because multiple sample amplicons may be
selectively hybridized to controlled sites in the electronic array,
the gene expression profiles of the polynucleotide populations from
different sources can be directly compared in an array format using
electronic hybridization methodologies. Also provided in the
invention are methods for detecting the level of sample amplicons
using electronically assisted primer extension detection, and
utilizing individual test site hybridization controls. The
hybridization data collected utilizing the improved methods of the
present invention will allow the correlation of changes in mRNA
level with the corresponding expression of the encoded protein in
the biological source material, and thus aid in studying the role
of gene expression in disease.
Inventors: |
Weidenhammer, Elaine M.;
(San Diego, CA) ; Xu, Xiao; (San Diego, CA)
; Heller, Michael J.; (Encinitas, CA) ; Kahl,
Brenda F.; (San Diego, CA) |
Correspondence
Address: |
LYON & LYON LLP
633 WEST FIFTH STREET
SUITE 4700
LOS ANGELES
CA
90071
US
|
Assignee: |
Nanogen, Inc.
San Diego
CA
92121
|
Family ID: |
24853035 |
Appl. No.: |
10/075579 |
Filed: |
February 12, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10075579 |
Feb 12, 2002 |
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09710200 |
Nov 9, 2000 |
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6379897 |
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10075579 |
Feb 12, 2002 |
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09490965 |
Jan 24, 2000 |
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09490965 |
Jan 24, 2000 |
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08271882 |
Jul 7, 1994 |
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6017696 |
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Current U.S.
Class: |
435/6.11 ;
257/E21.705; 435/6.12; 435/6.13; 435/91.2; 702/20 |
Current CPC
Class: |
C07K 1/045 20130101;
G11C 19/00 20130101; C12Q 2527/113 20130101; B01L 3/5027 20130101;
B01J 19/0046 20130101; B01J 2219/00659 20130101; B01J 2219/00653
20130101; B82Y 5/00 20130101; C12Q 1/6825 20130101; C12Q 1/6837
20130101; C40B 40/06 20130101; B01J 2219/00585 20130101; B01J
2219/00317 20130101; G11C 13/0014 20130101; B01J 2219/0059
20130101; B82Y 10/00 20130101; C40B 60/14 20130101; B01J 19/0093
20130101; H01L 2924/0002 20130101; B01J 2219/00689 20130101; C12Q
1/6816 20130101; C12Q 1/6813 20130101; H01L 25/50 20130101; H01L
2924/0002 20130101; C07H 21/00 20130101; C12Q 1/6837 20130101; C12Q
1/6837 20130101; B01J 2219/00713 20130101; G11C 13/0019 20130101;
C12Q 2565/515 20130101; B01J 2219/00722 20130101; C12Q 2565/607
20130101; H01L 2924/00 20130101 |
Class at
Publication: |
435/6 ; 702/20;
435/91.2 |
International
Class: |
C12Q 001/68; G06F
019/00; C12P 019/34; G01N 033/48; G01N 033/50 |
Claims
We claim:
1. A method for detecting the relative amounts of at least two mRNA
sequences in at least one biological sample, the method comprising:
(a) isolating mRNA from at least one biological sample; (b)
amplifying at least two mRNA transcripts from each biological
sample to produce amplicons, wherein the amplicons are less than
about 300 bases in length, and wherein the amplification comprises
a linear amplification step; (c) electronically hybridizing the
amplicons produced in step (b) to at least two probes bound to a
support at predetermined locations; and (d) detecting the amounts
of each amplicon hybridized to the bound probes at the
predetermined locations.
2. The method of claim 1, wherein the relative amounts of at least
two mRNA sequences are detected in at least two samples, wherein
each set of amplicons produced for each sample in step (b) is
selectively electronically hybridized to an electronically
controlled set of predetermined locations on the support.
3. The method of claim 2 wherein all sets of amplicons for each
sample are electronically hybridized prior to the detection step
(d).
4. The method of claim 2 wherein the relative amounts of at least
two mRNA sequences are detected in at least 10 samples.
5. The method of claim 4 wherein the relative amounts of at least
two mRNA sequences are detected in at least 50 samples.
6. The method of claim 1 wherein the relative amounts of at least 5
mRNA sequences are detected.
7. The method of claim 1 wherein the relative amounts of at least
10 mRNA sequences are detected.
8. The method of claim 1 wherein the relative amounts of at least
20 mRNA sequences are detected.
9. The method of claim 1 wherein the relative amounts of at least
40 mRNA sequences are detected.
10. The method of claim 1 wherein the relative amounts of at least
50 mRNA sequences are detected.
11. The method of claim 1 wherein total mRNA is isolated from the
biological sample in step (a).
12. The method of claim 1 wherein polyA mRNA is isolated from the
biological sample in step (a).
13. The method of claim 1 wherein the amplification step (b)
comprises a reverse-transcription step in which a cDNA library is
generated from the mRNA isolated in step (a).
14. The method of claim 13 wherein the amplification in step (b)
further comprises a DNA polymerase amplification step in which
members of the cDNA library are amplified with at least two
chimeric primers, wherein each chimeric primer comprises a RNA
polymerase recognition site upstream of a sequence specific for one
of the mRNA sequences of interest.
15. The method of claim 14 wherein the DNA polymerase amplification
step is linear.
16. The method of claim 15 wherein the amplification in step (b)
further comprises a geometric DNA polymerase amplification step, in
which members of the cDNA library are amplified with at least one
additional primer, wherein at least one primer is complementary to
a portion of each mRNA sequence of interest and at least one primer
is the same as a portion of each mRNA sequence of interest.
17. The method of claim 16 wherein the additional primer is a set
of random primer sequences.
18. The method of claim 16 wherein the additional primer is a
polydeoxythymine primer sequence.
19. The method of claim 16 wherein the additional primer is a set
of primer sequences specific for each mRNA sequence of
interest.
20. The method of claim 14 wherein the DNA polymerase amplification
step is geometric, in which members of the cDNA library are
amplified with at least one additional primer, wherein at least one
primer is complementary to a portion of each mRNA sequence of
interest and at least one primer is the same as a portion of each
mRNA sequence of interest.
21. The method of claim 20 wherein the additional primer is a set
of random primer sequences.
22. The method of claim 20 wherein the additional primer is a
polydeoxythymine primer sequence.
23. The method of claim 20 wherein the additional primer is a set
of primer sequences specific for each mRNA sequence of
interest.
24. The method of claim 14 wherein the chimeric primers are labeled
with an affinity moiety.
25. The method of claim 24 wherein the affinity moiety is
biotin.
26. The method of claim 25 wherein the amplification in step (b)
further comprises immobilizing the DNA produced in the DNA
polymerase amplification step on a surface comprising a protein
selected from the group consisting of streptavidin and avidin.
27. The method of claim 14 wherein the amplification in step (b)
further comprises in vitro transcription of the DNA produced in the
DNA polymerase amplification step.
28. The method of claim 14 wherein the amplified mRNA sequences of
interest comprise a type IIs endonuclease recognition site.
29. The method of claim 28 wherein the amplification in step (b)
further comprises a type IIs endonuclease digestion step.
30. The method of claim 29 wherein the amplification in step (b)
further comprises an in vitro transcription step.
31. The method of claim 1 wherein the amplification in step (b)
comprises an in vitro transcription step.
32. The method of claim 1 wherein each amplicon is between about 50
and about 300 nucleotides in length.
33. The method of claim 1 wherein each amplicon is between about 50
and about 200 nucleotides in length.
34. The method of claim 1 wherein each amplicon is between about 50
and about 100 nucleotides in length.
35. The method of claim 1 wherein the amplicons differ in length by
less than 20 bases.
36. The method of claim 1 wherein the amplicons differ in length by
less than 10 bases.
37. The method of claim 1 wherein the detection in step (d) is by
an enzymatic extension of the probes to which the amplicons are
hybridized to incorporate a labeled nucleotide.
38. The method of claim 1 wherein the detection in step (d) is by
detecting a labeled nucleotide incorporated into the amplicons.
39. The method of claim 1 wherein the detection in step (d) is by
i) electronically or passively hybridizing a labeled reporter probe
to the amplicon hybridized to the bound probe, and ii) detecting
the labeled reporter probe.
40. The method of claim 1 wherein the probes are bound to a
permeation layer over an electrode of a semiconductor chip
device.
41. The method of claim 1 wherein the detection in step (d) is by
fluorometry, colorimetry, or luminometry.
42. The method of claim 41 wherein the detection is by
fluorometry.
43. A method of preserving and reusing a nucleic acid library
produced from a patient biological sample, the method comprising:
(a) isolating mRNA from a patient biological sample; (b)
reverse-transcribing the mRNA from step (a) to produce a cDNA
library; (c) amplifying the cDNA library from step (b) by a DNA
polymerase reaction utilizing at least one chimeric primer
comprising a RNA polymerase recognition site upstream of a sequence
specific for a mRNA transcript of interest and a fill-in primer for
the complementary nucleic acid strand chosen from the group
consisting of sequence specific primers and random primers, wherein
at least one of the primers used is chosen from the group
consisting of 5' affinity-moiety labeled chimeric primers and 5'
affinity-moiety labeled sequence specific fill-in primers; (d)
binding the amplification products from step (c) to a solid support
coated with an affinity-binding moiety; (e) utilizing the bound
amplification products from step (d) as a template for an in vitro
transcription reaction; (f) separating the in vitro transcription
products from step (e) from the amplification products bound to the
solid support; and (g) utilizing the bound amplification products
from step (f) as a template for at least one additional in vitro
transcription reaction, wherein the amount of in vitro
transcription product produced is not significantly less than that
produced in step (e).
44. The method of claim 43 further comprising repeating steps (f)
and (g).
45. The method of claim 44 further comprising repeating steps (f)
and (g).
46. The method of claim 45 further comprising repeating steps (f)
and (g).
47. The method of claim 43 wherein the amplified sequence comprises
a type IIs endonuclease recognition site, the method further
comprising the step of digesting the amplification products with a
type IIs endonuclease prior to the in vitro translation of step
(e).
48. The method of claim 43 wherein the fill-in primer is a sequence
specific primer.
49. The method of claim 43 wherein the fill-in primer is a random
sequence primer.
50. The method of claim 43 wherein the affinity moiety is selected
from the group consisting of biotin, haptens, and an antigenic
moiety.
51. The method of claim 43 wherein the affinity moiety is
biotin.
52. The method of claim 51 wherein the affinity-binding moiety is
selected from the group consisting of streptavidin and avidin
53. The method of claim 43 wherein the solid support is selected
from the group consisting of paramagnetic beads, polymer beads, and
metallic beads.
54. A method of detecting the extent of hybridization of a nucleic
acid in a sample to a probe nucleic acid sequence, the method
comprising: (a) electronically hybridizing the nucleic acid in a
sample to a nucleic acid probe bound to a support at a
predetermined location; (b) utilizing the hybridized nucleic acid
as a template in a nucleic acid polymerase reaction to extend the
bound probe, whereby a labeled nucleotide is incorporated into the
extended probe; and (c) detecting the labeled nucleotide
incorporated into the extended bound probe at the predetermined
location.
55. The method of claim 54 wherein the labeled nucleotide comprises
a labeling moiety selected from the group consisting of fluorescent
moieties, colorigenic moieties, chemiluminescent moieties, and
affinity moieties.
56. The method of claim 55 wherein the labeled nucleotide comprises
a fluorescent moiety.
57. The method of claim 54 wherein the nucleic acid polymerase
reaction is a DNA polymerase reaction.
58. The method of claim 54 wherein the nucleic acid polymerase
reaction is a reverse-transcriptase reaction.
59. A method of providing an internal control for an individual
test site in a nucleic acid hybridization reaction assay to
determine the presence of at least one nucleic acid sequence of
interest in at least one nucleic acid containing sample, wherein
the nucleic acid hybridization assay is performed on an
electronically controlled microarray comprising at least two test
sites, the method comprising: (a) attaching a mixed nucleic acid
probe consisting of a first nucleic acid probe specific for a first
nucleic acid sequence known to be present in the sample, and a
second nucleic acid probe specific for a second nucleic acid
sequence of interest to a first test site on the electronically
controlled microarray; (b) attaching a mixed nucleic acid probe
consisting of the first nucleic acid probe and a third nucleic acid
probe specific for a third nucleic acid sequence of interest,
wherein the third nucleic acid sequence of interest may be the same
as or different than the second nucleic acid sequence of interest,
to a second test site on the electronically controlled microarray;
(c) electronically hybridizing the sample nucleic acids from at
least one sample to the nucleic acid probes on the first and second
test sites; (d) specifically detecting the extent of hybridization
of the sample nucleic acids to the first nucleic acid probe at the
first and second test sites; (e) specifically detecting the extent
of hybridization of the sample nucleic acids to the second and
third nucleic acid probes at the first and second test sites; (f)
comparing the hybridization values obtained for the first nucleic
acid probe at the first and second test sites to obtain a
normalization factor; and (g) normalizing the hybridization values
obtained in (e) for the second and third probes using the
normalization factor obtained in (f).
60. The method of claim 59 wherein the first nucleic acid sequence
is a sequence which encodes, or is complementary to a sequence
which encodes, a housekeeping gene.
61. The method of claim 59 wherein the first nucleic acid sequence
is an exogenous nucleic acid sequence which has been added to the
sample.
62. The method of claim 59 wherein the specific detection in (d) is
by detecting a labeled nucleotide which has been specifically
incorporated into the sample nucleic acids which contain the first
nucleic acid sequence by a nucleic acid polymerase reaction.
63. The method of claim 59, further comprising the step of
electronically or passively hybridizing a first reporter nucleic
acid comprising a detectable moiety specific for the first nucleic
acid sequence to the sample nucleic acids which are hybridized to
the first nucleic acid probe at the first and second test sites,
wherein the specific detection in (d) is by detecting the
detectable moiety.
64. The method of claim 59, further comprising the step of
extending the first nucleic acid probe by utilizing the sample
nucleic acids which have hybridized to the first nucleic acid probe
as a template for a nucleic acid polymerase reaction, wherein the
specific detection in (d) is by detecting a labeled nucleotide
which has been incorporated into the extended first nucleic acid
probe by the polymerase reaction.
65. The method of claim 64, wherein the first nucleic acid probe
attached in (a) comprises a protecting group that prevents
enzymatic extension of the probe.
66. The method of claim 59 wherein the specific detection in (e) is
by detecting a labeled nucleotide which has been specifically
incorporated into the sample nucleic acids which contain the second
and third nucleic acid sequences of interest by a nucleic acid
polymerase reaction.
67. The method of claim 59, further comprising the step of
electronically or passively hybridizing a second reporter nucleic
acid probe comprising a detectable moiety to the sample nucleic
acids which are hybridized to the second nucleic acid probe at the
second test site, wherein the second reporter probe is specific for
the second and third nucleic acid sequences of interest, and
wherein the second reporter probe contains one or more reporter
probe sequences, wherein the specific detection in (e) is by
detecting the detectable moiety.
68. The method of claim 59, further comprising the step of
extending the second and third nucleic acid probes by utilizing the
sample nucleic acids which have hybridized to the second nucleic
acid probe as a template for a nucleic acid polymerase reaction,
wherein the specific detection in (e) is by detecting a labeled
nucleotide which has been incorporated into the extended second and
third nucleic acid probes by the polymerase reaction.
69. The method of claim 68, wherein the second nucleic acid probe
attached in (b) comprises a protecting group that prevents
enzymatic extension of the probe.
70. The method of claim 59, wherein a first detectable moiety is
detected in step (d), and a second detectable moiety is detected in
step (e).
71. The method of claim 70 wherein the first and second detectable
moieties are independently selected from the group consisting of
fluorescent moieties, colorigenic moieties, chemiluminescent
moieties, and affinity moieties.
72. The method of claim 71 wherein the first and second detectable
moieties are fluorescent moieties.
Description
FIELD OF THE INVENTION
[0001] The present invention presents methods for gene expression
monitoring that utilize microelectronic arrays to drive the
transport and hybridization of nucleic acids. Procedures are
described for generating mRNA expression samples for use in these
methods from populations of cells, tissues, or other biological
source materials, that may differ in their physiological and/or
pathological state. Provided in the invention are methods for
generating a reusable nucleic acid transcript library from mRNA in
a sample of biological material. In order to improve gene
expression monitoring on the microelectronic arrays, the
transcripts are amplified to produce sample nucleic acid amplicons
of a defined length. Because multiple sample amplicons may be
selectively hybridized to controlled sites in the electronic array,
the gene expression profiles of the polynucleotide populations from
different sources can be directly compared in an array format using
electronic hybridization methodologies. Also provided in the
invention are methods for detecting the level of sample amplicons
using electronically assisted primer extension detection, and
utilizing individual test site hybridization controls. The
hybridization data collected utilizing the improved methods of the
present invention will allow the correlation of changes in mRNA
level with the corresponding expression of the encoded protein in
the biological source material, and thus aid in studying the role
of gene expression in disease.
BACKGROUND OF THE INVENTION
[0002] The human genome contains approximately 100,000 genes. These
genes are expressed at vastly different levels; the majority of
species, over 90%, are present at low abundance, i.e. at five to
fifteen copies per cell, while a few high abundance genes are
expressed at thousands of copies per cell. In addition to the
different levels of basal expression, gene expression is modulated
in response to cell state, cell type, extracellular environment,
disease, etc. Thus, information on changes in the levels of genes
will enable a greater understanding of the pathological and/or
physiological state of the organism under conditions of
interest.
[0003] A number of methods currently exist for analyzing the
expression levels of different messenger RNA (mRNA) species.
Subtractive hybridization was used early in the history of
monitoring of gene expression to analyze differences in levels of
gene expression in different cell populations (Scott, et al.). This
technique is not sufficiently sensitive to detect messages present
at low levels in a polynucleotide population. Representational
difference analysis is a more recent modification that includes
amplification after subtraction, in order to detect mRNAs that are
expressed at low levels (Hubank and Schatz). While this method
allows identification of differentially expressed messages that are
present at low levels, the amplification step makes quantification
difficult.
[0004] Adaptations of the polymerase chain reaction (PCR) have
proven valuable in the field of gene expression. Reverse
transcription coupled with competitive PCR (Competitive RT-PCR)
involves co-amplifying a known amount of an exogenous RNA
competitor with the target mRNA sequence (Gilliland, et al.). The
amount of target is extrapolated from a titration curve based on
the concentration of competitor. The difficulties with this
technique lie in the limited dynamic range of the assay and the
tedium of constructing separate competitors for each target of
interest.
[0005] Real-time PCR is a powerful approach for gene expression
monitoring. The original method detected accumulation of double
stranded species during amplification using ethidium bromide and an
adapted thermocycler (Higuchi, et al.); detection of non-specific
products was a drawback that was subsequently overcome by designing
of probes that generate signal only if the target of interest is
amplified (Holland, et al.; Lee, et al.). This approach requires
that the linear ranges of amplification are similar for abundant
internal controls and endogenous target mRNAs that may be present
at much lower levels. In addition, primer design is critical and
requires special software programs for optimal efficiency.
[0006] Differential display PCR (dd-PCR) is also a PCR-based method
that has been adapted for monitoring gene expression. The original
protocol used sets of random, anchored primers to amplify all mRNAs
in two different cell populations; differences in levels are
visualized by separating the PCR product on denaturing
polyacrylamide gels (Liang and Pardee). Many variations on this
original technique have been devised. In general, however, the
PCR-based amplification of these methods results in a lack of
quantitative correlation of band intensity with message abundance,
variable reproducibility, and a high level of false positives.
Results generated by dd-PCR must therefore be confirmed by other
methods.
[0007] Serial analysis of gene expression (SAGE) is another
technique for gene expression monitoring. Short sequence tags that
uniquely identify the mRNA transcripts in a given cell population
are isolated, concatenated, cloned and sequenced (Velculescu, et
al.). The frequency of any given tag reflects the abundance of the
corresponding transcript. This technique, while powerful, is rather
complicated, requires generation and analysis of large amounts of
sequence data, and the amplification event can skew
quantitation.
[0008] The most recent developments in the field are in the area of
microarrays (Schena, et al.; DeRisi, et al.; Zhao, et al.).
Gene-specific probes are individually arrayed on a solid matrix and
incubated with labeled cDNAs from control and experimental
populations. Comparison of the intensity of probe hybridization
with cDNA targets from the distinct samples reveals differences in
expression of the corresponding mRNAs. Because these arrays are
hybridized passively in a low stringency buffer, differences in
availability of relevant target sequences to the complimentary
probes on the array may not be uniform. In addition, hybridization
characteristics of each probe will vary, due to T.sub.m
considerations and the affinity of probe-target interactions.
Therefore, while these high-density microarrays offer
high-throughput, the hybridization kinetics may not be optimal for
all different probe-target combinations.
[0009] Although great strides have been made in methods to detect
alterations in gene expression, each of the procedures has
drawbacks as well as advantages, as indicated above. All of the
above approaches are either time consuming, complicated, labor
intensive, or a combination of all three. Rapid, sensitive
approaches that allow simultaneous monitoring of multiple mRNAs are
still needed.
SUMMARY OF THE INVENTION
[0010] The present invention provides a method that allows
efficient electronic hybridization of amplified nucleic acids
generated from target mRNAs to complementary probes in a microarray
format. The use of electric fields to transport and drive
hybridization of nucleic acids allows the rapid analysis of
polynucleotide populations. Utilizing electronic hybridization
devices, such as those described in U.S. Pat. No. 5,605,662,
hybridization assays may be accomplished in as little as 1-5
minutes. Additionally, because each site on the microarray is
individually controlled, targets from different samples can be
analyzed on the same matrix under optimized conditions, an aspect
unique to this technology. By improving the use of electronic
hybridization methods and devices in gene expression monitoring
applications, the disclosed methods will dramatically increase the
ability of those in the art to rapidly generate gene expression
information with a minimum of sequence-specific optimization.
[0011] The methods of the invention facilitate the use of
electronically hybridized gene expression monitoring for both
research and clinical applications in several ways. First, through
the use of shortened amplicons of uniform size, the methods of the
invention allow the rapid, simultaneous monitoring of dozens of
genes in comparative and quantitative procedures with minimal
interference from cross-hybridization and secondary structure
formation. Because the individual test sites in the electronic
array may be selectively controlled, several samples may be
screened on the same microarray in the same experiment. Preferred
embodiments of the method for determining the level of mRNA
expression in the cells of a biological sample include the steps of
(a) isolating mRNA from at least one biological sample, (b)
quantitatively amplifying from the isolated mRNA population at
least two gene sequences of interest to produce shortened amplicons
of less than about 300 bases in length, (c) electronically
hybridizing the amplicons to at least two probes bound to a support
at predetermined locations, and (d) determining the amount of each
amplicon hybridized to each probe at the predetermined
locations.
[0012] Although several equally desirable embodiments of the
general method of the invention are provided, it is preferred that
the quantitative amplification step of the method comprise a linear
amplification step in which the sequences of interest are amplified
from a fixed amount of template generated from the reverse
transcription of the mRNA population isolated from the biological
sample. Exemplary preferred processes include single primer DNA
polymerase amplification and in vitro transcription amplification.
The amplicons are preferably shortened during the amplification
process through the use of matched sets of "bookending" primers
which generate amplicons of a defined length, or by the utilization
of an endogenous or introduced type IIs endonuclease site to cleave
the amplicons at some point in the amplification process. The
shortened amplicons produced for use in the methods of the present
invention are preferably about 50 to about 300 bases in length,
more preferably about 50 to about 200 bases in length, and most
preferably about 50 to about 100 bases in length.
[0013] As the electronic hybridization processes of the method may
be carried out on arrays of individually electronically controlled
test sites, multiple genes may be monitored in multiple samples
during a single experiment on the same electronic array device. At
least two, at least ten, and even fifty or more samples may be
assayed in a single experiment. Similarly, at least, 5, 10, 20, 40,
or 50 or more different genes may be simultaneously monitored in an
experiment. As electronic microarray devices with tens of thousands
of test sites have been produced, and the electronic hybridization
process can be completed in as little as 1-5 minutes, an experiment
in which 80 genes are monitored in 100 different samples
sequentially hybridized to rows of test sites on the array may be
completed in a few hours.
[0014] Detection methods which may be used in the gene expression
monitoring methods of the present invention include all commonly
employed nucleic acid hybridization interaction detection methods
such as primer extension labeling, amplicon labeling, reporter
probe detection, and even intercalating dyes. The detectable moiety
in these labeling methods may be a fluorophore, chemiluminescent,
colorigenic, or other detectable moiety. Fluorophore moiety labels
are preferred for use in the present invention because of their
widespread availability and relative ease of use.
[0015] In as second aspect, the present invention provides methods
for the use of reusable bead libraries produced from mRNA samples
to extend the effective amount and life of precious biological and
patient samples by allowing re-amplification of the same sample
nucleic acids. Preferred embodiments of this method of the
invention include the steps of: (a) isolating mRNA from a patient
sample; (b) reverse transcribing a cDNA library from the mRNA
isolate; (c) amplifying the cDNA library with a primer containing
an upstream RNA polymerase promoter site upstream of a sequence
specific for the mRNA of interest and a fill-in primer, wherein at
least one of the primers comprises an affinity moiety; (d) binding
the amplification products from (c) to a solid support coated with
an affinity-binding moiety; (e) utilizing the bound amplification
products as a template for an in vitro transcription reaction; (f)
separating the in vitro transcription products from step (e) from
the amplification products bound to the solid support; and (g)
utilizing the bound amplification products from step (f) as a
template for at least one additional in vitro transcription
reaction, wherein the amount of in vitro transcription product
produced is not significantly less than that produced in step
(e).
[0016] In more preferred embodiments, steps (f) and (g) are
repeated one, two, or even three or more times. As observed by
applicants, the amount of transcript produced in successive rounds
of in vitro transcription does not decrease significantly as
compared to the amount of transcript produced in the proceeding
round. Preferably, at least about 70%, more preferably at least
about 80%, and most preferably at least about 90% of the amount of
transcript produced in a preceding round of transcription is
produced in a succeeding round.
[0017] Preferred affinity moieties for use in the reusable library
method of the invention include biotin, haptens, and antigenic
moieties. Biotin is particularly preferred, and in embodiments
where biotin is the affinity moiety, streptavidin and avidin are
preferred affinity-binding moieties. Preferred solid supports for
use in the reusable library method include beads, microtiter wells,
pins, and the like. Exemplary preferred beads include paramagnetic
beads, polymer beads, and metallic beads.
[0018] In a third aspect, the present invention provides rapid
detection methods for detecting the hybridization of target
sequences to the electronic microarray without the need for
additional reporter probes, or labeling of the target sequences,
using primer extension reactions. Preferred embodiments of this
method of the invention comprise the steps of (a) electronically
hybridizing a nucleic acid in a sample to a nucleic acid probe
bound to a support at a predetermined location; (b) utilizing the
hybridized nucleic acid as a template in a nucleic acid polymerase
reaction to extend the bound probe, thus incorporating a labeled
nucleotide into the extended probe; and (c) detecting the labeled
nucleotide incorporated into the extended bound probe. Preferred
labeling moieties for the labeled nucleotide include fluorescent
moieties, colorigenic moieties, chemiluminescent moieties, and
affinity moieties. Fluorescent moieties are particularly preferred.
Nucleic acid polymerase reactions which may be used in the method
include DNA polymerase reactions (where the hybridized nucleic acid
is DNA) and reverse-transcriptase reactions (where the hybridized
nucleic acid is RNA).
[0019] A fourth aspect of the present invention is a method of
providing an internal control for individual test sites on an
electronically controlled microarray for use in nucleic acid
hybridization reaction assays for determining the presence of
nucleic acid sequences in nucleic-acid-containing samples. Such
internal controls are useful for real-world applications of
microarray technology because of the inherent irregularities
introduced by the microfluidics systems which distribute the sample
and reagents to the surface of the microarray. Preferred
embodiments of the method comprise the steps: (a) attaching a mixed
nucleic acid probe consisting of a first nucleic acid probe
specific for a first nucleic acid sequence known to be present in
the sample (e.g., endogenous or spiked), and a second nucleic acid
probe specific for a second nucleic acid sequence of interest to a
first test site on the electronically controlled microarray; (b)
attaching a mixed nucleic acid probe consisting of the first
nucleic acid probe and a third nucleic acid probe specific for a
third nucleic acid sequence of interest, which may be the same as
or different than the second nucleic acid sequence, to a second
test site; (c) electronically hybridizing the sample nucleic acids
to the nucleic acid probes on the first and second test sites; (d)
specifically detecting the extent of hybridization of the sample
nucleic acids to the first nucleic acid probe at the first and
second test sites; (e) specifically detecting the extent of
hybridization of the sample nucleic acids to the second and third
nucleic acid probes at the first and second test sites; (f)
comparing the hybridization values obtained for the first nucleic
acid probe at the first and second test sites to obtain a
normalization factor; and (g) normalizing the hybridization values
obtained in (e) for the second and third probes using the
normalization factor obtained in (f).
[0020] Preferred embodiments of the internal control methods of the
invention utilize an endogenous "housekeeping" gene sequence, which
is known to be maintained at a steady-state level across the
relevant sample cell types, as the first control sequence.
Alternatively, exogenous nucleic acid sequence may be added to the
sample at known concentrations. The detection methods utilized to
specifically detect the hybridization of the sample nucleic acids
to the first and the second and third nucleic acid probes may be
independently chosen from any standard detection method, including
the labeling of amplified sample nucleic acids through sequence
specific primers, primer extension detection, hybridization of
reporter probes to bound sample nucleic acids, or a combination of
these methods. In order for hybridization to the first nucleic acid
probe to be distinguishably detectable from hybridization to the
second and third nucleic acid probes, it is desirable to use two
easily distinguishable detectable moieties. Preferred detectable
moieties for use in the internal control method are fluorescent
moieties with different emission wavelengths. Alternatively, the
extent of hybridization to the first (control) probe may be
determined first using a detectable moiety after performing a first
selective labeling method, and then the extent of hybridization to
the second and third probes determined after a second selective
labeling method with the same detectable moiety by determining the
increase in the detectable signal.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1: A schematic of two alternative methods for
generating shortened amplicons of a defined size for use in the
mRNA gene expression monitoring methods of the invention.
[0022] FIG. 2: A schematic of an electronically addressable
microarray device which is similar to the device used in the
Examples. The depicted device is described in detail in U.S. Pat.
No. 5,605,662. Note that in this device the test sites on the array
are defined in the plane of the array by the electrodes, but are
separated from the electrodes by a permeation layer, to which
nucleic acid probes may be attached.
[0023] FIG. 3: Shown are the agarose gel electrophoresis-separated
RNA products of a 1.sup.st, 2.sup.nd, and 3.sup.rd round of in
vitro transcription from the same double stranded amplified cDNA
library immobilized on magnetic streptavidin-coated beads. The far
left lane contains a molecular size marker.
[0024] FIG. 4: Shown are grayscale hybridization fluorescence data
for five separate gene probes, using 50, 100, 250, and 500
nucleotide amplicons. As can be seen from the best results were
obtained using amplicons of around 100 bases in length.
[0025] FIG. 5a: Shown are grayscale hybridization fluorescence data
for serial dilutions of amplified sample plasmid nucleic acids,
hybridized to a .beta.-1a capture probe. NS denotes no capture
sequence attached to the test sites in the far right column. Note
the reproducibility of the hybridization signal across the rows of
the electronic array.
[0026] FIGS. 5b & 5c: Shown are grayscale hybridization
fluorescence data for defined amounts of sample nucleic acid
sequence, and a graphical representation of a concentration curve
derived from these data.
[0027] FIG. 6: Shown are grayscale hybridization fluorescence data
for four separate gene probes and a control sequence (NS) on twelve
test sites on a NanoChip.TM.. mRNA was isolated from LPS induced
and uninduced monocytes; amplification, hybridization and reporting
were carried out as described in Example 5. The treated monocytes
clearly show an altered mRNA expression level in IL1, TGF.beta.,
and JUN, while the expression level of IL6 is only slightly
increased.
[0028] FIG. 7: A graphical illustration of the results shown in
FIG. 6.
DEFINITIONS
[0029] As used herein, "nucleic acid" refers to any nucleic acid
polymer, including deoxyribonucleic acid and ribonucleic acid
polymers, including modified DNA or RNA, including synthetic
oligonucleotides, peptide nucleic acids (PNA), pyranosyl nucleic
acids (pRNA), and derivatives of these nucleic acids.
[0030] "Amplicon," as used herein, denotes an amplified nucleic
acid sequence which may be either RNA or DNA, depending on the
amplification method used (e.g., DNA PCR produces a DNA amplicon,
while in vitro transcription produces an RNA amplicon.) The
amplified nucleic sequence comprises either the same or
complementary sequence as that of the original nucleic acid
sequence which was amplified. "Shortened amplicon" denotes an
amplicon which contains a portion of the full endogenous nucleic
acid sequence rather than the whole sequence.
[0031] "Electronic hybridization" means the use of electric fields
to concentrate nucleic acids within a certain area in order to
reduce the time necessary to hybridize complementary nucleic acids.
Electronic hybridization is advantageously performed using
electronically controlled microarray devices, such as those
described in U.S. Pat. No. 5,605,662. These devices comprise
individually controllable electrodes which are covered by a
permeation layer, or layer of material which permits ion exchange
between the buffer and the electrode, but which inhibits contact
between nucleic acids in the buffer and the electrode. Nucleic acid
capture probes may be advantageously attached to binding moieties
within or on the permeation layer of these electronic array
devices.
[0032] "Gene," as used herein, means an organismal nucleic acid
sequence encoding a protein, such as genomic DNA or mRNA (including
splicing variants), or a copy or portion thereof produced by
molecular genetic manipulations, such as cDNA, in vitro transcribed
RNA, cloned DNA, etc. As used herein "target" generally refers to a
gene of interest.
[0033] "Amplification," as used broadly herein, encompasses various
processes and sets of processes (including: reverse transcription,
DNA polymerase reactions, ligase reactions, in vitro transcription,
and combinations thereof) for copying nucleic acids (either in
their original deoxyribonucleotides or ribonucleotides, or other
natural or artificial nucleotides) through molecular genetic
manipulations. "Linear amplification" or "quantitative
amplification" refers to processes and sets of processes for
producing copies of nucleic acids from a substantially constant
amount of template, so that the number of copies of the nucleic
acid, or its complement, increases linearly as a function of time,
rather than exponentially.
DETAILED DESCRIPTION OF INVENTION
[0034] The methods described are designed to determine the
abundance of target genes in a distinct polynucleotide population,
particularly as compared to the abundance of those target genes
within a different polynucleotide population. The methods of this
invention are particularly suited for gathering data to correlate
differences in expression patterns with specific physiological
and/or pathological states. Electronic nucleic acid hybridization
techniques and devices (such as those described in U.S. Pat. No.
5,605,662, incorporated herein in its entirety), are capable of
dramatically reducing the time necessary to perform various nucleic
acid hybridization procedures, and are thus an extremely useful
tool for a wide variety of biological assay methods. The methods of
this invention enable those of skill in the biochemical arts to
more fully exploit this tool to study the expression of genes in
organisms under various physiological conditions.
[0035] In the methods of this invention, target genes are
quantitatively amplified from mRNA populations derived from any
biomaterial, including, but not limited to, cells from unicellular
organisms, cells derived from in vitro cultured cell lines, and
cells or tissues from multicellular organisms. In one preferred
embodiment, the amplified target genes (amplicons) are
enzymatically shortened using a type IIs restriction endonuclease.
In another preferred embodiment, generation of shortened amplicons
is mediated via sequence specific oligonucleotides which "bookend"
the amplified sequence. Shortened amplicons are analyzed by
electronic hybridization to capture probes comprising complementary
nucleic acids that are specific for each target gene of interest.
These capture probes are preferably immobilized in a microarray
format. Detection of the hybridized targets may be performed by
primer extension of the capture probe, or by hybridization of a
second complementary species that is labeled, or by other
means.
[0036] An important aspect of the invention is the generation of
target nucleic acid sequences which are of similar size and/or
reduced length. The efficiency and uniformity of electronic
hybridization is increased when targets are limited in size. In
addition, efficient analysis of different targets is facilitated if
the target species are similar in size because variations in
electronic transport and hybridization of different targets are
reduced. Further, when the target(s) being examined comprise single
stranded nucleic acids, particularly ribonucleic acids, secondary
structure considerations are minimized by reduced length. Gene
expression monitoring on electronic microarrays is thus improved by
utilization of shortened targets of relatively uniform size, as is
demonstrated in the data of Table 1.
[0037] mRNA Isolation and cDNA Library Preparation
[0038] The initial step in the subject invention is isolation of a
sample of polynucleotides, usually mRNA, from populations of
interest. The polynucleotide populations may be derived from a
variety of sources, including but not limited to different cell
types from in vitro cultured cell lines or different cell types
from organs or tissues of multicellular organisms, or the same cell
type from different organisms of the same species. The
polynucleotide populations may also be derived from the same cell
type from in vitro cultured cell lines or from an organ or tissue
of a multicellular organism, at different stages of development,
disease, or treatment.
[0039] After isolation by standard means well known to those of
skill in the molecular genetics arts, reverse transcription is
performed to generate single-stranded cDNA from the mRNA
population. cDNA synthesis can be performed by any method known to
those of skill in the art. The various dNTPs, buffer medium, and
enzyme with reverse transcriptase activity may be purchased
commercially from various sources. Applicants have found the
Superscript.TM. enzyme system to be well suited for the production
of an initial cDNA library. First strand synthesis may be directed
by an oligo(dT) primer that hybridizes to all polyadenylated RNA
species. The oligo(dT) primer is usually 10-30 bases long, more
preferably 12-18 bases long, and may comprise a mixture of primers
of different lengths. Other suitable polythymine primers include
the non-replicable dT primer described in U.S. Pat. No. 6,027,923,
and oligo(dT).sub.nV (V=A, C, or G) primers.
[0040] Quantitative Shortened Amplicon Generation: Primer Extension
and in vitro Translation Techniques
[0041] In transcription-amplification embodiments of the invention,
the generation of amplicons is accomplished through the use of a
chimeric oligonucleotide specific to each target of interest.
Following cDNA synthesis, the target(s) of interest may be linearly
amplified by primer extension of a chimeric oligonucleotide(s)
using DNA polymerase. Linear amplification either by primer
extension, as here, or by other means (such as in vitro
transcription, used below), is necessary in order to allow
quantitative comparisons between different samples. The sequence
specific portion of the chimeric oligonucleotides are sufficiently
specific as to anneal to the complementary target sequence under
conditions of primer extension by a polymerase reaction (DNA
polymerase if produced from a cDNA library), which are familiar to
those of skill in the molecular biology arts. In order to generate
target amplicons for the gene sequences of interest, one chimeric
oligonucleotide specific to each target of interest is used. These
oligonucleotides contain an RNA polymerase promoter sequence at the
5' end; this sequence may be the consensus binding site for T7, T3,
SP6, or another RNA polymerase. Adjacent to the RNA polymerase
promoter site is a sequence specific to the target gene of
interest. This gene-specific sequence is not restricted in length;
however, to promote efficient annealing of the chimeric
oligonucleotide(s) to the target(s) of interest, the gene-specific
sequence will preferably be between 15 and 35 bases in length. The
oligonucleotides may contain a biotin moiety at the 5' end, or may
be unmodified. In embodiments utilizing a type IIs endonuclease for
shortened amplicon generation, the chimeric primer will also
contain, at the 3' end, the recognition sequence for a type IIs
restriction endonuclease.
[0042] The sets of chimeric oligonucleotides that are used in
target preparation will generally represent at least two distinct
target species but may represent 10, 20, 40, or even up to 50
distinct target species. Above about 50 different target species,
amplification efficiency and the quantitative nature of the results
may be compromised, utilizing currently available amplification
techniques. However, the use of the present invention with improved
amplification techniques to measure, potentially, 80, 90 or over
100 specific genes simultaneously is also contemplated by the
present methods. The chimeric oligonucleotides are typically chosen
to represent targets that are known or suspected to be
differentially expressed under the experimental conditions (e.g.,
cell type, or two different stimuli for a single cell type), or may
represent targets for which no expression data are available. One
of the chimeric oligonucleotides in the set is chosen to allow
analysis of levels of a housekeeping gene that is known to be
expressed at similar levels in the different conditions being
examined, or of an added exogenous control sequence.
[0043] Examples of commonly used housekeeping genes include
glyceraldehyde phosphate dehydrogenase (GAPDH), .beta.-actin, and
ribosomal RNAs. Controls can also comprise exogenous sequences that
are added into the starting material at known concentration and
processed along with the targets of interest. An example of such
sequence is the .beta.-lactamase gene, a prokaryotic gene that
confers ampicillin resistance. (For review, see Reischl, U. and
Kochanowski, B. (1999) "Quantitative PCR" Quantitative PCR
Protocols (pp.3-30). Humana Press., Totowa, N.J.; and also Ferre F.
(1992) Quantitative or semi-quantitative PCR: Reality versus myth.
PCR Methods Appl. 2, 1-9.) The selection of a particular control
sequence for use in an application of the described methods will be
case specific, depending on the organism used, the particular cells
studied, and the familiarity of the researcher with a particular
constitutively expressed sequence. However, using the guidelines
presented, one of ordinary skill in the art could readily select an
endogenous or exogenous sequence for use as a control in the
present methods.
[0044] Single-primer extension may be directed by a thermophilic
DNA polymerase, usually a 3'-5' exonuclease-minus derivative
polymerase, e.g. Vent.RTM..sub.R(exo-) DNA polymerase. Multiple
copies of the target(s) are generated during the extension
reaction, in which repeated cycles of denaturation, oligonucleotide
primer annealing, and DNA polymerase-directed primer extension are
performed. Following generation of multiple single-stranded copies
of target(s) from the cDNA pool, the complementary strands are
generated. In one embodiment, generation of the complementary
strand is mediated by a common primer that binds to all amplified
targets. This primer may be the oligo(dT) oligonucleotide used for
first strand cDNA synthesis, a fraction of which is carried over
from the first strand cDNA synthesis reaction into the primer
extension reaction. Alternatively, the primer may be a mixture of
random short polynucleotide sequences, e.g. random hexamer primers.
In this embodiment, the primer is allowed to anneal at a
temperature corresponding to the T.sub.m of the primer used for
second strand synthesis. In another embodiment, a gene-specific
oligonucleotide for each target of interest is utilized, yielding a
"bookended" product, as described further below. This primer will
generally be 15-30 bases in length and will hybridize at a specific
site within the amplified target such that the second strand
produces an amplicon of a defined length. The primers may contain a
biotin moiety at the 5' end, or may be unmodified. Primers are
allowed to anneal at or slightly below the lowest T.sub.m among the
primers in the set. After allowing sufficient time for the primers
to anneal, the temperature is increased to allow efficient
extension of the primers by the thermophilic DNA polymerase.
[0045] Shortening of Amplicons by Type IIs Endonuclease Cleavage or
Bookending
[0046] Amplicons for use in the present invention preferably are
less than about 300 bases, more preferably less than about 200
bases, and most preferably less than about 100 bases. Because the
amplicon must be of a sufficient size to hybridize specifically to
the capture probe, amplicons are preferably greater than 15 bases
in length, and are more preferably at least about 50 bases in
length. In addition, although the amplicons for use in the methods
of the invention may differ significantly in length, it is
preferred that they be of similar length, preferably differing in
length by less than 20 bases, and more preferably differing in
length by less than 10 bases. The generation of shortened amplicons
may be accomplished through the use of Type IIs endonucleases, or
by the use of primers designed to "bookend" a stretch of the target
sequence. Although both methods are described below, other methods
may be devised by those of skill in the art. The use of shortened
amplicons made by alternative amplification strategies in the
methods of the present invention is contemplated to be within the
scope of those methods, so long as a shortened, quantitatively
amplified nucleic acid is produced from the sample mRNA.
[0047] In a first strategy, shortening of the target nucleic acid
sequences is performed by digestion with a type IIs restriction
endonuclease. Type IIs restriction endonucleases cleave at a
defined distance from the recognition site, thus producing a
gene-specific "tag" that can subsequently be used to isolate and
quantify each target. For this embodiment, a type IIs restriction
endonuclease recognition site is incorporated at or near the 3' end
of the chimeric oligonucleotide used for amplification, supra.
Where possible, this sequence will be present in the target(s) of
interest, and the chimeric oligonucleotide(s) will be designed to
utilize the endogenous restriction endonuclease recognition
sequence. In this case, the gene-specific portion of the
oligonucleotide(s) will anneal to the restriction endonuclease
recognition site and sufficient adjacent sequence 5' of the
recognition site to produce an amplicon of the desired length upon
cleaving.
[0048] Where an endogenous type IIs restriction enzyme recognition
site is not present in the target of interest, a sequence within
the target will be chosen that resembles the desired site as
closely as possible, with preference given to sequences that
perfectly match the 3' end of the endonuclease recognition site.
The chimeric oligonucleotide will then be designed to generate the
desired restriction endonuclease recognition site by altering the
target sequence during primer extension. Such alterations may
include changing, inserting or deleting nucleotides as necessary to
generate a type IIs restriction endonuclease recognition site into
the amplified target.
[0049] The proximity of the restriction endonuclease recognition
site to the 3' end of the chimeric oligonucleotide will in part be
determined by whether a site exists within the targets of interest
or whether such site must be generated within the chimeric
oligonucleotide. If an endogenous site is used, the chimeric
oligonucleotide may end at the last base pair of the restriction
endonuclease recognition site or may include additional
target-specific sequence. In the case where the target sequence
must be altered to generate a restriction endonuclease recognition
site, the chimeric oligonucleotide will generally, although not
necessarily, extend beyond the restriction endonuclease recognition
site in order to permit the mutated oligonucleotide sequence to
efficiently anneal to the target. The extension, if any, will
preferably be only a few bases in length, in order to ensure that
sufficient target sequence is retained upon cleavage by the Type
IIs endonuclease.
[0050] Digestion with the type IIs restriction endonuclease will
yield target fragments that contain the RNA polymerase promoter
sequence followed by the target-specific sequence contained within
the chimeric primer, plus the target-specific sequence that lies
between the type IIs restriction enzyme recognition site and the
type IIs restriction enzyme cleavage site. If the chimeric
oligonucleotide contains a biotin moiety at the 5' end, the
digested fragments may at this point be isolated using
streptavidin-coupled magnetic beads; alternatively, the
Vent-amplified material can be applied to the streptavidin-coupled
magnetic beads for purification prior to restriction enzyme
digestion. If a biotinylated 3' gene-specific oligonucleotide is
used to fill in the amplified single-stranded products, targets can
be applied to streptavidin-coupled magnetic beads prior to
digestion and thereby isolated from any nonspecific materials.
Subsequent to enzymatic digestion, the 5' ends of the targets can
be isolated away from the bead-bound 3' ends of the amplified
targets.
[0051] Subsequent to digestion, target fragments are used as
templates in in vitro transcription reactions mediated by a RNA
polymerase, the promoter sequence for which was incorporated via
the chimeric oligonucleotide. Standard methods for in vitro
transcription are known to those of skill in the art. In the case
where biotinylated chimeric oligonucleotides were used to generate
amplified target, multiple rounds of in vitro transcription can be
performed; the bead-bound templates can also be stored for future
in vitro transcription reactions. In vitro transcribed templates
are then cleaned and desalted by any standard method including, but
not limited to, gel filtration columns.
[0052] In an alternative strategy, shortened targets are generated
via oligonucleotide primers rather than by endonuclease digestion.
In the case where oligo(dT), random primers, or gene specific
oligonucleotides were utilized to fill in the single-stranded
amplified target(s), in vitro transcription reactions are performed
after the fill-in reaction. When a gene-specific oligonucleotide is
used to generate the second strand, this primer is designed to
hybridize to a sequence within the target at a defined length from
the 5' end, generally less than about 300 base pairs away. In this
"early bookending" embodiment, the RNA produced in the in vitro
transcription reaction is sufficiently shortened such that the
amplified material can be directly analyzed by electronic
hybridization. Alternatively, the shortened RNA products may be
subjected to another round of cDNA production and DNA polymerase
mediated amplification utilizing gene-specific or random
primers.
[0053] In the case where oligo(dT) or a set of primers of random
sequence is utilized to fill in the amplified target(s), in vitro
transcription will yield RNAs of variable length, some of which may
be too short to be recognized by the capture oligonucleotide used
in electronic hybridization, as described infra, and some of which
may be too long to be efficiently electronically transported and
hybridized. In order to improve detection of these target(s), the
RNA derived from the in vitro transcription reaction is used as a
template for a second cDNA synthesis reaction. For this method, a
gene-specific oligonucleotide specific for each target of interest
is used to prime cDNA synthesis. This primer will generally be
15-30 bases in length and will hybridize at a specific site within
the amplified target such that the second strand is of a defined
length, generally less than about 300 base pairs, more preferably
about 50 to about 300 bases, more preferably from about 50 to about
200 bases, and most preferably from about 50 to about 100 bases.
This reverse transcription reaction is performed using reagents and
methods known to those of skill in the art, as described supra. The
products of this reaction will comprise shortened amplicons for
each target of interest
[0054] Electronic Hybridization of Shortened Amplicons
[0055] Analysis of target(s) is performed on microarrays using
electronic hybridization. The specific electronic hybridization
procedures and probe attachment will vary slightly from device to
device, and may be modified from the discussion below by one of
ordinary skill in the biochemical arts. For the purposes of
illustration, hybridization and detection procedures will be
described with reference to the NanoChip.TM. electronic
hybridization device, similar to that pictured in FIG. 2. However,
other electronically addressable devices for microscale biochemical
reactions may be utilized to carry out the methods of the
invention.
[0056] Capture probes are immobilized via interaction with a
permeation layer on the electronic microarray surface, as described
in U.S. Pat. No. 5,605,662, incorporated fully herein by reference.
The stable interaction of the probes may be accomplished by a
variety of different methodologies including, but not limited to,
streptavidin-biotin interactions in which the capture probes
contain a biotin moiety at the 5' end and streptavidin is
incorporated within the permeation layer.
[0057] Biotinylated capture probes specific to each target of
interest are immobilized at different position, or test site, on
the microarray using electric field transport (i.e., electronically
addressed to the test sites). When primer-extension detection
methods will be used to quantify hybridized amplicons, the biotin
label should not be on the 3' phosphate of the capture probe, so
that the phosphate is available for extension in the polymerase
reaction. In embodiments where Type IIs endonuclease shortening is
used, these capture probes are designed to be complementary to the
target sequence that is between the type IIs restriction
endonuclease recognition site and the type IIs restriction
endonuclease cleavage site. The capture probes may contain
additional target-specific sequence including the type IIs
restriction endonuclease recognition sequence and upstream
sequence. In embodiments where bookending primers are used, capture
probes are designed to hybridize to a region flanked by the binding
sites for the chimeric oligonucleotide and the gene-specific
oligonucleotide used to fill in single-stranded amplified products
and/or generate cDNA. Generally the capture probes will comprise
about 18 to about 30 bases, although shorter or longer capture
probes may be utilized with electronic hybridization procedures.
Capture probes may include non-amplicon-complementary sequences for
use in zip-code addressing of the probes, or for other purposes
(e.g., restriction endonuclease sites for selective cleavage).
[0058] In a preferred embodiment, a capture probe complementary to
a control sequence is addressed to one or more location(s) on the
microarray. This control sequence may be a housekeeping gene that
is expressed at similar levels under the different conditions being
examined, or may be an exogenous sequence which is added to the
sample nucleic acid mixture prior to or after amplification. In a
preferred embodiment, the capture probe complementary to the
housekeeping gene is combined with each target-specific capture
probe, and the two capture probes are co-addressed to a given
position(s) on the microarray. This embodiment allows normalization
within each position of the microarray, allowing better
quantitative results. The microfluidics utilized to feed a sample
nucleic acid solution introduce variations in exposure of each
individual test site to the sample nucleic acid solution because of
flow patterns, pooling, etc. Thus, the use of an internal control
probe at each test site position allows for the normalization of
assay data for each individual test site microenvironment.
[0059] The pool of shortened amplicons (in vitro transcribed target
template(s), or cDNA copies thereof) is subsequently electronically
hybridized to the immobilized capture probes. Because sites on the
array are electronically controlled, hybridization can be
restricted to a subset of locations within the microarray. Thus,
different shortened amplicon pools, derived from different samples,
can be analyzed on the same microarray. For these experiments, one
pool of shortened amplicons from a given condition, cell type, or
other source is injected into the electronic hybridization device,
electronically transported to a subset of locations within the
microarray, and allowed to hybridize with specific immobilized
capture probes at those locations. Subsequently, this target pool
is removed, unbound nucleic acids are washed away, and a second
pool of targets from a different condition, cell type, or other
source is injected and electronically transported to a distinct
subset of locations within the microarray that contains the same
immobilized target-specific capture probes. This second target pool
is allowed to hybridize and unbound material is removed by washing.
Subsequently, hybridized target species are detected by one of a
variety of reporting methodologies.
[0060] This procedure, which allows direct analysis of a set of
targets from different target samples on the same microarray,
represents a particular strength of electronic hybridization in the
area of gene expression profiling. Using the methods of the
invention, it is easy for one of skill in the art to assay at least
two, at least ten, and even fifty or more samples in a single
experiment. The results of this process are illustrated in FIG. 6,
which shows the hybridization of two groups of mRNA amplicons on
the same chip: one sample was isolated from monocytes which have
been treated with lipo-polysaccharide, and the other sample was
isolated from untreated monocytes. Alternatively, different
electronic microarrays can be used to analyze expression patterns
in different amplicon samples.
[0061] Detection of Hybridized Amplicons on Electronic
Microarrays
[0062] Following electronic hybridization of target(s) to
immobilized capture probes, the bound target(s) may be detected by
several means, including include all commonly employed nucleic acid
hybridization interaction detection methods such as primer
extension labeling, amplicon labeling (preferably through labeled
sequence-specific primers), reporter probe detection, and even
intercalating dyes. The detectable moiety in these labeling methods
may be a fluorophore, chemiluminescent, colorigenic, or other
detectable moiety. Fluorophore moiety labels are preferred for use
in the present invention because of their widespread availability
and relative ease of use.
[0063] In one preferred embodiment, the hybridized amplicons are
detected by hybridization of a reporter species, such as a distinct
target-specific oligonucleotide, that is labeled such that a
detectable signal is produced, either directly or in combined
action with an additional component(s) of a signal-producing
system. An example of a directly detectable label is a fluorescent
moiety that is present on the reporter, including, but not limited
to, bodipy dyes such as Bodipy Texas Red and cyanine dyes such as
Cy3 and Cy5. In a preferred embodiment in which the capture probe
complementary to the housekeeping gene, or the control, is combined
with each target-specific capture probe and co-addressed to a given
position on the microarray, the reporter for the said housekeeping
gene is labeled with a fluorophore emitting at one wavelength,
while the reporters for the amplicons of the genes of interest are
labeled with a different fluorophore that emits at least one other
wavelength. Analysis of signal generated at each wavelength allows
detection of all species hybridized on the microarray and
subsequent normalization using the expression data from the
housekeeping gene.
[0064] In another preferred embodiment, detection of at least a
portion of the hybridized amplicons of the genes of interest is
accomplished by enzymatic reporting. In this variation, after
electronic hybridization, the microarray is incubated with reagents
that allow primer extension of the capture probe using the bound
RNA or cDNA amplicon target(s) as a template. For RNA targets, the
extension is performed using an enzyme with reverse transcriptase
activity, e.g. Superscript.RTM. reverse transcriptase, that uses
RNA-DNA hybrids, but not RNA-RNA or DNA-DNA hybrids, as a template.
Utilization of such enzyme will reduce non-specific signal that
otherwise may be produced from extension of self-annealed regions
of either capture probes or target molecules. For cDNA targets, an
enzyme with DNA polymerase activity is used. In either case, one or
more of the nucleotides present in the reaction will be labeled.
The nucleotide species may be either deoxynucleotide(s) or
dideoxynucleotide(s) and may be labeled with any detectable moiety,
usually a fluorescent moiety. Preferred fluorescent moieties for
use in the primer extension methods of the invention include
cyanine dyes, e.g. Cy5 and Cy3, and other fluorescent dyes such as
Bodipy Texas Red, rhodamine, fluorescein, and cumarin.
Incorporation of the labeled nucleotide(s) into the extended
capture probe will allow target-specific hybridization of the
amplicons to be detected as a fluorescent signal at the individual
test sites in the array. Other reagents included in the reaction
include buffer medium for optimal enzymatic activity; such medium
is commercially available and known to those of skill in the
art.
[0065] In embodiments where the capture probe for the housekeeping
target is co-addressed with target-specific capture probes, these
two species must be differentiated at the reporting step. In one
variation, a reporter specific for the housekeeping gene is allowed
to hybridize prior to, concurrent with, or after the primer
extension of capture probes specific for the genes of interest. The
reporter is modified (e.g., by phosphoramidite chemistry or other
means known in the art) at the 3' end to prevent extension of the
reporter species during the primer extension reaction. The
housekeeping gene-specific reporter is labeled such that the signal
from the hybridized reporter can be distinguished from the signal
generated from the extended target-specific capture probes, e.g.
the reporter is labeled with Cy3 and the labeled NTP(s) used in the
primer extension reaction is labeled with Cy5. In a preferred
variation, the capture probe for the housekeeping gene is also
modified at the 3' end, e.g. with an amino-blocking group, to
prevent primer extension. Use of a blocked capture probe allows
simultaneous hybridization of the reporter complementary to the
housekeeping gene and primer extension of the target-specific
capture probes, since the blocked capture probe hybridized to the
housekeeping gene will not be extended.
[0066] The hybridization patterns are analyzed after signal
detection. The signal generated by the housekeeping gene, which is
expressed at equivalent levels in the different samples tested, is
used to normalize differences in total nucleic acid concentration,
electronic transport, and electronic hybridization efficiencies
between samples, and to account for micro-environmental variations
between test sites. Differences in intensities in the target
amplicon signals after normalization is an indication of altered
expression levels in the original mRNA in the sample nucleic acids
under the conditions examined.
[0067] Applications of the Present Methods
[0068] The methods of the present invention may be readily applied
to a wide variety of gene expression experimental models for use in
studying, for example, disease and oncogenesis, physio-chemical
cellular responses to stimuli, and cell growth and differentiation.
The disclosed methods are ideal for these applications because of
their speed, reproducibility, and flexibility as to the number,
kind, and concentration of gene probes used in the hybridization
experiment. As generally outlined below, the disclosed methods can
greatly increase the ease and rapidity of common expression
experiments.
[0069] For example, the methods of the present invention may easily
be used to titrate the amount of amplified mRNA present in a
sample. As illustrated in FIG. 5, a sample of amplified nucleic
acids may be serially diluted. Each dilution may then be
specifically hybridized with a subset of electronically activated
test sites on a NanoChip.TM. device. By producing a concentration
curve from the serial dilution data, the original concentration of
the amplified mRNA sequence in the sample may be determined. If a
specific amount of control sequence is added to the sample prior to
amplification for use as a control, this amplified mRNA
concentration may also be utilized to determine the original mRNA
concentration in the sample. Thus, the methods of the invention may
be used to obtain absolute quantitative measurements, as well as
relative quantitative measurements of gene expression.
[0070] The methods of the present invention may also be used
advantageously to compare, side by side, the expression levels of
mRNA in a cell type that has undergone two different physical or
chemical stimuli. For instance, in Example 5, the expression levels
of four genes in monocyte cells which were treated with LPS
(lipopolysaccharide) or untreated (control) were examined. As shown
in FIG. 6, a marked change in several genes was observed between
the LPS treated monocytes and the untreated monocytes. Similar
experiments may easily be devised by those of skill in the art for
screening potential chemical inducers or repressors of gene
expression for use in combinatorial-library high-throughput drug
discovery, while monitoring the effects of the compound on
non-target gene expression pathways to minimize side effects.
[0071] Because of the flexibility of being able to perform multiple
sample tests on the same electronically assisted hybridization
device, the specific format of an assay may also be easily changed
to suit new directions in a particular research project. For
instance, a researcher may initially use a 100 test site
NanoChip.TM. to screen 50 different genes in two samples from a
cell line that has and has not been exposed to a chemical agent.
Upon identifying 5 genes of particular interest, the researcher may
then strip off the old probes from the streptavidin permeation
layer, reconfigure the 100 test sites with using just 5 of the
original 50 probes, and verify the result with a larger sample set
of 5 controls and 15 stimulated cell samples, using 3 groups of
cells stimulated with different concentrations of the chemical
agent. Once an apparent rough critical concentration of chemical
agent has been identified, four cell samples may be stimulated with
concentrations of the agent centered around the rough critical
concentration. The amount of amplified mRNA produced at each
concentration may then be determined from four serial dilutions of
each sample, utilizing the NanoChip.TM. as configured for screening
the 20 samples above. As each screening step may be accomplished in
10 minutes to an hour, the entire project may be completed in a
manner of days utilizing the methods of the present invention,
depending on the stimulation period allotted for the cell
samples.
[0072] The following examples are offered to further illustrate the
various aspects of the present invention, and are not meant to
limit the invention in any fashion. Based on these examples, and
the preceding discussion of the embodiments and uses of the
invention, several variations of the invention will become apparent
to one of ordinary skill in the art. Such self-evident alterations
are also considered to be within the scope of the present
invention.
EXAMPLES
Example 1
General Protocol for Gene Expression Monitoring with Shortened
Amplicons
[0073] A. cDNA Synthesis.
[0074] Total RNA (5 .mu.g) or poly(A.sup.+) RNA(0.5 .mu.g) was used
as template in cDNA synthesis reactions with Superscript.TM. II
RNase H.sup.- Reverse Transcriptase (Life Technologies, Rockville,
Md.) per manufacturer's instructions, with the inclusion of 10U
RNase Inhibitor (Roche Diagnostics, Mannheim, Germany). Synthesis
was primed with either 500 ng oligo(dT).sub.12-18 (Life
Technologies), or with oligo(dT).sub.17V (where V=A, G, or C), or
with an oligo(dT.sub.18T.sub.modN.sub.3) containing a
non-replicable 1,3-propane diol moiety on the fourth base from the
3' end (U.S. Pat. No. 6,027,923). When an exogenous target gene was
used as a control, 100-500 pg poly(A.sup.+) RNA for the target was
included in this initial cDNA synthesis reaction.
[0075] B. Linear Amplification Using DNA Polymerase.
[0076] One-fourth to one-half of the cDNA synthesis reactions was
used in Vent.sub.R.RTM.(exo.sup.-) DNA polymerase (New England
Biolabs, Beverly, Mass.) mediated primer extension reactions.
1.times. Thermopol Buffer (New England Biolabs), 200 nM each dNTP
(Roche Molecular Biochemicals, Indianapolis, Ind.), 50 nM each
biotinylated chimeric T7-target specific oligonucleotide [See Table
2], and 0.04U/ml reaction Vent.sub.R.RTM.(exo.sup.-) DNA polymerase
were used. Final Mg.sup.++ concentration was generally 2.3 mM, but
was occasionally adjusted to as much as 4.6 mM. Chimeric primers
for one to nine specific targets were added, in addition to a
chimeric oligonucleotide specific for the internal (exogenous or
endogenous) control. Samples were denatured at 95.degree. C. 10
min, then cycled 30-50 times at (95.degree. C. 30 sec,
50-60.degree. C. 60 sec, 72.degree. C. 60 sec). After the last
cycle, samples were held at 72.degree. C. for 5 min. 50 ng random
hexamer primers (Life Technologies) or 200 nM target-specific
oligonucleotide(s) were added and annealed to the amplified targets
at 42.degree. C., 10 min. Annealed primers were extended by
increasing the temperature to 70.degree. C., 10 min.
[0077] C. Binding of Amplified Targets to Streptavidin-Coated
Magnetic Beads and in vitro Transcription Reactions.
[0078] Amplified targets were isolated from other components of the
amplification reaction mixture by binding to streptavidin-coated
magnetic beads (Dynalo.RTM., Oslo, Norway) 30-60 min at 37.degree.
C. with occasional agitation. Beads were then isolated on a
magnetic separator (Dynal) and the bound target was used as
template for in vitro transcription reactions using the T7
MegaShortScript in vitro transcription kit (Ambion, Inc., Austin,
Tex.). Reactions were incubated as per manufacturer's instructions
2-4 hr at 37.degree. C. Transcribed RNA was purified from excess
NTPs by isolation with Chromaspin-30 columns (Clontech, Palo Alto,
Calif.) as per manufacturer's instructions. The RNA targets were
used directly for analysis on microelectronic arrays, or used as
template in a second cDNA synthesis reaction as described
below.
[0079] D. Second Round cDNA Synthesis
[0080] 20 .mu.l of in vitro transcribed RNA was used as template in
a second cDNA synthesis reaction. 50 ng random hexamer primers
(Life Technologies) (sometimes used in multiplex analysis
amplification) or 200 nM target-specific oligonucleotide(s) were
annealed to RNA targets at 70.degree. C. 10 min. 1.times. First
strand buffer, 10 mM DTT, and 0.5 mM dNTPs (Roche Molecular
Biochemicals, Indianapolis, Ind.) were added; samples were
incubated at 42.degree. C. 2min prior to addition of 300U
Superscript.TM. II RNase H.sup.- reverse transcriptase (Life
Technologies). Reactions were incubated at 42.degree. C. 50-75 min.
1 .mu.g RNaseA (Ambion, Inc.) and 1U RnaseH (Life Technologies)
were added per reaction and samples were incubated at 37.degree. C.
10 min. Samples were then desalted on either Chromaspin-30 columns
(Clontech) or Bio-Spin 6 columns (Bio-Rad Laboratories, Hercules,
Calif.) and used on microelectronic arrays.
[0081] N.B. When restriction digestion is done, we use RNA target,
we don't convert it into cDNA. The choice of random hexamer v. gene
specific oligo for cDNA synthesis is based on the advantage random
hexamers provide in multiplexing experiments weighed against the
possible reduction in amount of detectable cDNA generated.
[0082] E. Electronic Capture Addressing and Target
Hybridization.
[0083] 500 nM target-specific biotinylated capture oligonucleotide
in 50 mM histidine was electronically addressed to specific sites
on the NanoChip.TM., using the Nanogen Molecular Biology
Workstation. Probes were transported using 2.0V constant voltage
for 1 minute; typically, 2-10 pads were addressed simultaneously.
Unbound oligonucleotides were removed by washing with 50 mM
histidine. When an internal control was included within the sample,
500 nM capture for this species was co-addressed with
target-specific capture oligonucleotide; this internal control
capture is blocked at the 3' end with an amino modifier to prevent
extension in the subsequent reporting reaction.
[0084] RNA or cDNA targets in 50 mM histidine were heat-denatured
by incubation at 95.degree. C. 5 min and quick-chilled on ice.
Targets were electronically hybridized to specific capture
oligonucleotides using 2.0V for 2-3 min; typically, 2-10 sites on
the array were addressed simultaneously. Unhybridized nucleic acids
were removed by washing with 50 mM histidine.
[0085] F. Reporting
[0086] The internal control target is detected by hybridization
with 0.5-1 .mu.M 3.degree. Cy3-labeled oligonucleotide that binds
directly adjacent to the capture oligonucleotide; this reporter
oligonucleotide can be included in the enzymatic reporting mixture.
Targets of interest that are hybridized to capture oligonucleotides
are detected by extension of the capture probes in the presence of
Cy5-labeled dCTP. For RNA targets, after the final hybridization
reaction and washing was completed, the NanoChip.TM. was incubated
with 1.times. First strand buffer (Life Technologies); 10 mM DTT
(Life Technologies); 0.25 mM each dATP, cGTP, and dTTP (Roche
Molecular Biochemicals, Indianapolis, Ind.); 0.125 mM Cy5-dCTP
(Amersham Pharmacia Biotech, Buckinhamshire, England); and 200U
Superscript.TM. II RNase H.sup.- reverse transcriptase (Life
Technologies) for 10 min at 37.degree. C. For cDNA targets, the
NanoChip was incubated in 50 mM NaPO.sub.4 for 10 min at 37.degree.
C. prior to enzymatic extension of the capture oligonucleotide(s).
Reporting of hybridized cDNA targets was performed as described for
RNA targets, except that 1.times. Second strand buffer (Life
Technologies) and 20U DNA polymerase I (Life Technologies) were
used in place of First strand buffer and reverse transcriptase.
Following the enzymatic extension reaction, NanoChips.TM. were
washed with 50 mM NaPO.sub.4 Due to the high salt content of the
enzymatic reporting mixture, the included Cy-3 labeled internal
control probe hybridizes to the housekeeping sequence fairly
rapidly at the 37.degree. C. incubation temperature. Fluorescence
from the Cy3-labeled internal control reporter and from the
incorporated Cy5-dCTP was detected on the Nanogen Molecular Biology
Workstation.
[0087] For quantitation of targets, the signal from the Cy3 labeled
internal (exogenous or endogenous) control gene, the expression of
which is not expected to change under the conditions being
examined, is equalized, and a normalization factor is thus
obtained. Target-specific signal (Cy 5) is then adjusted by this
normalization factor. The amount of change in target gene
expression level under the experimental condition may then be
determined by comparing these adjusted hybridization/fluorescence
values.
Example 2
Enzymatic-Mediated Shortening of Targets
[0088] cDNA synthesis and amplification were as described above.
Following Vent.sub.R.RTM.(exo.sup.-) DNA polymerase-mediated
amplification and fill-in reactions, the targets were enzymatically
shortened by digestion with the type IIs restriction endonuclease
BpmI, utilizing a BpmI recognition site in the endogenous sequence
of IL6, TGF.beta.1, and p53 and chimeric primer altered sites in
COX1 and GAPDH, as described in Example 4. Digestion was performed
at 37.degree. C. for 1.5-2 h. Streptavidin-coated magnetic beads
were then added and incubation was continued at 37.degree. C. for
an additional 30-60 min. The bead-bound targets were used in in
vitro transcription reactions as described above, and RNA targets
were analyzed on NanoChip.TM. microarrays.
Example 3
Re-Use of Targets Bound to Streptavidin-Coated Magnetic Beads for
Multiple in vitro Transcription Reactions.
[0089] After the initial cDNA synthesis reaction and
Vent.sub.R.RTM.(exo.sup.-) DNA polymerase mediated amplification,
as described above, double stranded target(s) are immobilized on
streptavidin-coated magnetic beads via the biotin moiety introduced
by the chimeric T7 RNA polymerase recognition site-gene specific
oligonucleotide. These targets are then used as templates in in
vitro transcription reactions, as described above. Following in
vitro transcription, the bead-bound target can be stored at
4.degree. C. and re-used in subsequent in vitro transcription
reactions. This aspect of the protocol allows re-analysis of
targets of interest from a given sample pool without having to
obtain more of the initial cellular RNA.
[0090] FIG. 3 demonstrates the results obtained when a single pool
of bead-bound targets is utilized in three separate rounds of in
vitro transcription. Note that, unexpectedly, the amount of
transcript produced decreases only slightly between the first and
third rounds of transcription, and the decrease in the amount of
transcript is not significant between transcription rounds. Several
additional rounds of in vitro transcription are possible, with a
minimal iterative decrease in the amount of transcript
produced.
Example 4
Demonstration of the Effect of Amplicon Size on Hybridization
Efficiency in Electronic and Passive Hybridization Systems
[0091] Cellular messenger RNA was isolated from cultured U937
monocyte cells (see Example 5) and reverse transcribed into cDNA
with oligo(dT).sub.12-17. The hybridization of COX1, GAPDH, IL6,
TGF.beta.1, and p53 amplicons of approximately 50, 100, 250, and
500 bases in size were determined utilizing both electronic (as
described above) and passive hybridization procedures (outlined
below), using a TPOX control for normalization of the hybridization
values. One primer for each target contained the T7 RNA polymerase
recognition site upstream of gene specific sequence; these primers
were designed to incorporate a BpmI site at the 3' end. The other
primers were designed to generate products that were 100 bp, 250
bp, or 500 bp in length. A portion of the 100 bp amplified target
was digested with BpmI to generate 50 bp fragments. After
amplification, similarly sized targets were pooled and used as
template in in vitro transcription reactions with the T7 MegaScript
or MegaShortScript kits (Ambion, Inc.). The different pools of RNA
targets were then hybridized passively or electronically to
microarray sites co-addressed with target-specific and control
(TPOX)-specific capture oligonucleotides. Hybridized targets were
detected by enzymatic extension of the hybridized capture
oligonucleotides in the presence of Cy5-dCTP. In the case of the
internal control TPOX, this reporter was labeled with Cy3.
[0092] A) Exponential Amplification of Target Sequences
[0093] For exponential amplification of cDNA, one-tenth (2 .mu.l)
of a standard cDNA synthesis reaction was utilized. Amplification
was performed in the presence of 1.times. PCR buffer II (Perkin
Elmer, Foster City, Calif.); 0.2 .mu.M each primer; 200 .mu.M dNTP
mix; and 2.5U AmpliTaq Gold (Perkin Elmer). cDNA was denatured at
95.degree. C. 10 minutes, then amplified by cycling (95.degree. C.
30 sec; 50-60.degree. C. 60 sec; 72.degree. C. 120 sec).
Amplification reactions were generally performed for 25-30 cycles.
The annealing temperature was chosen to be 5.degree. C. lower than
the T.sub.m of the primers.
[0094] B) Passive Hybridization of RNA targets
[0095] 10 nM RNA targets were passively hybridized to
capture-loaded microarrays by incubation in 4.times. SSC, 1.times.
Denhard's, and 100 .mu.g yeast tRNA. Hybridization was performed by
placing the microarray on a moist piece of filter paper in a small
petri dish. The dish was sealed with Parafilm (American National
Can, Neenah, Wis.) and the microarray was incubated 14-16 hr at
37.degree. C. The hybridization solution was subsequently removed
and the unbound target removed by washing with 50 mM
NaPO.sub.4.
[0096] C) Passive Hybridization of Labeled Reporter
Oligonucleotides
[0097] In the case where fluorescently labeled reporter
oligonucleotides exclusively are used to detect target species on
the microarray, 1 .mu.M each reporter species is passively
hybridized to the immobilized target in 50 mM NaPO.sub.4/500 mM
NaCl. This hybridization reaction is performed at 23.degree. C. for
5 min; unbound reporters are then removed by washing with 50 mM
NaPO.sub.4 prior to imaging of the microarray.
[0098] The hybridizations utilized to generate the electronic
hybridization data are shown in FIG. 4.
1TABLE 1 Effect of target size on hybridization efficiency Target
Gene: 50 nt 100 nt 250 nt 500 nt ELECTRONIC HYBRIDIZATION: (After
2-3 minutes) COX2 36.6 45.8 21.0 4.62 GAPDH 77.2 95.0 39.5 2.22 IL6
46.9 69.2 47.3 49.2 TGF.beta. 206 187 131 16.9 p53 17.5 19.4 1.65
ND PASSIVE HYBRIDIZATION: (After 14-16 hours) COX1 34.4 28.0 6.06
5.51 GAPDH 68.1 75.0 6.89 2.20 IL6 71.3 26.4 12.9 6.91 TGF.beta.1
150 150 38.2 6.86 p53 23.0 14.6 6.17 6.76
[0099] These numbers represent fluorescence units
[0100] As can be seen from the above data, the length of the
amplicons used in the hybridization procedure had a marked effect
on hybridization efficiency.
Example 5
Demonstration of Gene Expression Monitoring in LPS Stimulated
Monocyte Cells
[0101] The monocytic cell line U937 was cultured to
4.times.10.sup.7 cells/75 cm.sup.2 flask in RPMI media (American
Type Culture Collection, Manassas Va.) containing 10% Fetal Bovine
Serum (American Type Culture Collection) and 200 units/g/ml
penicillin/streptomycin (Life Technologies) in two or more flasks.
Cells were collected and resuspended in either RPMI/FBS/antibiotics
or in RPMI/FBS/antibiotics+phorbol myristate acid (Life
Technologies) at a final concentration of 10 ng/ml. 72 h after PMA
treatment, cells were collected and resuspended in RPMI media
containing FBS and antibiotics; PMA-treated cells were induced by
treatment with 500 ng/ml lipopolysaccharide (Sigma, St. Louis,
Mo.). Cells were harvested 6 h after addition of LPS. Uninduced
cells that had been maintained in RPMI/FBS/antibiotic medium were
also collected ("Uninduced"). Untreated and treated cells were snap
frozen in liquid nitrogen until needed for mRNA isolation. mRNA
isolation was performed with the Poly(A) Pure Isolation kit
(Ambion, Inc.). Cellular poly(A.sup.+) RNA was isolated and cDNA
was generated using the propane diol-modified oligo dT primer.
Limited amplification reactions were performed in duplex reactions
as follows: the biotinylated chimeric T7-gene specific
oligonucleotides for Interleukin I beta (IL1) and Transforming
Growth Factor beta-2 (TGF.beta.2) were used in one amplification
reaction; and the biotinylated chimeric T7-gene specific
oligonucleotides for Interleukin 6 (IL6) and c-jun were used in a
second amplification reaction. Single stranded targets were filled
in by addition of gene specific oligonucleotides and used in in
vitro transcription reactions. The RNA generated was used as
template for second cDNA syntheses. Electronic capture addressing,
target hybridization, and enzymatic extension with DNA polymerase
were carried out as described in Example 1.
[0102] The hybridization results of the experiment are shown in
FIG. 6, and graphically represented in FIG. 7. cNS represents a
non-specific biotinylated oligonucleotide that does not contain
sequence complimentary to any of the amplified targets. As shown,
the addition of LPS to the monocyte cell culture provoked
dramatically increased mRNA expression for IL1, moderately
increased expression of TGF.beta.2 and c-jun message, and minimally
increased expression of IL6. Thus, as illustrated, altered
expression of genes between two sample cell populations can be
easily monitored using the methods of the present invention.
Example 6
Demonstration of Serial Dilution and Quantitative Methods Utilizing
the NanoChip.TM. Electronic Hybridization Device
[0103] A) Detection of Serially Diluted Short RNA Targets on the
NanoChip.TM.
[0104] The .beta.-1a target gene was amplified from plasmid DNA
using chimeric gene specific oligonucleotides, as described above.
The 5' primer (pT7Amp.s1) contains a T7 RNA polymerase recognition
sequence upstream of gene specific sequence; the 3' primer contains
a poly(dT) tract downstream of gene specific sequence. This PCR
product, representing the full-length 3-1a gene, was used as
template in in vitro transcription reactions with the T7 Megascript
kit per manufacturer's instructions (Ambion, Inc.). The target was
reverse transcribed to generate cDNA, and the cDNA product was used
in a Vent.sub.R.RTM.(exo.sup- .-) DNA polymerase-mediated
amplification reaction with a second, internal chimeric T7 RNA
polymerase recognition site/gene-specific oligonucleotide
(pT7AmpBpm.s1). The amplified target was enzymatically digested
with BpmI and used in a second in vitro transcription reaction
using the T7 MegaShortScript kit (Ambion, Inc.). The resulting
short RNA target was serially diluted as indicated and
electronically hybridized to a .beta.-1a specific capture
oligonucleotide. The electronic hybridization data is shown in FIG.
5a.
[0105] B) Detection of Exogenous Target in a Pool of Cellular
RNA.
[0106] The full length .beta.-1a target was generated as described
above. This RNA was added in defined amounts (0 to 800 pg) to 250
ng cellular messenger RNA. The entire pool of RNA was then reverse
transcribed into cDNA, and the .beta.-1a target was amplified in a
Vent.RTM. (exo.sup.-) DNA polymerase-mediated amplification
reaction. The amplified target was enzymatically digested with BpmI
and used in a second in vitro transcription reaction using the T7
MegaShortScript kit (Ambion, Inc.). The resulting short RNA target
was electronically hybridized to a .beta.-1a specific capture
oligonucleotide. The hybridization data is shown in FIG. 5b, and
graphically represented in FIG. 5c.
2TABLE 2 Oligonucleotides Used in the Above Experimental
Procedures, Organized by Target Gene Target Primer name Gene and
description Primer sequence Angioten- bAt7AngBpm.s1--
BiotinTAATACGACTCACTATAGG sinogen biotinylated
GAGACACAGAACTGGATGTTGCT chimeric T7 GCTGGAG promoter/gene specific
oligonucleotide Angioten- cAng.a1--capture
BiotinCATGAACCTGTCAATCTTCT sinogen for RNA Angioten- pAng.a1--3'
gene GGAAGGTGCCCATGCCAGAGA sinogen specific primer Cathepsin
bAt7CathBpm.s1-- BiotinTAATACGACTCACTATAGG G biotinylated
GAGAGCTGCCTTCAAGGGGGATT chimeric T7 CTGGAG promoter/gene specific
oligonucleotide Cathepsin pCath.a1--3' gene
AGCTTCTCATTGTTGTCCTTATCC G specific primer Cathepsin
cCath.a1--capture BiotinTGTTACACAGCAGGGGGCC G for RNA T c-jun
bT7jun.s1-- BiotinTAATACGACTCACTATAGG biotinylated
GAGACGGCCAACATGCTCAGGG chimeric T7 AACAGGT promoter/gene specific
oligonucleotide c-jun cjun.s1--capture BiotinCAAACATTTTGAAGAGAGA
for cDNA CCGTCG c-jun pjun.a1--3' gene TTTTTCTTCGTTGCCCCTCAGCC
specific primer COX1 pcox1.as.3--gene TGCCCAGGATTGATTCACAGG
specific primer for generation of 500 bp fragment COX1
pcox1.as.2--gene AGGCCAGAAGGAATGATGGG specific primer for
generation of 250 bp fragment COX1 pcox1.as.1--gene
CTAAGCCCAAAGTGTGGATC specific primer for generation of 100 bp
fragment COX1 T7.cox1.s-- GAAATTAATACGACTCACTATAG chimeric T7/gene
GGAGAACCCTTTTCTCAGGACCT specific CTGGAGG oligonucleotide COX1
cCOX1bpm.a1-- BiotinACAGAGGTCCTGAGAAAAG capture for RNA GGTCT COX2
bAt7COX2bpm. BiotinTAATACGACTCACTATAGG s2--biotinylated
GAGACTATGAATCATTTGAAGAA chimeric T7 CTTACTGGAG promoter/gene
specific oligonucleotide COX2 cCOX2.a2--capture
BiotinCTGCAGACATTTCCTTTTCT for RNA COX2 pCOX2.a2--3' gene
GCATCTGGCCGAGGCTTTTCTAC specific primer GAPDH pGAPs.2--gene
GTTCGACAGTCAGCCGCATCTTC specific primer for generation of 500 bp
fragment GAPDH pGAPs.3--gene TGATGCCCCCATGTTCGTCATGG specific
primer for generation of 100 bp fragment GAPDH pGAPs.4--gene
CTTCCAGGAGCGAGATCCCTCC specific primer for generation of 250 bp
fragment GAPDH T7GAPbpm.a1-- GTAATACGACTCACTATAGGGCG chimeric
T7/gene GGGTGCTAAGCAGTTGGTGGTGC specific TGGAG oligonucleotide
GAPDH cGAPbpm.s1-- BiotinCAGCCTCAAGATCATCAGC capture for aRNA
AATGCCT GAPDH bAt7GAPbpm.s2-- BiotinTAATACGACTCACTATAGG
biotinylated GAGACTCAAGGGCATCCTGGGCT chimeric T7 ACACTGGAGCAC
promoter/gene specific oligonucleotide GAPDH pGAPDH.a6--3'
GAGGTCCACCACCCTGTTGCTGT gene specific AG primer GAPDH cGAPbpm.a2--
BiotinGTTGAAGTCAGAGGAGACC capture for RNA ACCTGGTGCT HMG-17
bAt7HMG17Bpm. BiotinTAATACGACTCACTATAGG s1--biotinylated
GAGAGGAATAACCCTGCAGAAA chimeric T7 CTGGAG promoter/gene specific
oligonucleotide HMG-17 pHMG17.a1--3' CCCTTCCCCCAAAAACAACAATG gene
specific A primer HMG-17 cHMG17.a1-- BiotinCCTGGTCTGTTTTGGCATCT
capture for RNA Interleukin pIL6s.4--gene ATTCTGCCCTCGAGCCCACCGGG 6
specific primer for generation of 500 bp fragment Interleukin
pIL6S.3--gene CAAACAAATTCGGTACATCCTCG 6 specific primer for
generation of 250 bp fragment Interleukin pIL6S.2--gene
TGGATTCAATGAGGAGACTTGCC 6 specific primer for generation of 100 bp
fragment Interleukin T7IL6bpm.a1-- GTAATACGACTCACTATAGGGCG 6
chimeric T7/gene CCTCACTACTCTCAAATCTGTTCT specific GGAG
oligonucleotide Interleukin cIL6bpm.s1- BiotinGGAGTTTGAGGTATACCTA 6
capture for aRNA GAGTACCT Interleukin bT7IL6.s1--
BiotinTAATACGACTCACTATAGG 6 biotinylated GAGACCTGAGGGCTCTTCGGCAA
chimeric T7 ATGTAG promoter/gene specific oligonucleotide
Interleukin cIL6.s1-- BiotinAATGGGCATTCCTTCTTCTG 6 capture for cDNA
GTCAG Interleukin pI16.a1--3' gene GAACAACATAAGTTCTGTGCCCA 6
specific primer GTG Interleukin bT7IL1.s1--
BiotinTAATACGACTCACTATAGG 1 beta biotinylated
GAGACAGAAAACATGCCCGTCTT chimeric T7 CCTGG promoter/gene specific
oligonucleotide Interleukin cIL1.s1--capture
BiotinGCGGCCAGGATATAACTGA 1 beta for cDNA CTTCAC Interleukin
pI11.a1--3' gene TCCACATTCAGCACAGGACTCTC 1 beta specific primer TG
LD78 bAt7LD78Bpm. BiotinTAATACGACTCACTATAGG s1--biotinylated
GAGAAGTGACCTAGAGCTGAGTG chimeric T7 CCTGGAG promoter/gene specific
oligonucleotide LD78 pLD78.a1--3' gene CTCTCAGAGCAAACAATCACAAA
specific primer CACAC LD78 cLD78.a1--capture
BiotinTCGAAGCTTCTGGACCCCT for RNA Osteo- bAt7OstBpm.s1--
BiotinTAATACGACTCACTATAGG pontin biotinylated
GAGAGAGGTGATAGTGTGGTTTA chimeric T7 TGGACTGGAG promoter/gene
specific oligonucleotide Osteo- pOst.a1--3' gene
CAACGGGGATGGCCTTGTATGC pontin specific primer Osteo-
cOst.a1--capture BiotinAACTTCTTAGATTTTGACCT pontin for RNA p53
pp53s.3--gene ACAGAAACACTTTTCGACATAG specific primer for generation
of 500 bp fragment p53 pp53s.2--gene AAAGGGGAGCCTCACCACGAGC
specific primer for generation of 250 bp fragment p53 pp53.s1--gene
CGTGAGCGCTTCGAGATGTTCC specific primer for generation of 100 bp
fragment p53 T7p53bpm.a1-- GTAATACGACTCACTATAGGGCG chimeric T7/gene
ACCCTTTTTGGACTTCAGGTGGCT specific GGAG oligonucleotide p53
cp53bpm.s1-- BiotinGAGCCAGGGGGGAGCAGG capture for aRNA GCTCACT
TGF.beta.1 pTGFb1S.3--gene GGGATAACACACTGCAAGTGGAC specific primer
for generation of 500 bp fragment TGF.beta.1 pTGFb1s.2--gene
CCACGAGCCCAAGGGCTACCATG specific primer C for generation of 250 bp
fragment TGF.beta.1 pTGFb1.s1--gene CGCTGGAGCCGCTGCCCATCGTG
specific primer TA for generation of 100 bp fragment TGF.beta.1
T7TGFb1bpm.a1-- GTAATACGACTCACTATAGGGCG chimeric T7/gene
GGCGGGACCTCAGCTGCACTTGC specific TGGAG oligonucleotide TGF.beta.1
cTGFb1bpm.s1-- BiotinCAGCTGTCCAACATGATCG capture for aRNA TGCGCT
TGF.beta.2 bT7TGFb.s1-- BiotinTAATACGACTCACTATAGG biotinylated
GAGACTCTGCCTCCTCCTGCCTGT chimeric T7 CTGC promoter/gene specific
oligonucleotide TGF.beta.2 cTGFb.s1--capture
BiotinCGGCATCAAGGCACAGGGG for cDNA ACCAGT TGF.beta.2 pTGFb.a1--3'
gene CTTCAACAGTGCCCAAGGTGCTC specific primer AA TPOX TPOX9C--
BiotinTTAGGGAACCCTCACTGAA biotinylated TGAATGAATGAATGAATGAATGA
synthetic target ATGAATG TPOX TPOXcapcomp-- CATTCATTCATTCAGTGAGGGTT
Cy3 labeled reporter CC for TPOX9C Vimentin bAt7VimBpm.s2--
BiotinTAATACGACTCACTATAGG biotinylated GAGACATCGACAAGGTGCGCTTC
chimeric T7 CTGGAG promoter/gene specific oligonucleotide Vimentin
pVim.a1--3' gene CGCGGGCTTTGTCGTTGGTTAG specific primer Vimentin
cVim.a2--capture BiotinCAGGATCTTATTCTGCTGCT for RNA .beta.-Actin
bAt7Actin.s-- BiotinTAATACGACTCACTATAGG biotinylated
GAGACCCCTTTTTGTCCCCCAACT chimeric T7 GGAGA promoter/gene specific
oligonucleotide .beta.-Actin cActin.a--capture
BiotinCCAAAAGCCTTCATACATCT for RNA .beta.-Actin pbAa.4--3' gene
AAGGTGTGCACTTTTATTCAACT specific primer GGTCTCAAG .beta.-1a
pT7AmpBpm.s1 -- TAATACGACTCACTATAGGCTGG chimeric T7/gene
CTGGTTTATTGCTGATAAATCTG specific oligo- GAG nucleotide for
generation of short RNA .beta.-1a pAmp.a2--3'
T.sub.30CCAATGCTTAATCAGTGAGGC primer with ACCTATCTC poly(dT) tract
.beta.-1a cAmp.a1--capture biotin- for RNA CGAGACCCACGCTCACCGGCT
.beta.-1a pT7Amp.s1-- TAATACGACTCACTATAGGGCAC chimeric T7/gene
CCAGAAACGCTGGTGAAAGTAA specific AAG oligonucleotide for generation
of full-length gene .beta.- bAt7Throm.s1--
BiotinTAATACGACTCACTATAGG Thrombo- biotinylated
GAGAGGAAAACTGGGTGCAGAG globulin- chimeric T7 GGTTCTGGAG like
promoter/gene protein specific gene oligonucleotide .beta.-
pbThrom.a1--3' GGCAACCCTACAACAGACCCACA Thrombo- gene specific C
globulin- primer like protein gene .beta.- cbThrom.a1--
BiotinAGCCCTCTTCAAAAACTTCT Thrombo- capture for RNA globulin- like
protein gene
[0107]
Sequence CWU 1
1
73 1 49 DNA Homo sapiens modified_base (1)..(1) Biotinylated 1
taatacgact cactataggg agacacagaa ctggatgttg ctgctggag 49 2 20 DNA
Homo sapiens modified_base (1)..(1) Biotinylated 2 catgaacctg
tcaatcttct 20 3 21 DNA Homo sapiens 3 ggaaggtgcc catgccagag a 21 4
48 DNA Homo sapiens modified_base (1)..(1) Biotinylated 4
taatacgact cactataggg agagctgcct tcaaggggga ttctggag 48 5 24 DNA
Homo sapiens 5 agcttctcat tgttgtcctt atcc 24 6 20 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 6 tgttacacag cagggggcct 20 7 48
DNA Homo sapiens modified_base (1)..(1) Biotinylated 7 taatacgact
cactataggg agacggccaa catgctcagg gaacaggt 48 8 25 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 8 caaacatttt gaagagagac cgtcg
25 9 23 DNA Homo sapiens 9 tttttcttcg ttgcccctca gcc 23 10 21 DNA
Homo sapiens 10 tgcccaggat tgattcacag g 21 11 20 DNA Homo sapiens
11 aggccagaag gaatgatggg 20 12 20 DNA Homo sapiens 12 ctaagcccaa
agtgtggatc 20 13 53 DNA Homo sapiens 13 gaaattaata cgactcacta
tagggagaac ccttttctca ggacctctgg agg 53 14 24 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 14 acagaggtcc tgagaaaagg gtct
24 15 52 DNA Homo sapiens modified_base (1)..(1) Biotinylated 15
taatacgact cactataggg agactatgaa tcatttgaag aacttactgg ag 52 16 20
DNA Homo sapiens modified_base (1)..(1) Biotinylated 16 ctgcagacat
ttccttttct 20 17 23 DNA Homo sapiens 17 gcatctggcc gaggcttttc tac
23 18 23 DNA Homo sapiens 18 gttcgacagt cagccgcatc ttc 23 19 23 DNA
Homo sapiens 19 tgatgccccc atgttcgtca tgg 23 20 22 DNA Homo sapiens
20 cttccaggag cgagatccct cc 22 21 51 DNA Homo sapiens 21 gtaatacgac
tcactatagg gcggggtgct aagcagttgg tggtgctgga g 51 22 26 DNA Homo
sapiens modified_base (1)..(1) Biotinylated 22 cagcctcaag
atcatcagca atgcct 26 23 54 DNA Homo sapiens modified_base (1)..(1)
Biotinylated 23 taatacgact cactataggg agactcaagg gcatcctggg
ctacactgga gcac 54 24 25 DNA Homo sapiens 24 gaggtccacc accctgttgc
tgtag 25 25 29 DNA Homo sapiens modified_base (1)..(1) Biotinylated
25 gttgaagtca gaggagacca cctggtgct 29 26 47 DNA Homo sapiens 26
taatacgact cactataggg agaggaataa ccctgcagaa actggag 47 27 24 DNA
Homo sapiens 27 cccttccccc aaaaacaaca atga 24 28 20 DNA Homo
sapiens modified_base (1)..(1) Biotinylated 28 cctggtctgt
tttggcatct 20 29 23 DNA Homo sapiens 29 attctgccct cgagcccacc ggg
23 30 23 DNA Homo sapiens 30 caaacaaatt cggtacatcc tcg 23 31 23 DNA
Homo sapiens 31 tggattcaat gaggagactt gcc 23 32 51 DNA Homo sapiens
32 gtaatacgac tcactatagg gcgcctcact actctcaaat ctgttctgga g 51 33
27 DNA Homo sapiens modified_base (1)..(1) Biotinylated 33
ggagtttgag gtatacctag agtacct 27 34 48 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 34 taatacgact cactataggg
agacctgagg gctcttcggc aaatgtag 48 35 25 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 35 aatgggcatt ccttcttctg gtcag
25 36 26 DNA Homo sapiens 36 gaacaacata agttctgtgc ccagtg 26 37 47
DNA Homo sapiens modified_base (1)..(1) Biotinylated 37 taatacgact
cactataggg agacagaaaa catgcccgtc ttcctgg 47 38 25 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 38 gcggccagga tataactgac ttcac
25 39 25 DNA Homo sapiens 39 tccacattca gcacaggact ctctg 25 40 49
DNA Homo sapiens modified_base (1)..(1) Biotinylated 40 taatacgact
cactataggg agaagtgacc tagagctgag tgcctggag 49 41 28 DNA Homo
sapiens 41 ctctcagagc aaacaatcac aaacacac 28 42 19 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 42 tcgaagcttc tggacccct 19 43
52 DNA Homo sapiens modified_base (1)..(1) Biotinylated 43
taatacgact cactataggg agagaggtga tagtgtggtt tatggactgg ag 52 44 22
DNA Homo sapiens 44 caacggggat ggccttgtat gc 22 45 20 DNA Homo
sapiens modified_base (1)..(1) Biotinylated 45 aacttcttag
attttgacct 20 46 22 DNA Homo sapiens 46 acagaaacac ttttcgacat ag 22
47 22 DNA Homo sapiens 47 aaaggggagc ctcaccacga gc 22 48 22 DNA
Homo sapiens 48 cgtgagcgct tcgagatgtt cc 22 49 51 DNA Homo sapiens
49 gtaatacgac tcactatagg gcgacccttt ttggacttca ggtggctgga g 51 50
25 DNA Homo sapiens modified_base (1)..(1) Biotinylated 50
gagccagggg ggagcagggc tcact 25 51 23 DNA Homo sapiens 51 gggataacac
actgcaagtg gac 23 52 24 DNA Homo sapiens 52 ccacgagccc aagggctacc
atgc 24 53 25 DNA Homo sapiens 53 cgctggagcc gctgcccatc gtgta 25 54
51 DNA Homo sapiens 54 gtaatacgac tcactatagg gcgggcggga cctcagctgc
acttgctgga g 51 55 25 DNA Homo sapiens modified_base (1)..(1)
Biotinylated 55 cagctgtcca acatgatcgt gcgct 25 56 47 DNA Homo
sapiens modified_base (1)..(1) Biotinylated 56 taatacgact
cactataggg agactctgcc tcctcctgcc tgtctgc 47 57 25 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 57 cggcatcaag gcacagggga ccagt
25 58 25 DNA Homo sapiens 58 cttcaacagt gcccaaggtg ctcaa 25 59 49
DNA Homo sapiens modified_base (1)..(1) Biotinylated 59 ttagggaacc
ctcactgaat gaatgaatga atgaatgaat gaatgaatg 49 60 25 DNA Homo
sapiens 60 cattcattca ttcagtgagg gttcc 25 61 48 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 61 taatacgact cactataggg
agacatcgac aaggtgcgct tcctggag 48 62 22 DNA Homo sapiens 62
cgcgggcttt gtcgttggtt ag 22 63 20 DNA Homo sapiens modified_base
(1)..(1) Biotinylated 63 caggatctta ttctgctgct 20 64 48 DNA Homo
sapiens 64 taatacgact cactataggg agaccccttt ttgtccccca actggaga 48
65 20 DNA Homo sapiens modified_base (1)..(1) Biotinylated 65
ccaaaagcct tcatacatct 20 66 32 DNA Homo sapiens 66 aaggtgtgca
cttttattca actggtctca ag 32 67 49 DNA Homo sapiens 67 taatacgact
cactataggc tggctggttt attgctgata aatctggag 49 68 60 DNA Homo
sapiens 68 tttttttttt tttttttttt tttttttttt ccaatgctta atcagtgagg
cacctatctc 60 69 21 DNA Homo sapiens modified_base (1)..(1)
Biotinylated 69 cgagacccac gctcaccggc t 21 70 48 DNA Homo sapiens
70 taatacgact cactataggg cacccagaaa cgctggtgaa agtaaaag 48 71 51
DNA Homo sapiens modified_base (1)..(1) Biotinylated 71 taatacgact
cactataggg agaggaaaac tgggtgcaga gggttctgga g 51 72 24 DNA Homo
sapiens 72 ggcaacccta caacagaccc acac 24 73 20 DNA Homo sapiens
modified_base (1)..(1) Biotinylated 73 agccctcttc aaaaacttct 20
* * * * *