U.S. patent application number 10/212476 was filed with the patent office on 2003-08-21 for customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable.
Invention is credited to Chik, Valentine, Drobyshev, Aleksei, Fotin, Alexander, Guschin, Dmitry Y., Lysov, Yuri, Mirzabekov, Andrei, Yershov, Gennadiy.
Application Number | 20030157509 10/212476 |
Document ID | / |
Family ID | 22992014 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157509 |
Kind Code |
A1 |
Mirzabekov, Andrei ; et
al. |
August 21, 2003 |
Customized oligonucleotide microchips that convert multiple genetic
information to simple patterns, are portable and reusable
Abstract
This invention relates to using customized oligonucleotide
microchips as biosensors for the detection and identification of
nucleic acids specific for different genes, organisms and/or
individuals in the environment, in food and in biological samples.
The microchips are designed to convert multiple bits of genetic
information into simpler patterns of signals that are interpreted
as a unit. Because of an improved method of hybridizing
oligonucleotides from samples to microchips, microchips are
reusable and transportable. For field study, portable laser or bar
code scanners are suitable.
Inventors: |
Mirzabekov, Andrei; (Darien,
IL) ; Guschin, Dmitry Y.; (Rockville, MD) ;
Chik, Valentine; (Woodridge, IL) ; Drobyshev,
Aleksei; (Elektrosol, RU) ; Fotin, Alexander;
(Cambridge, MA) ; Yershov, Gennadiy; (Hinsdale,
IL) ; Lysov, Yuri; (US) |
Correspondence
Address: |
BARNES & THORNBURG
2600 CHASE PLAZA
10 SOUTH LASALLE STREET
CHICAGO
IL
60603
US
|
Family ID: |
22992014 |
Appl. No.: |
10/212476 |
Filed: |
August 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10212476 |
Aug 5, 2002 |
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09261115 |
Mar 3, 1999 |
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6458584 |
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10212476 |
Aug 5, 2002 |
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08780026 |
Dec 23, 1996 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/6.12 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 1/6881 20130101; C12Q 1/6837 20130101; G16B 25/30 20190201;
G16B 25/00 20190201; B01J 2219/00644 20130101; C12Q 1/689 20130101;
C12Q 2600/156 20130101; C12Q 1/6837 20130101; C12Q 2565/607
20130101; C12Q 1/6837 20130101; C12Q 2565/513 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Goverment Interests
[0002] The U.S. Government has rights to the invention pursuant to
Contract W-32-109-ENG between the U.S. Department of Energy and the
University of Chicago representing (Argonne National Laboratory).
Claims
We claim:
1. A method for using a reusable microchip to identify at least one
nitrogen base sequence in nucleic acids of a sample, said method
comprising: (a) providing a customized matrix of oligonucleotides
on the microchip in an arrangement that is an ordered scheme
designed to present a detectable pattern as a whole that identifies
the nitrogen base sequences of nucleic acids in the sample; (b)
hybridizing nucleic acids from the sample on said microchip; and
(c) identifying at least one nitrogen base sequence in said sample
by the pattern detected as a whole on the microchip, said pattern
resulting from the locations of oligonucleotides which hybridized
to the sample nucleic acids.
2. The method of claim 1, wherein said nitrogen base sequence is in
a DNA molecule.
3. The method of claim 1, wherein said nitrogen base sequence is in
a 16S RNA, mRNA or other RNA molecule.
4. The method of claim 1, wherein the customized matrix of
oligonucleotides on the reusable microchip is formed by a plurality
of gel elements, wherein the number of elements is determined by
the number of oligonucleotides in the matrix and wherein each gel
element contains one oligonucleotide of a desired nitrogen base
sequence length and concentration, each gel element being separated
from another by hydrophobic glass spaces, and wherein the gel
elements have a vertical height above the plane of the interstitial
spaces of not more than about 30 .mu.m, said oligonucleotides being
positioned in specific locations.
7. The method of claim 1, further comprising: (e) adding a label to
the nitrogen base sequences in said sample before hybridizing them
to oligonucleotides on the microchip.
8. The method of claim 7, wherein the label is a fluorescent
dye.
9. The method of claim 7, wherein the label is a plurality of
different dyes.
11. A diagnostic assay for the presence of a mutation in a gene in
a sample said assay comprising: (d) designing a customized reusable
microchip biosensor comprising at least one oligonucleotide that
hybridizes to a gene having the mutation, said designing producing
an overall pattern that is detectable in the presence of said
hybridization of the mutation; (e) contacting the sample to the
customized microchip biosensor under conditions that allow
hybridization of the gene sequence that is a mutation to
oligonucleotides on the microchip; and (f)determining whether
hybridization occurs by analyzing the microchip pattern from which
presence of the mutation in the gene is determined.
12. A method using non-equilibrium melting curves to detect
mismatches between a nitrogen base sequence on a microchip and a
nitrogen base sequence to be tested, said method comprising: (g)
generating the non-equilibrium melting curves by simultaneously
monitoring hybridization between the nitrogen base sequence that
matches or mismatches with sequences on the microchip at a series
of temperatures; (h) selecting the temperature at which maximum
discrimination occurs between the match and the mismatch; and (i)
determining the degree of mismatch of the nitrogen base sequence to
be tested at the selected temperature.
13. The method of claim 12, wherein the selected temperature is
that temperature at which the signal intensity of a mismatched
sequence is at least one tenth of the signal intensity of a matched
sequence.
14. A customized oligonucleotide microchip for the detection of a
betaglobin mutation, said microchip containing seven
oligonucleotides having the following nitrogen base sequences:
8 Identifier Sequences SEQ ID NO: IVS (N) CCTGGGCAGGTTGGTATCA; 48
IVS I/2 T/A CCTGGGCAGGaTGGTATCA; 49 IVS I/1 G/A
CCTGGGCAGaTTGGTATCA; 50 IVS I/6 T/C CCTGGGCAGGTTGcTATCA; 51 IVS 1/5
G/T CCTGGGCAGGTTGtTATCA; 52 CD26 (N) GTTGGTGGTGAGGCCCTGG; 53 CD26
G/A GTTGGTGGTaAGGCCCTGG 54.
15. A reusable customized oligonucleotide microchip for
quantitation of the expression of a gene, wherein the design of the
microchip presents a pattern corresponding to the degree of
expression.
16. A customized oligonucleotide microchip for the detection of HLA
polymorphism, wherein the design of the microchip presents a
pattern in which HLA probes are positioned to answer a specific
question when hybridization occurs.
17. The customized oligonucleotide microchip of claim 16,
comprising the following oligonucleotides;
9 AGGCAACGTG (1); (SEQ 1D:55) AGGCGACGTG (2); (SEQ ID:56)
GGTGAACTGG (3); (SEQ ID:57) TAAATCTGCG (4); (SEQ ID:58) AGGCAACATG
(5); (SEQ ID:59) CAAAACCTCC (6); (SEQ ID:60) GCAAACACCA (7); (SEQ
ID:61) TACACCATAA (8); (SEQ ID:62) ACTGCTCATC (9); (SEQ ID:63)
CAATGTCTTC (10); (SEQ ID:64) CTCCTCATCT (11); (SEQ ID:65)
TGCCGGTCAA (12); (SEQ ID:66) TTAGGACAGC (13); (SEQ ID:67)
ACACCACAAG (14); (SEQ ID:68) CACAATGCCT (15); (SEQ ID:69)
CAGCAGTAGA (16); (SEQ ID:70) TGCGGGTCAA (17); (SEQ ID:71)
TTAGCACACC (18). (SEQ TD:72)
18. A method for designing a reusable genetic microchip to answer a
specific question about a test sample, said answer provided by
detection of hybridization patterns of the nitrogen base sequences
of nucleic or ribonucleic acids of molecules present in the sample,
on the microchip as a whole, said method comprising: (a)
determining the specific question; and (b) designing the microchip
so that hybridization of nucleic acids present in the sample
provides a pattern that when detected as a whole provides an answer
to the question; and (c) detecting the pattern.
19. The method of claim 18, wherein the sample is water, and the
question is what pathogens are present.
20. The method of claim 18, wherein the sample is blood, and the
question is what genetic mutations are present.
21. The method of claim 18, wherein the sample is a food, and the
specific question is what containments are present.
22. The method of claim 18, wherein the pattern is a bar code
pattern and detecting the pattern is done by a bar code laser
scanner.
Description
[0001] This application is a divisional of U.S. Ser. No. 09/261,115
filed Mar. 3, 1999 and is a continuation-in-part of U.S. patent
application Ser. No. 08/780,026 filed Dec. 23, 1996 now
abandoned.
[0003] A novel microchip is customized to answer specific questions
and has oligonucleotides positioned on the microchip so that
multiple bits of information are evidenced to a simpler pattern. A
new method of hybridization to a microchip is also presented.
BACKGROUND OF THE INVENTION
[0004] Differences in nucleotide and amino acid sequences may be
exploited to analyze environmental, food or biological samples.
Detection and identification of microorganisms is important for
clinical purposes and for determination of contaminated food, air,
water or soil. Studies in environmental microbiology are often
limited by the inability to unambiguously identify and directly
quantify the enormous diversity of natural populations. This
problem is now changing with increasing use of molecular techniques
to directly measure different genetic features. (Mobarry et al.,
1996; Stahl, 1995; Wagner et al, 1995) For example, DNA probes are
now commonly used to detect by hybridization, genes encoding
proteins involved in specific catabolic functions, and to resolve
different genetic populations in the environment. In particular,
the use of group-specific DNA probes complementary to the small
subunit (SSU) 16S rRNA has provided a comprehensive framework for
studies of microbial population structure in complex systems.
Sequencing of this subunit revolutionized microbial classification
and led to the discovery of archebacteria. (Woese, 1987) A large
number of the sequences for different organisms has been collected.
(Maidak et al., 1996) Every microorganism species is characterized
by a specific DNA sequence within a variable region of its
ribosomal RNA gene or other genes. A highly efficient procedure for
microorganism classification and for construction of their
evolutionary trees is based on these observations. Identification
of specific sequences in ribosomal DNA is a reliable microbial
analysis that can be carried out by direct DNA sequencing. However
DNA sequencing is a rather complicated, expensive and time
consuming procedure to use for serial microbial analysis on a
commercial scale for environmental or medical applications.
Consequently, new methods are needed to make sequence matching
commercially feasible.
[0005] Also, methods are needed that are transportable to the
field. A nucleic acid hybridization is a highly specific and
sensitive procedure that allows a specific sequence to be detected
and identified among other millions of sequences in a genome of
higher organisms, or among a mixture of different organisms. The
principle of hybridization is that sequences hybridize as a
function of the similarity of their linear nucleotide sequence. The
hybridization of DNA or RNA extracted from even a very complicated
mixture to a specific oligonucleotide probe has resulted in
unambiguous identification of specific microorganisms in an
environmental sample, for example. In the course of such an
analysis, RNA or DNA is extracted from a sample of microorganisms
isolated from water solutions, air or soil, immobilized on a filter
and then hybridized successively with several oligonucleotide
probes for different microorganisms. However, for this purpose, the
sample needs to be checked for the presence of hundreds or
thousands of different oligonucleotides corresponding to various
microorganisms which is prohibitively laborious and expensive using
present methods and yields results that must be interpreted by a
computer in order to decipher the identification. What is needed is
a simplified pattern to provide rapid answers to specific
questions, e.g. are any known pathogens in a water sample?
[0006] The scope of applications of nucleotide hybridization is
often limited by the nature of the assays, generally involving the
independent hybridization and interpretation of multiple
environmental samples to multiple DNA probes. In addition, some
detection assays require amplification of the target nucleic acid,
for example, via PCR. This may contribute to quantitative biases.
Thus, there is need for assays that provide for greater sample
through-put capacity and greater sensitivity, rapid read-out of
results.
[0007] Another area in which specific DNA or RNA sequences are of
interest is mutation and polymorphism analyses. The number of base
changes discovered (mutations) in different genes is growing
rapidly. These changes are associated with genetic diseases, with
disease predispositions and cancers, with development of drug
resistance in microorganisms, and with genetic polymorphisms.
Polymorphisms are useful for determining the source of a sample,
e.g. in forensic analyses. Polymorphisms such as in the HLA system
are essential to predict success of tissue transplants. The ability
to simultaneously analyze many mutations in a gene in a simple,
fast, and inexpensive way is essential in clinical medicine and
this need has stimulated the development of different methods for
screening mutations, but all have serious limitations. What is
needed are kits that are transportable and interpretable, e.g. for
use in clinics without high technology microscopes.
[0008] Hybridization of filter-immobilized DNA with allele-specific
oligonucleotides was suggested as a way to screen for mutations.
(Conner et al., 1983) However, the number of alleles that can be
assayed at one time is limited, the filters are usable only for a
few times, and there is little opportunity for complex analysis or
easy interpretation of results.
[0009] A possible solution to large scale hybridization is to use
microchips for DNA sequence hybridizations (SHOM, sequencing by
hybridization with oligonucleotides in a microchip) (e.g. Khrapko,
1996; Yershov, 1996). The development of an array of hundreds or
thousands of immobilized oligonucleotides, the so-called
"oligonucleotide chips", permits simultaneous analysis of many
mutations (for a review, see Mirzabekov, 1994). Such arrays can be
manufactured by a parallel synthesis of oligonucleotides (Southern
et al., 1992; Fodor et al., 1991; Pease et al., 1994; Matson et
al., 1995) or by chemical immobilization of presynthesized
oligonucleotides (Khrapko et al., 1991; Lamture et al., 1994; Ghu
et al., 1994). Glass surfaces (Southern et al., 1992; Fodor et al.,
1991; Ghu et al., 1994), glass pores (Beattie et al., 1995),
polypropylene sheets (Matson et al., 1995), and gel pads (Khrapko
et al., 1991; Yershov et al., 1996) have been used as solid
supports for oligonucleotide immobilization. However
"Oligonucleotide array technology has not yet lived up to its
promise." Southern, 1996 p. 115.
[0010] Some of the deficiencies in the art are unpredictability of
the results, lack of knowledge of optimum conditions, and failure
to demonstrate accuracy and commercial feasibility. Moreover,
analysis of the results of hybridization requires computer programs
capable of assimilating and interpreting multiples bits of
information, and high technology microscopes. The microchips are
neither portable, reusable, nor easily interpreted.S
SUMMARY OF THE INVENTION
[0011] This invention embodies applications of oligonucleotide
microchip technology wherein the microchip is a biosensor and
customized oligonucleotide microchips are designed for specific
applications of nucleic acid hybridization.
[0012] Hybridization is a process by which, under defined reaction
conditions, partially or completely complementary nucleic acids are
allowed to join in an antiparallel fashion to form specific and
stable hydrogen bonds.
[0013] Aspects of the invention include:
[0014] 1. microchips designed so that multiple bits of genetic
information are converted to a pattern, which is interpreted as a
unit, wherein the appearance of the pattern provides answers to
specific questions; this construction facilitates providing easily
interpretable answers provided by hybridization patterns and
removes some need for high technology instruments to interpret the
results of hybridization; and
[0015] 2. improved methods of hybridizing oligonucleotides in a
sample to oligonucleotides on a customized microchip do not require
a washing step but rather measure non-equilibrium melting curves
(temperature curves) that do not require washing with a solution
that removes immobilized oligonucleotides from microchips; this
means that microchips are reusable because the oligonucleotides
anchored within the gel elements, do not wash away, and are
available for reuse. (Microchips with samples are generally kept in
solution, however, microchips can be dried and stored for many
months before being reused.)
[0016] The patterns exhibited after hybridization to a microchip
generally are not directly related to the nature of the
hybridizations and are not simply converting a "yes" or a "no"
signal, or a "positive" or "negative" signal to a binary outcome,
nor are the patterns of the present invention converting a
gradation of quantities to another form of gradation, e.g.
colorimetric gradations. The deliberate organization of the
oligonucleotides on the microchips themselves does not transmit
information; only after hybridization with a test nucleic acid will
the hybridization signal itself form the pattern. The pattern is
then detected by a detection means which can include visual
interpretation without the aid of additional detection
instrumentation.
[0017] By choosing ordered schemes of oligonucleotide positioning
on the microchips, visual signals are simplified and enhanced, e.g.
the letter "P" is observed if certain pathogenic groups are
present; columns of gel elements on the chip that include the same
oligonucleotide probes, will be readily detectable as a positive
linear column, if the matching oligonucleotides are in the test
sample. The visual appearance may be strong enough to see with the
naked eye, may be determined with a UPC (Universal Product Code or
"bar code") laser scanner, or with a laser gun. The wavelength of
the scanner and the sensor that accepts the signal for a bar code
must be concordant with the dye or label used to hybridize the
DNA.
[0018] Of course, aspects one and two do not have to be used
together. Designs that result from converting multiple amounts of
genetic information obtained by large numbers of hybridizations of
oligonucleotides to simpler, readily interpretable patterns, could
be done on microchips constructed and analyzed by the methods used
prior to the present invention.
[0019] Similarly, the improved methods of providing hybridization
results on microchips could be used on microchips that are not
designed to convert multiple pieces of genetic information into a
simpler pattern.
[0020] Other aspects of the invention include improved
predictability, increased accuracy, and standardized factors for
detection and identification of nucleotide sequences. The
improvements result from optimizing conditions, methods and
compositions for microchip hybridization. Deliberate ordered
schemes that are designed to answer specific questions and that
convert complex data to simpler patterns, are followed so that much
hybridization information can be readily obtained from a single
scan of a microchip to detect hybridization of immobilized
oligonucleotides by nucleic acids in a sample to be investigated.
Samples include air, water, soil, blood, cells, tissue, tissue
culture and a food. An aspect of the invention is that the same
microchip can be used for hybridization for more than 20-30 times,
without any noticeable deterioration of the hybridization signal
because immobilized oligonucleotides are not washed out or
stripped. Customized sets of microchips are obtained for specific
applications. Also, parallel hybridization of nucleic acids in a
sample to many oligonucleotides on a microchip is possible,
allowing replication and standardization. For example, the sequence
diversity of SSU rRNAs recovered from different microbial
populations of varying abundances is analyzed by a single
hybridization to a microchip. A large number of HLA alleles, are
assayed by a single hybridization to a microchip.
[0021] The invention relates a method for identifying a nucleotide
sequence in a sample using a microchip, said method comprising:
[0022] (a) providing a customized matrix of oligonucleotides on the
microchip designed to identify genetic sequences in the sample,
wherein an ordered scheme positions oligonucleotides to provide a
pattern to answer specific questions after hybridization;
[0023] (b) hybridizing nucleic acids extracted from the sample as
such or after amplification on said microchip; and
[0024] (c) identifying the nucleotide sequences represented in said
sample by analyzing the pattern of the oligonucleotides which
hybridized to the sequences, said pattern provided by signals.
[0025] The nucleic acids suitable for the practice of the invention
include DNA, mRNA, 16S rRNA sequences and other RNA species.
[0026] Customized oligonucleotide microchips are aspects of the
invention. The microchip includes a gel-matrix affixed to a
support, said matrix is formed by a plurality of gel pad element
sites. The number of sites is determined by the number of
oligonucleotides in the array. Each gel element contains one
chemically immobilized oligonucleotide of a desired sequence,
length and concentration; the gel elements being separated from one
another by hydrophobic glass spaces and the gel portions having a
vertical height above the plane of the interstitial spaces of
generally not more than 30 .mu.m. In some applications, the same
type of oligonucleotides may be immobilized to different gel pads
to form a pattern.
[0027] The invention relates screening nucleic acid preparations
for genes, RNA transcripts or any other unique nucleotide
sequences, for example those that encode microbial 16S ribosomal
RNAs. Ratios of DNA/RNA or any other unique nucleotide sequences
specific for certain types of organisms are suitable. Multiple
labeling allows simultaneous detection and quantitative comparison
of different nucleic acid sequences that are hybridized to a
microchip.
[0028] The methods of the present invention include labeling the
oligonucleotide sequence in said sample before bringing it in
contact with the array. A suitable label is a fluorescent dye. A
plurality of different dyes may be used concurrently.
Oligonucleotides immobilized on a customized microchip include
those complementary to the beta globin gene, sequences specific for
Salmonella, or polymorphic HLA allele sequences.
[0029] An oligonucleotide microchip for the detection and
classification of nitrifying bacteria has a customized design
wherein identifying labels in the cells of the microchip refer to
oligonucleotides selected from a class of bacteria, and the
selection is designed to answer specific questions regarding
classification.
[0030] An embodiment of an application of the present invention is
detecting and identifying microorganisms in samples obtained from
the environment, e.g. water, air or soil samples to check for
pollutants; biological samples obtained for medical diagnosis; or
food samples to check for contamination. Other applications include
forensic testing to identify DNA in samples obtained for criminal
investigations, and detection of chromosomal fragments, or single
gene mutations e.g. for diagnosing genetic diseases such as
.beta.-thalassemia or types of cancers. Tissue typing for
polymorphic HLA alleles for transplantation or studying human
diversity is facilitated.
[0031] The nucleic acid preparations are made from samples
collected in any type of environment, where detection and
identification of the microorganisms in that environment is of
interest, or where it is likely that new (previously unidentified)
organisms may be discovered.
[0032] DNA and RNA molecules in a sample can be separated from each
other during their isolation and labeled with different fluorescent
dyes. These RNA and DNA molecules are simultaneously hybridized
with oligonucleotides on a microchip that is specific to the sample
to be tested. The quantitative monitoring of the simultaneous
hybridization of differently labeled DNA and RNA with a microscope
that can discriminate multicolors at several wave lengths allows
the calculation of DNA/RNA ratios in the sample. For bacterial
samples, this ratio determines the state of vitality and
physiological activity of the bacterium. In an embodiment, the
ratio of RNA/DNA is used to discriminate the dead bacterium cells
and spores from the active state of microbial growth. In the same
way, a DNA or RNA molecule of a bacterial strain stained with one
dye can be added in a calculated amount as an internal standard to
a sequence or sequences under investigation in which the sequences
being investigated stained with a different (second) dye. The
fluorescence measurements of hybridization intensities at different
wave lengths for the standard and investigated sequences (probes)
allow relative quantitative ratios to be determined.
[0033] Hybridization on microchips allows unambiguous typing of
different groups of chosen bacteria in a sample. Microchip
hybridization is a simple, fast, inexpensive and reliable method
for bacterial typing.
[0034] An aspect of the invention is that there is no limitation on
the number of sequences that can be checked or the number of types
of microorganisms that can be detected. Instead of multiple
sequential hybridizations with different probes of, e.g. a 16S rRNA
preparation, only one round of hybridization is required to find
out what different sequences are in a sample. The volume of
hybridizations is dramatically reduced and the assay requires much
less RNA or DNA compared with standard techniques. An advantage is
that culturing of bacteria and gene amplification can be
avoided.
[0035] Methods of the invention significantly reduce sample
preparation time, avoid the culturing of organisms collected from
field situations, and allow the identification of all species of
microorganisms contained in a particular sample. Portable
microchips are available for field work.
[0036] For example, oligonucleotides complementary to small subunit
rRNA sequences of selected microbial groups, encompassing key
genera of nitrifying bacteria, were shown to selectively retain or
hybridize with labeled target nucleic acid derived from either DNA
or RNA forms of the target sequences. Methods and compositions of
the present invention discriminate among the Genera, Nitrosomonas,
Nitrobacter and Nitrosovibrio sp. using fluorescently labeled
nucleic acid probes that hybridize to 16S rRNA sequences. Each
species has specific DNA sequences within the variable region of
its rRNA genes. Since the rRNAs are naturally amplified, often
present in thousand of copies per cell, they provide greater
sensitivity, eliminating the need for amplification in many
applications.
[0037] The invention facilitates identification of organisms from
environmental samples in a faster, and more economical approach
than presently available. In addition, new species may be
discovered that would be highly informative regarding taxonomic
status of known as well as newly discovered organisms.
[0038] A diagnostic assay of the present invention for a mutation
in a gene, includes the following steps:
[0039] (a) designing a customized oligonucleotide microchip
biosensor comprising oligonucleotides that hybridize to a gene
having the mutation, wherein the oligonucleotides are positioned on
the microchips so that patterns result depending on what
oligonucleotides are in the sample to answer a specific
question(s);
[0040] (b) contacting a nucleic acid sample to the customized
oligonucleotide microchip biosensor under conditions that allow
hybridization of the nucleic acid to the microchip; and
[0041] (c) determining the pattern of hybridization from which
observation the presence of specific nucleic acid sequences is
inferred and the specific question is answered.
[0042] For diagnostic assays for genetic diseases, sequence
analysis of DNA is carried out by hybridization of PCR amplified
DNA or its RNA transcripts with oligonucleotide array microchips.
Polyacrylamide gel pads containing allele-specific immobilized
oligonucleotides are fixed on a glass slide of the microchip. The
RNA transcripts of PCR-amplified genomic DNA are optionally
fluorescently labeled by enzymatic or chemical methods and
hybridized with the microchip. In the field, the chemical methods
are preferred because results are obtained faster, and some
chemicals will fragment DNA at the same time which is needed for
the sample.
[0043] When melting curve experiments are performed, both matching
and mismatching oligos can be immobilized in the gel pads, and both
matching and mismatching nucleic acids can be in the sample. The
biochips are reusable in two types of embodiments: 1) the sample or
test nucleic acids can be removed or stripped off the chip and a
different test sample can be introduced and 2) the same melting
point curve experiments can be run and re-run without any
washing.
[0044] When experiments are performed with a different test sample,
the original sample is removed from the chip by a washing or
stripping procedure using distilled water at 60.degree. C. with an
hour (or up to overnight) incubation. If the melting curve
experiments are repeated (or reused) then the same sample is left
in contact with the chip and appearance and disappearance of
hybridization signal is observed over a variety of temperatures,
usually ranging from 0.degree.-50.degree. C.
[0045] When the chips are incubated, in order to remove the sample
nucleotides, virtually none of the immobilized oligos are removed
in the process. This is because the oligos are covalently linked to
the gel matrix of the gel pads that form the microchip.
[0046] Repeated reuse of the chips in which different samples are
applied after sequential removal is usually limited to about 50
uses, because eventually the amount of non-specific or background
hybridization signal is greater than one-tenth of a mismatch
hybridization signal. The conditions under which a chip would not
be reusable (up to 50 times) are very few. Such conditions include
allowing the chips to be cooled to -20.degree. C. or performing
experiments where the chips are heated to above 70.degree. C.,
conditions that have been shown to cause degradation of the chips,
thus rendering them unstable.
[0047] The simultaneous measurement in real time of the
hybridization and melting curves on the entire oligonucleotide
array is carried out with a fluorescence microscope with a laser
light source equipped with CCD camera or a special laser scanner.
Some work only with dried microchips. The monitoring of the
hybridization specificity for duplexes with different stabilities
and AT content is enhanced by its measurement at optimal
discrimination temperatures on melting curves. Microchip
diagnostics are optimized by choosing the proper allele-specific
oligonucleotides from among the set of overlapping oligomers. The
accuracy of mutation detection can be increased by simultaneous
hybridization of the microchip with at least two differently
labeled samples of normal and mutated alleles, and by parallel
monitoring their hybridization with a multi-wavelength fluorescence
microscope. The efficiency and reliability of the sequence analysis
was demonstrated by diagnosing .beta.-thalassemia mutations and HLA
polymorphisms. Determining levels of gene expression is an aspect
of the invention.
[0048] Because the methods of the present invention require only a
simple procedure of hybridization and because only one round of
hybridization is necessary, it is fast and inexpensive. Because the
invention allows a lot of information to be obtained from one
experiment, in a simple pattern as compared to the analysis of
hundreds of data points, it has increased efficiency. The invention
is reliable because the microchips are reusable. Immobilized
oligonucleotides are not washed out. There is no waste of
hybridization probes, therefore the microchip hybridization is
inexpensive and non-isotopic detection simplifies all
procedures.
[0049] Effective and precise sequence analysis by the hybridization
of a probe with rather short microchip-immobilized oligonucleotides
depends on many factors. Major factors are the reliability of the
discrimination of perfect duplexes from duplexes containing
mismatches, differences in stability of AT- and GC-rich duplexes,
the efficiency of the hybridization, and simplicity in the
preparation of the labeled samples for hybridization.
[0050] Identification of base variations is significantly improved
by parallel measuring of the melting curves of the duplexes formed
on the entire oligonucleotide array, as well as by monitoring the
simultaneous hybridization of two differently labeled samples at
two wavelengths and by choosing proper allele-specific
oligonucleotides.
[0051] Other factors to be considered for operation of the
invention include (1) regulating the flow of the fluid containing a
sample to be tested over the microchip during the hybridization;
and (2) control of the temperature of the microchip gel layer and
the fluid layer, in a differential manner, by placing a cooling and
heating apparatus adjacent to the gel layer and the top fluid
layer. The gel layer temperature is controlled in a uniform or
gradient manner by a heating/cooling device attached to the glass
plate substrate of the gels. For field work, the optimum
temperature for a particular question is determined previously in a
laboratory.
[0052] A definition of "customized microchip" is a microchip of gel
elements on a support, wherein the oligonucleotides are immobilized
in gel elements according to an ordered scheme such that multiple
bits of information are ordered to a simpler pattern to answer a
specific question.
[0053] Removal of test or sample nucleic acids from microchip is
accomplished by an incubation step carried out using distilled
water for at least one hour (up to overnight) at 60.degree. C.
(This procedure is analogous to the step of "stripping" a filter
for re-use in the standard technique of probing a Southern blot.)
The immobilized oligonucleotides in the gel matrix are not removed
by this incubation as the oligonucleotides are covalently linked to
the gel substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 shows non-equilibrium melting curves of duplexes of
RNA with microchip oligonucleotides.
[0055] FIG. 2 shows an example of four melting curves for
75-nt-long RNA fragments hybridized with the microchip
oligonucleotides. The RNA was derived from a patient having the IVS
I/2 T/A mutation in the .beta.-globin gene. The curves were
normalized to the initial hybridization signals. Melting curves 1
and 3 correspond to perfect duplexes; curves 2 and 4 correspond to
duplexes containing internal T-T or G-T mismatches, respectively.
The curves for the perfect and mismatched duplexes are shifted by
about 10.degree. C. from each other.
[0056] FIG. 3 shows hybridization of fluorescein labelled 16S rRNAs
to a microchip. The microchip with immobilized probes (see Table 1
and Table 2) was hybridized sequentially to in vitro transcribed
16S rRNA of Nitrosovibrio tenuis (A), Nitrosomonas europaea (B), E.
coli (C), and with E. coli rRNA recovered from isolated ribosomes
(D). The panels to the right display the number of mismatches
between each probe and the RNA.
[0057] FIGS. 4A and 4B show hybridization of the mixture of
differently labelled E. coli and Nitrosovibrio tenuis rRNAs to the
microchip at 10.degree. C. and 40.degree. C., measured
simultaneously by multicolor detection. A. The microchip was
hybridized with a mixture of fluorescein labelled Nitrosovibrio
tenuis and tetramethylrhodamine labelled E. coli 16S rRNA and
washed serially at the indicated temperatures, a.u.--arbitrary
units of fluorescence intensities. B. The ratio of the
hybridization intensities of Nitrosovibrio tenuis (I.sub.Nt) to E.
coli (I.sub.E.coli) 16S RNA measured at 10.degree. C. and
40.degree. C. R=(INt/.sub.IE. coli).
[0058] FIG. 5 illustrates the concentration effect of the
immobilized oligonucleotides on the hybridization intensities. A
microchip with different concentrations of immobilized
oligonucleotides was hybridized with N. tenuis 16S rRNA labelled
with fluorescein and washed at 20.degree. C. Curve 1 corresponded
to Nsv443 (nitrosovibrio-like) probe, curve 2--Bac338 (Bacteria),
curve 3--Nso1225 (ammonia oxidizers), curve 4--Uni1390 (all life),
and curve 5--Nsm-156 (nitrosomonas), a.u.--arbitrary units of
fluorescence intensities.
[0059] FIG. 6 shows the sequences of .beta.-globin alleles
specifying oligonucleotides that were immobilized on a
microchip.
[0060] FIG. 7 shows the experimental design to detect .beta.-globin
mutations using oligonucleotide microchips.
[0061] FIG. 8 shows results of gene expression studies.
[0062] FIG. 9 shows 18 short HLA oligonucleotides.
[0063] FIG. 10 shows HLA oligonucleotides hybridized to the
microchips.
[0064] FIG. 11 illustrates a closed microchamber 1 containing a
microchip with a gel array 3 on a glass support 4; ports 2 are used
merely to provide wetting solution.
[0065] FIG. 12 shows an ordered scheme in which a letter "P" will
be detected if there is a group of hybridizations of
oligonucleotides from a sample that are oligonucleotides from
pathogens.
[0066] FIG. 13 illustrates an ordered scheme on a microchip wherein
the presence of B. anthracis.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0067] This invention relates to using customized oligonucleotide
microchips as biosensors for the detection and identification of
nucleic acids specific for different genes, organisms and
individuals in the environment, in food and in biological samples.
"Environment" includes water, air and soil. Biological samples
include blood, skin, tumors amniotic fluid, tissues, cells and cell
cultures. Detection of sequences in nucleic acids is used to
identify microorganisms in a sample, to diagnose genetic defects or
polymorphisms, to detect gene expression and for forensic
studies.
[0068] Means for detecting a pattern generated by signals from
hybridization within individual gel elements in a microchip of the
present invention include a laser scanner, e.g. a laser "gun" such
as used to scan bar codes, a CCD camera coupled to a fluorescent
microscope. In the field, the naked eye or a scanner is used.
[0069] The invention relates to a deliberate and informative
arrangement of oligonucleotides immobilized on a microchip, such
that upon hybridization with oligonucleotides in a test sample, a
pattern is produced that can be interpreted with a suitable means.
Hybridization may be detected by letter (FIG. 12), design or bar
code pattern (FIG. 13) wherein columns "1" and "3" are dark bars
signifying the presence of a pathogen, and the specific pathogen in
"1 " is a different anthrax species from that in "3." By
immobilizing all of one type of oligonucleotide in a column, for
example, the pattern is readily detected as a linear column, as
contrasted to detecting hybridization in a single small gel element
or elements, which requires a microscope to detect it, and computer
programs to analyze it.
[0070] A nucleic acid hybridization is a highly specific and
sensitive procedure and allows a specific sequence to be detected
and identified among other millions of sequences in a genome of an
organism. However, nucleic acid hybridization is a useful but quite
a cumbersome procedure. This drawback can be overcome by using
oligonucleotide microchips as biosensors for different
microorganisms. Within a small area of a few square millimeters or
centimeters, hundreds and thousands of synthetic oligonucleotide
probes are immobilized that are specific to ribosomal DNA or to
other specific nucleic acids. Subsequent hybridization of a DNA or
RNA molecule to the microchip enables a menu of oligonucleotides to
be identified in a sample. Instead of having to interpret hundreds
or thousands of individual hybridizations, a relative simple
pattern produced by hybridizations is analyzed.
[0071] For bacterial assays, pure culture microorganisms, purified
target nucleic acid or even synthetic oligonucleotides are useful
as internal standards, serving to estimate the efficiency of
nucleic acid isolation or the absolute amount of target nucleic
acid recovered.
[0072] The customized oligonucleotide microchips are produced by
chemical immobilization of presynthesized oligonucleotides, or by
direct synthesis of oligonucleotides on a microchip. If a microchip
contains rather long oligo-nucleotides, the former methods are the
methods of choice because before immobilization, the
oligonucleotides are purified and checked for their quality.
[0073] Methods and technologies have been developed for microchip
manufacturing, hybridization of fluorescently labeled DNA and RNA
with the microchips and monitoring the hybridization with a
fluorescence microscope equipped with CCD-camera, computer and
proper software (see U.S. Pat. No. 5,552,270 herein incorporated by
reference).
[0074] The oligonucleotide microchips consist of many
polyacrylamide gel pad elements generally of the size of
40.times.40.times.20 .mu.m and larger. The elements are chemically
fixed on a glass surface. Each microchip gel element contains a
specific presynthesized oligonucleotide that is immobilized through
a covalent bond. Hundreds of microchips containing hundreds and
thousands of different immobilized oligonucleotides can be
manufactured by a specially devised robot. The gel array also
offers several advantages over formats using an in situ synthesis
of the oligonucleotide array. The synthetic oligonucleotides are
purified by gel electrophoresis or HPLC prior to immobilization on
the microchip. This provides for stringent quality control of
oligonucleotide purity and insures high specificity. The
polyacrylamide gel support has a capacity of immobilized
oligonucleotides from 0.03 pmol up to 10 pmol per
100.times.100.times.20 .mu.m gel pad. This offers improved
quantification and better discrimination between perfect and
mismatched duplexes. It also provides a way to normalize
differences in hybridization signal intensities.
[0075] Oligonucleotide microchip technology for sequencing by
hybridization is available to identify the presence of
microorganisms in a sample of any type, or to find new species. As
shown in the examples herein, the hybridization of DNA or RNA
extracted from even a very complicated mixture to a specific
oligonucleotide probe has resulted in unambiguous identification of
microorganisms. The nucleotide sequence of the microorganisms for
genes encoding a small subunit of ribosomal 16S rRNA forms the
basis for a microchip biosensor. Instead of direct sequencing of
the gene, hybridization analysis of DNA or RNA samples with
oligonucleotides specific for the microorganisms is performed. This
new technology provides efficient microbial analysis and
environmental monitoring. Fluorescently labeled DNA and RNA samples
from microorganisms are hybridized with microchips containing
oligonucleotides specific for several microorganisms. These
microorganisms are reliably identified by microchip hybridization
patterns. Microorganism biosensor technology is developed, reusable
customized microbial oligonucleotide microchips are produced by
methods of the present invention, and methods are developed for
simultaneous quantitative and qualitative microchip analysis of
hundreds and thousands of microorganisms in a sample and for
discovery of new ones.
EXAMPLES
[0076] The following examples are presented as illustrations of
aspects of the invention, rather than limitations on the invention.
Other applications include detection of genetic mutations such as
are characteristic of hemoglobin disorders; detection of genetic
polymorphisms such as HLA; investigation of gene expression;
detection of causative agents of diseases; forensic studies; and
detection of microbial pollutants.
Example 1
[0077] Preparation of an Oligonucleotide Microchip Biosensor
[0078] Oligonucleotides are synthesized using a 394 DNA/RNA
synthesizer (Applied Biosystems). The synthesis of oligonucleotides
for immobilization began with 3-methyluridine at the 3'-terminal
position.
[0079] In one embodiment, fluorescently labeled RNA was prepared
using T7 RNA polymerase. Template DNA (133 and 75 bp long) for in
vitro transcription was prepared by PCR amplification with the
nested primers T7-V2L-45,
5'-GGAATTCCTAATACGACTCACTATAGGGAC[C]ACC-ATGGTGCACCTGACTCC-3'(S- EQ
ID NO: 5), as well as with the common reverse primer T7-V2L-103
5'-GGAATTCCTAATACGACTCACTATAGGGAGGTGAACGTGGATGAAGTT GG-3'(SEQ ID
NO: 16) AND 5'-TCTCCTTAAACCTGTCTTGTAACC-3' (SEQ ID NO: 17).
Templates were purified using a QIAquick PCR purification kit
(QIAGENE) according to the manufacturer's protocol. The RNA
polymerase reaction was performed using the MEGAshortscript.TM. T7
kit (Ambion) with fluorescein 12-UTP (Molecular probes).
Fluorescently labeled ssDNA (single stranded DNA) fragments were
prepared by single primer reamplification.
[0080] A polyacrylamide gel micromatrix was prepared by
photopolymerization of a solution of 4% acrylamide
(acrylamide/bisacrylamide 19/1), 40% glycerol, 0.0002% methylene
blue, and 0.012% TEMED in 0.1 M sodium-phosphate buffer, pH 7.0.
The mixture was applied to an assembled polymerization chamber
illuminated with U.V. light.
[0081] Two types of microchip matrices (micromatrices) were
routinely prepared with gel pad elements of about
60.times.60.times.20 .mu.m and 100.times.100.times.20 .mu.m that
were spaced by 120 and 220 .mu.m, respectively. About 1 nl of
activated oligonucleotide solution was transferred to a gel element
using either a robot or a simple manual device.
[0082] The device includes a Peltier thermostated pin placed under
a binocular lens in conjunction with a micromanipulated holder, a
power supply, and a refrigerated circulator.
[0083] The manufacture of microchips of gel-immobilized
oligonucleotides basically consists of three steps; shaping the
desired topology of oligonucleotides on a gel micromatrix; loading
microvolumes of oligonucleotide solutions onto the micromatrix, and
immobilizing within the gel oligonucleotides containing the active
3' or 5' terminal aldehyde or amine groups.
[0084] To avoid the exchange of different oligonucleotide solutions
applied on adjacent gel pads, the pads are separated on the
micromatrix by a hydrophobic glass surface. Two-dimensional
scribing or laser evaporation is used for micromatrix preparation,
but these procedures require rather complex equipment and
experienced personnel. The photopolymerization method significantly
simplifies the procedure and makes it accessible to a biochemical
laboratory.
[0085] Microfabrication by mask-directed photopolymerization (e.g.,
a photoresist method in microelectronics) is a well developed
technique. From several acrylamide photopolymerization techniques
tested, modified--methylene-blue induced photopolymerization
produced the best results for micromatrix manufacture. The gel
matrix consists of gel pads photopolymerized on a glass slide. The
gel pads are formed according to the mask topology due to the lack
of photopolymerization in places covered by a nontransparent
grid.
[0086] The microchip is manufactured by applying the activated
oligonucleotide solutions onto the micromatrix of gel elements
containing active hydrazide or aldehyde groups. A simple device
exists for manual loading of up to 100 different oligonucleotides
on a micromatrix. The transfer is carried out by the hydrophilic
upper surface of a pin that is first immersed into, and is wetted
with, an oligonucleotide solution, and then is withdrawn from the
solution and brought into contact with the gel surface. This
transfers about 1 nl of oligonucleotide solution with a
reproducibility of <10%. The temperature of the pin is
maintained near the dew point of the ambient air to avoid the
evaporation of this microvolume solution in the course of
transfer.
[0087] The oligonucleotides are positioned according to a design
wherein hybridization pattern data will be reduced to a readily
interpretable pattern.
Example 2
[0088] The Hybridization of Microchips with DNA and RNA using a
Hybridization Buffer.
[0089] Fluorescently labeled DNA or RNA (5 .mu.l, 0.1-1 pmol/.mu.l
were hybridized to a microchip at +5.degree. C. in a hybridization
buffer containing 1 M NaCl, 1 mM EDTA, 1% Tween-20, and 10 mM
sodium phosphate at a pH of 7.0, for between about 2-24 h. The
microchip was covered with a cover glass or a Teflon sheet so that
a 300-.mu.m space is above. Then the hybridization solution
containing DNA or RNA fragments was substituted with 10 .mu.l of
cooled hybridization buffer. The microchip with the cover glass was
placed on a thermostabilized table. Hybridization was monitored
quantitatively using a specially constructed multicolor
epifluorescent microscope with a 4.times.4 mm observation field
equipped with a CCD camera and suitable software.
Example 3
[0090] Analysis of Melting Curves; a Hybridization Buffer is Not
Required.
[0091] The polyacrylamide gel used on a microchip provides more
than 100 times higher capacity for three-dimensional immobilization
of oligonucleotides than does a two-dimensional glass surface. The
high concentration of immobilized oligonucleotides facilitates the
discrimination of mismatched duplexes and enhances the sensitivity
of measurements on the microchips. This allows the use of a
CCD-camera-equipped fluorescence microscope (Yershov et al., 1996)
although it is less sensitive than laser scanning systems (Lipshutz
et al., 1995), but offers the advantage of monitoring the
hybridization on a microchip at different temperatures in real time
for measurement of the melting curves. Melting curves are defined
herein as produced by plotting the amount of duplexes [fluorescent
intensity] versus temperature. The procedure, the software, and the
hybridization microchamber (Yershov et al., 1996) have all been
developed for recording melting curves at a wide range of
temperatures simultaneously for perfect and mismatched duplexes
formed upon hybridization of a probe with all microchip
oligonucleotides.
[0092] A significant amount of time is needed for the microchips
hybridized with rather long RNA or DNA probes to achieve
equilibrium. Therefore, non-equilibrium dissociation melting curves
were measured. However, they are not far away from equilibrium
where some difference in heating rate did not significantly affect
the results. The melting curves for hybridization of, for example,
synthetic 19-mers with the microchip oligonucleotides reached
equilibrium under the same conditions that were used for measuring
non-equilibrium RNA and DNA melting curves. The melting curves can
also be measured after a few minutes, far away from equilibrium, if
an internal standard is added to a tested sample. This standard can
be a differently labeled RNA of a normal allele. This significantly
speeds up the identification of nucleic acid base changes.
Example 4
[0093] Choice of Optimum Melting Temperatures for Non-Equilibrium
Hybridization.
[0094] This invention embodies an improvement in the SHOM
technology in which hybridizations between an array of
gel-immobilized nucleotides (a microchip) and the unknown
nucleotides to be tested are measured at optimal, discriminatory
melting temperatures. This improvement is achieved by parallel
measuring of the melting curves of the duplexes formed by
hybridization on the entire oligonucleotide array, as well as by
monitoring the simultaneous hybridization of two samples of
nucleotides labeled with different fluorochromes, and judicious
choice of proper allele-specific oligonucleotides as the
immobilized probes. The fluorochromes chosen for the labeling emit
light of sufficiently differing wavelengths, that both types of
labels can be measured in the same reaction mixture.
[0095] The greatest discrimination between perfect and mismatched
duplexes was achieved at a temperature at which the intensity of
the hybridization signal from a perfect duplex dropped to one-tenth
of its initial value; at such a temperature, the hybridization
intensities from mismatched duplexes usually approached the
background level. The temperature at which the initial signal of
hybridization drops by a factor of 10 is termed the discrimination
temperature (Td.).
[0096] In the case of beta-thalassemia mutation detections
described in Example 6 herein: (1) RNA transcripts of PCR-amplified
DNA were hybridized with immobilized oligonucleotides; (2) the Td
values for perfect 40% and 70% GC-rich duplexes were 52.degree. and
64.degree., respectively; (curves 1 and 3 in FIGS. 1A and 1B); (3)
the immobilized oligonucleotides were chosen from among a set of
overlapping sequences; and (4) the two samples included in the
reaction mixture were a mutated allele RNA labeled with one
fluorochrome and a sample of the normal allele RNA labeled with a
different fluorochrome.
[0097] The Td is determined by hybridization with an RNA sample if
an allelic DNA is available. If such DNA is unavailable, the Td can
be measured from the hybridization data resulting from experiments
performed with synthetic oligonucleotides corresponding to the
mutated allele of interest.
[0098] The dissociation curves for perfect and mismatched duplexes
are parallel at the range of about 10.degree. (in the middle of the
curves) when plotted on a semilogarithmic scale. At this 10.degree.
C. range, the ratios of the signals for perfect and mismatched
duplexes remain rather constant. This makes the discrimination
procedure robust to some inaccuracies in determining Td. The
discrimination temperature depends on experimental conditions (rate
of heating, ionic strength, probe concentration, extent of
fragmentation, and so forth) which can vary from one experiment to
another. However, these variations affect Td and the relative
intensities of the hybridization signals to a similar extend for
all microchip elements and therefore do not significantly distort
the discriminations. Therefore, to provide a reference Td, the
oligonucleotides CD26(N) and CD 26 G/A, which form perfect and
mismatched duplexes, respectively, with all RNAs tested, were
introduced into the microchip.
[0099] Since Td is robust to some inaccuracy in measurements,
19-mer oligodeoxynucleotides were used in these experiments instead
of more expensive 19-mer oligoribonucleotides. There are
differences in the stability of DNA-DNA homoduplexes relative to
DNA-RNA heteroduplexes (Lesnik and Freier, 1995). The pattern of
hybridization of the microchip with RNA derived from patients and
with 19-mers was rather similar to that from the 10-mers.
Hybridization with corresponding synthetic oligonucleotides is
preferred as a control when a mutation is identified in an RNA
sample by its hybridization with a diagnostic microchip.
[0100] A mixture of fluorescently labeled RNA samples was prepared
from two patients; the first sample was TMP-labeled RNA from a
patient that is homozygous for the normal CD26 area of the
beta-globin allele; the second sample is fluorescein-labeled RNA
from a patient that is heterozygous for the normal CD26 area and a
mutation CD26 G/A alleles. This mixture was hybridized with a
microchip consisting of two microchip elements that contained the
following immobilized oligonucleotides:
1 SEQUENCE A sample (element)-CD26(N-normal) 5'-GGCCTCACCA-3MeU-3'
(SEQ ID NO: 1) B sample (element)-CD26(G/A-mutant)
5'-GGCCTTACCA-3MeU-3' (SEQ ID NO: 2)
[0101] Usage of different filters during the registration of the
signal, allowed the independent, simultaneous registration of the
sample, which was marked with the different dyes; TMP (red) and
fluorescein (green), on the same element of the microchip. FIG. 2
demonstrates the interaction of sample 1 with the A microchip
element; Graph 2 demonstrates the interaction of sample 2 with the
A microchip element; Graph 3 demonstrates the interaction of sample
2 with the B microchip element; and Graph 4 demonstrates the
interaction of sample 1 interaction with the B microchip
element.
Example 5
[0102] Use of a Customized Microchip Matrix Biosensor to Identify
Nitrifying Microorganisms.
[0103] The results in this example were obtained using methods
previously available, not the non-equilibrium melting curves.
Microorganisms that degrade nitroaromatic compounds include
Pseudonomas, Arthrobacter, Nocarida, Myco-bacterium, and fungi.
[0104] Previously, methods for detection of these bacteria were
tedious and inaccurate. For example, to detect Pseudonomas capable
of degrading nitroaromatic compounds, 2-nitroluene was tested as a
sole carbon, energy and nitrogen source. It was difficult to
isolate the bacteria from soil samples to perform the test.
[0105] Nitrifying bacteria have proved particularly difficult to
study using cultivation techniques, such as most probable number
(MPN) and selective plating because of their long generation times
and poor counting efficiencies. Thus, a rapid and non-culture
dependent enumeration technique for nitrifiers could greatly
facilitate research in their ecology.
[0106] Microchips with 100.times.100.times.20 .mu.m gel pads
(alternatively 60.times.60.times.20 .mu.m), fixed on a glass
surface and containing a set of 10 oligonucleotides 15-20
bases-long were manufactured for bacterial typing experiments. The
set included oligonucleotides complementary to different regions of
16S ribosomal RNA. Since rRNA's are naturally amplified, and often
are present in thousands of copies per cell, they provide great
sensitivity and eliminate the need for amplification in many
applications. One oligonucleotide is represented in most living
organisms, another is typical for most of bacteria and the rest
belong to nitrosos (nitrifying) bacteria only. The group of
nitrosos bacteria oligonucleotides consists of two oligonucleotides
typical of nitrobacter, two typical of nitrosomonas and one typical
of nitrosovibrio. One oligonucleotide was complementary to an
antisense strand of rDNA for hybridization with ribosomal dsDNA,
that was PCR amplified from genomic or cDNA.
[0107] The following scheme for an ordered oligonucleotide loading
(placing on a chip) is useful for bacterial (or organism, species)
typing. In the micromatrix design shown in Table 1, the first
oligonucleotides characterize the highest order (i.e. to
distinguish a living organism). [Uni 1390-CIII]. Reducing the order
step by step down to the lowest level, i.e. from family, to genus,
to species provides further discrimination of oligonucleotides that
are present in a sample being investigated. For example, for
oligonucleotides used to classify nitrifying bacteria, a bacterial
oligonucleotide would be in the next position. [Bac 338--CI and
NonBac338--CII]. Oligonucleotides specific to nitrobacter
[Nb1000--AI and NIT3--AII] and ammonia oxidizers [NEU 23--AIII,
Nso190--AIV and Nso1225 --BI] follow in any order. Finally,
oligonucleotides specific to Nitrosomonas [Nsm156--BII] and
Nitrosovibrio [Nsv443--BIII] complete the micromatrix design.
[0108] The microchip was evaluated using three different rRNA
preparations (phenol extracts of cellular RNA, RNA isolated from
purified ribosomes, and in vitro transcripts of cloned ribosomal
DNA), and both fragmented double-stranded and single-stranded DNA.
Hybridizations were performed in a formamide buffer at low
temperature in order to enhance microchip durability and decrease
RNA degradation. Although all DNA and RNA preparations could be
used, the best discrimination was observed for in vitro transcribed
rRNAs using the hybridization conditions evaluated in this
study.
[0109] The hybridization of the microbial microchips was carried
out with five different preparations of target nucleic acids.
Ribosomal RNA and total RNA were recovered from cells. RNA
transcribed in vitro as well as single- and double-stranded
PCR-amplified 16S rDNA were obtained from plasmids containing the
cloned 16S rRNA gene. All of these sample types provided a
comparatively reliable identification of the microorganisms by
their hybridization with the microchip-immobilized oligonucleotides
and could be used for different purposes. For example, the rRNA
provides a naturally amplified target. Also, since cellular
ribosome content is well known to vary with growth rate, it is
generally thought that direct quantification of rRNA serves to
identify the more active environmental populations. In contrast,
analysis of PCR amplified rDNA provides a more general measure of
all microorganisms present in a sample. Alternatively, these
measures could be combined. For example, the RNA and DNA components
of an environmental sample could be isolated and labelled with
different fluorescent dyes. Following their combined hybridization,
the resulting ratio of RNA and DNA hybridizing to an individual gel
element could be used to infer the physiological status of the
corresponding microbial population.
[0110] Table 2 shows the sequences of the oligonucleotides and
other characteristics of them.
2TABLE 1 Micromatrix Design for Nitrifying Microorganisms. I II III
IV A Nb1000 NIT3 Nso190 B Nso1225 Nsm156 Nsv443 C Bac338 NonBac338
Uni1390
[0111]
3TABLE 2 Oligonucleotide Microchip Name and location Position
Sequence (5' to 3') Specificity Table 1 Td.sup.1C Nb1000 5'-tgc gac
cgg tca tgg-3' (SEQ ID NO: 6) Nitrobacter A-I 42.degree. NIT3 5'cct
gtg ctc cat gct ccg-3' (SEQ ID NO: 7) Nitrobacter A-II
66.degree..sup.2 NEU23 5'-ccc ctc tgc tgc act cta-3' (SEQ ID NO: 8)
Ammonia oxidizers A-III 66.degree..sup.2 NSO190 5'-cga tcc cct gct
ttt ctc-_3' (SEQ ID NO: 9) Ammonia oxidizers A-IV 62.degree.
NSO1225 5'-cgc gat tgt att acg tgt ga-3' (SEQ ID NO: 10) Ammonia
oxidizers B-I 51.degree. NSMO156 5'-tat tag cac atc ttt cga t-3' ?
(SEQ ID NO: 11) Nitrosomonas B-II 46.degree. NSV443 5'-ccg tga ccg
ttt cgt tcc-3' (SEQ ID NO: 12) Nitro-sospira-like B-III 52.degree.
BAC338 5'-gct gcc tcc cgt agg gat-3' (SEQ ID NO: 13) Bacteria C-I
54.degree. NonBAC338 5-'act cct acg gga ggc agc-3' (SEQ ID NO: 14)
Eub338 complementary strand C-II 54.degree. UNI1390 5'-gac ggg cgg
tgt gta caa-3' (SEQ ID NO: 15) all life (with a few exceptions)
C-III 44.degree. .sup.1Experimentally determined. .sup.2Estimated
from in situ hybridization.
[0112] A number of hybridization conditions were tested in terms of
efficiency and specificity of hybridization. Hybridization in
formamide containing buffer at low temperature gave good results.
Hybridizations were performed at 5.degree. centigrade in 33%
formamide. RNA samples and covalent bonding of oligonucleotides
with the support (hence durability of microchips) are more stable
at low temperatures. In addition, these conditions were favorable
from a point of view of RNA stability and microchip durability
similar to other RNA molecules at low temperatures of about
0.degree.-5.degree. C.
[0113] The hybridization on a microbial microchip was carried out
with in vitro RNA transcripts of 16S rDNA of different nitroso
bacteria, total RNA extracts and ribosomal RNA extracted form E.
coli and Desuifovibria vulgurus as well as PCR amplified double or
single stranded DNA of 16S rDNA.
[0114] The probes for ammonia oxidizing bacteria show different
discrimination specificity under different conditions. FIG. 3 shows
the fluorescence of individual gel elements on the microchip
following hybridization to the 16S rRNAs of Nitrosovibrio tenuis
(A), Nitrosomonas europaea (B), and E. coli, either in vitro
transcribed (C) or recovered from isolated ribosomes (D). The same
microchip was used for each hybridization following washing with
distilled water. Each microchip was routinely used for up to 20-30
hybridization experiments. The appropriate pattern of hybridization
was observed for all gel elements shown, despite a significant
difference in dissociation temperatures (T.sub.ds) previously
determined using membrane support hybridization (Table 1). For
hybrids of comparable stability, discrimination is generally
achieved by washing at increasing temperatures (described below) or
by simultaneously evaluating their melting characteristics, since
the fluorescence analyzer can monitor hybridization signals in real
time.
[0115] FIGS. 4A and 4B shows the results of an experiment
evaluating the effect of increasing washing temperature on target
RNA retention. A mixture of Nitrosovibrio tenuis and E. coli 16S
rRNA labelled with different fluorescent dyes (fluorescein and
tetramethylrhodamine, respectively) was hybridized to the chip at
5.degree. C. The hybridization solution was then replaced with
washing buffer and the retention of each RNA species was measured
following each 10.degree. C. incremental increase in temperature
(up to 60.degree. C.) using multicolor detection. Nonspecific
hybridization of E. coli rRNA to Nso1225 (ammonia oxidizer), Nsm156
(nitrosomonas), and NonBac338 (anti-sense) was observed following
the 10.degree. C. wash. However, this nonspecific hybridization was
significantly reduced following the 40.degree. C. wash. In like
manner, the 16S rRNA of Nitrosovibrio tenuis hybridized to
Nitrosomonas (Nsm156) at 10.degree. C., but was reduced to near
background (compared to NonBac338) following the 40.degree. C.
wash. Using the methods of the present invention, hybridization
buffer is not required. A more complete correction for differences
in stabilities of duplexes can be carried out by measuring the
equilibrium or non-equilibrium melting curves for all microchip
elements. This would provide a basis to compensate for the various
factors influencing individual duplex stability, e.g., their
length, GC-content, and competition with secondary and tertiary
structures in RNA and DNA.
[0116] FIG. 4B shows the ratios of hybridization intensities of
fluorescein labelled Nitrosovibrio tenuis to tetramethylrhodamine
labelled E. coli. with different microchip oligonucleotides at
10.degree. C. and 40.degree. C. (the ratios are derived from the
data presented on FIG. 4A. These ratios were not changed
significantly for oligonucleotides specific to bacteria and all
living organisms between 10.degree. C. and for more stringent
conditions at 40.degree. C. However, the ratio is dramatically
increased at 40.degree. C. (compared to 10.degree. C.) for
oligonucleotides specific to ammonia oxidizers and nitrosovibrio.
This increase reflects the greater duplex stability of
Nitrosovibrio tenuis RNA with the complementary oligonucleotides
compared with E. coli. RNA. Although the nitrosomonas ratio
increases, the signal originating from each labelled RNA is near
background. This experiment demonstrates that the inclusion of
second dye-labelled RNA, either isolated from cells or synthesized,
could be used as an internal standard for quantitative assessments
of hybridization patterns.
[0117] Variable hybridization to the different gel elements is the
expected consequence of using a single hybridization condition to
evaluate an array of probes, each having different kinetics of
association and dissociation. To some extent these difference can
be normalized by varying the concentration of oligonucleotides in
the individual gel elements. For example, the relatively low
hybridization signals of Nso1225(b-I) and Uni1390 (c-III) compared
to Nsv443 (b-III) could each be elevated by increasing the amount
of the corresponding oligonucleotides probes immobilized in the
gel. This approach was evaluated by synthesizing a microchip with
selected probes immobilized at several different concentrations, up
to 6 times higher than that used in the experiments previously
described. This was accomplished by multiple applications of the
standard loading solution (100 pmol/.mu.l probe) to each gel
element. Comparable hybridization of Nso1225 (ammonia oxidizer) and
Nsv443 (nitrosovibrio-like) was achieved following three
applications of the Nso1225 probe (FIG. 5). Similarly, two
applications of Bac338 (bacteria) and five of Uni1390 (all life)
resulted in hybridization comparable to Nsv443.
[0118] Strains used. Escherichia coli, Desulfovibrio vulgaris
strain PT2, Nitrosovibrio tenuis strain NV12, Nitrosomonas europaea
strain ATCC 19718, and Nitrosomonas strain C-56 were used as
sources of nucleic acid for these experiments.
[0119] RNA preparation. Total cellular RNA was isolated by
phenol/chloroform extraction. For some of the samples, a ribosome
enrichment was performed before RNA extraction. Forty ml of log
phase growth E. coli or D. vulgaris strain PT2 was centrifuged at
3500 g for 10 min. and resuspended in 4 ml of 4.degree. C. ribosome
buffer. Ribosome enrichment buffer consisted of 20 mM MgCl2, 50 mM
KCl, 50 mM Tris at pH 7.5, and 5 mM .beta.-mercaptoethanol in
diethyl pyrocarbonate treated double-distilled water. The cell
suspension was divided between 4 screwtop microfuge tubes and 0.5 g
of 0.1 mm ZrO2 beads were added. The cell suspensions were
disrupted for 2 min., put on ice for 5 min., and disrupted again
for 2 min. The cell suspensions were centrifuged at 14000 g for 10
minutes. The supernatant, which contained the ribosomes, was
recovered and transferred to ultracentrifuge tubes. Ribosomes were
pelleted by ultracentrifugation in ribosome buffer at 55,000 rpm
(201,000 g average) for 50 minutes in a Beckman Optima Series TL
swinging bucket rotor (Beckman, Fullerton, Calif.), for a Svedberg
sedimentation factor of 70S. After centrifugation, the supernatant
was discarded and the RNA was recovered from the pelleted ribosomes
by extraction with pH 5.1 phenol/chloroform. Quality and quantity
of extracted RNA was evaluated by polyacrylamide gel
electrophoresis and ethidium bromide staining.
[0120] Cloning of 16S rDNA and in vitro production of RNA
transcripts. DNA was extracted from E. coli, Desulfovibrio vulgaris
PT2, Nitrosovibrio tenuis NV12, Nitrosomonas europaea 19718, and
Nitrosomonas strain C-56 cell pastes using a guanidine/diatom
method. Near-complete 16S rDNA genes (ca. 1500 base pairs) were
recovered from each by PCR amplification using S-D-Bact-0011-a-S-17
(GTTTGATCCTGGCTCAG) (SEQ ID NO: 3) and S-D-Bact-1492-a-A-21
(ACGGYTACCTTGTTACGACTT) (SEQ ID NO: 4) as primers and a premixed
PCR amplification buffer (Pharmacia Biotech Inc. Piscataway, N.J.),
consisting of 0.2 mM Mg++, 2.5 mM each dATP, dCTP, dGTP, dTTP, 0.2
mM of each amplification primer, and 2.5 units of Taq DNA
polymerase (Pharmacia). Temperature cycling was done in an Idaho
Technology thermocycler (Idaho Falls, Id.) using 30 cycles of 15
sec at 94.degree. C., 20 sec at 50.degree. C., 30 sec at 72.degree.
C. The PCR-products were cloned in a pCR plasmid (Invitrogen, San
Diego, Calif.) according to manufacturers instructions. Plasmids
were isolated using the Wizard kit (Promega, Madison, Wis.) and
used for in vitro transcription of the cloned SSU rRNA genes.
[0121] DNA oligonucleotide probes. All probes were complementary to
the SSU rRNAs and previously characterized using a membrane
hybridization format. Five probes hybridize to different groups of
ammonia-oxidizing bacteria within the beta-subdivision of the
Proteobacteria. S-G-Nso-190-b-A-19 (Nso190) and S-G-Nso-1225-a-A-20
(Nso1225) encompass all sequenced ammonia-oxidizers of the
beta-subclass of Proteobacteria, probe S-G-Nsm-156-a-A-19 (Nsm156)
identifies members of the genus Nitrosomonas (also including
Nitrosococcus mobilis), probe S-G-Nsv-443-a-A-20 (Nsv443) is
specific for the Nitrosovibrio/Nitrosolobu- s/Nitrosospira group,
and probe S-G-Nsm-653-a-A-18 (NEU23) is specific for the
halotolerant members of Nitrosomonas. Probes for members of genus
Nitrobacter (nitrite oxidizing) were S-G-Nit-1000-b-A-15 (Nb1000)
and S-G-Nit-1035-a-A-18 (NIT3). Other probes used were
S-D-Bact-0338-a-A-18 (Bac338) which hybridizes to members of the
bacterial domain; S-D-NBac-0338-a-S-18 (NonBac338), complementary
to the antisense strand of the Bac338, and S-*-Univ-1390-a-A-18
(Uni1390) complementary to the SSU RNA of nearly all characterized
living organisms, with the exception of some protists.
[0122] RNA and DNA labeling and fragmentation. Single stranded DNA
was prepared by asymmetric PCR according to Ausubel et al. (1994)
using a 100 times excess of the forward primer. Briefly, DNA was
partially depurinated in 80% formic acid for 30 min. at 20.degree.
C., then incubated in 0.5 M ethylenediamine hydrochloride (pH 7.4)
for 3 hr at 37.degree. C., followed by 30 min. at 37.degree. C. in
the presence of 0.1 M NaBH4. Fluorescein isothiocyanate was
incorporated into fragmented DNA by incubation in absolute DMSO at
room temperature for 1 hr.
[0123] RNA was fragmented by base hydrolysis and dephosphorylated
with bovine phosphatase. Fragmented RNA was oxidized by NaIO4 and
labeled either by ethylenediamine mediated coupling of
6-carboxyfluorescein (FAM) succinamide or by direct incorporation
of tetramethylrhodamine-hydrazide (TMR).
[0124] Microchip fabrication. A matrix of glass-immobilized gel
elements measuring 60.times.60.times.20 or 100.times.100.times.20
.mu.m each and spaced apart by 120 or 200 .mu.m respectively was
prepared. The polyacrylamide gel was activated by substitution of
some amide groups with hydrazide groups by hydrazine-hydrate
treatment. Oligonucleotides were activated by oxidizing 3'-terminal
3-methyluridine using NaIO4 to produce dialdehyde groups for
coupling with hydrazide groups of the gel and coupled to each
micromatrix element by applying 0.5-1 nl of the activated
oligonucleotide solution (100 pmol/.mu.l) using a specially devised
robot.
[0125] Hybridization and image analysis. Probe binding was
quantified by measuring the fluorescence conferred by the binding
of fluorescently labeled DNA or RNA (tetramethyl rhodamine or
fluorescein) to the individual gel elements. Hybridization and
washing was controlled and monitored using a Peltier thermotable
(with a working range of -5.0.degree. C. to +60.0.degree. C.)
mounted on the stage of a custom-made epifluorescent microscope.
The microchip was hybridized at 5.degree. C., either overnight or
for 6 hr, in 2-5 .mu.l of the hybridization buffer [33% formamide,
0.9 M NaCl, 1 mM EDTA, 1% Tween-20, and 50 mM sodium phosphate (pH
7.0)] at a concentration of DNA and RNA between 0.2-2 pmol/.mu.l.
The hybridization mixture was replaced with 5-10 .mu.l
hybridization buffer without formamide immediately prior to
microscopic observation. Exposures were in the range of 0.1-10 sec
depending on the signal intensity, but were typically around 1 sec.
Fluorescence was monitored either at room temperature or using a
range of temperatures between 5-60.degree. C.
[0126] Conditions for the coupling of micromolecules to the
acrylamide gel were devised to rule out the possibility of liquid
evaporation during immobilization and to ensure that covalent
bonding of oligonucleotides with the gel matrix proceeds to
completion. After the microvolumes of the oligonucleotide solutions
have been applied to all cells of the matrix, the micromatrix gel
elements were swelled by condensing water from the ambient air.
Then the micromatrix surface was covered with a thin layer of an
inert nonluminescent oil, and chemical coupling of the activated
oligonucleotides to the activated polyacrylamide was carried out to
completion.
Example 6
[0127] Use of Microchip Biosensors as Diagnostic Assays
[0128] The microchip technology was successfully tested for
identification of single base changes in genomic DNA and RNA for
reliable diagnosis of human genetic diseases. A customized
microchip contained oligonucleotides specific to .beta.-thalassemia
normal and abnormal .beta.-globin genes. The hybridization with
PCR-amplified DNA or RNA samples derived from genomic DNA of
subjects allowed unambiguous identification of a mutation in a
sample to be tested. Reliability of the identification was enhanced
by using simultaneous hybridization with two samples of a normal
and mutated RNA stained with different fluorescence dyes and
monitoring the hybridization at different wavelengths; by
simultaneously measuring the melting curve for duplexes formed on a
microchip, and by using a proper set of several oligonucleotides
complementary to the mutated site of the DNA.
[0129] A number of the most commonly occurring .beta.-thalassemia
mutations with .beta.-globin gene were used in diagnostic assays
with oligonucleotide microchip biosensors. These mutations were
splice-site mutations for the 1.sup.st, 2.sup.nd, 5.sup.th, and
6.sup.th nucleotides in the first intron (IVS I) of the
.beta.-globin gene: IVS I/1 G/A (GIA=substitution of G for A), IVS
I/2 T/C, IVS I/5 G/T, IVS I/5 G/C, IVS I/6 T/C, and G/A
substitution in the 26.sup.th codon (GAG) of the first exon (FIG.
6), (also known as abnormal hemoglobin E) (see Diaz-Chico et al.,
1988 for terminology).
[0130] A microchip with 100H100H20 .mu.m gel elements (Yershov et
al., 1996) contained immobilized decadeoxyribonucleotides, that is,
10-mers that correspond to normal and mutant .beta.-thalassemia
alleles. These 10-mers discriminated mismatches less reliably than
8-mers, but were hybridized more efficiently than 8-mers. 10-mers
were, therefore, preferred for this assay. Table 3 shows the
sequences of the allele-specific oligonucleotides immobilized on
the microchips. It was expected that mismatches within the duplexes
would have a much higher destabilization effect than mismatches at
the terminal positions (Khrapko et al., 1991); therefore the
mutated bases were placed inside of the immobilized
oligonucleotides.
[0131] Single- and double-stranded PCR-amplified .beta.-globin DNA
fragments of different lengths and collected after a random
fragmentation were tested in assays for identification of some of
these mutations. However, the hybridization of RNA is preferred
over DNA hybridization. RNA fragments were derived from
PCR-amplified genomic DNA by transcription with T7 RNA polymerase
(Lipshutz et al., 1995). About 100 copies of unlabeled or
fluorescently labeled RNA transcripts are synthesized per DNA
molecule, providing a convenient way to prepare a sufficient amount
of the hybridization probes. RNA is fragmented and one fluorescent
dye molecule is introduced per fragment.
[0132] Table 3 shows the sequences of the microchip allele specific
10-mers. The oligonucleotides of microchip I are complementary to
the coding strand of DNA of the .beta.-globin gene of patients with
.beta.-thalassemia single-base mutations (G/A -substitution of A
for G) in the 1.sup.st, 2.sup.nd, 5.sup.th, or 6.sup.th nucleotides
of the first intron (IVS I/1, 2, 5, 6) of the .beta.-globin gene
and in the codon #-26 (CD-26) of the first exon. Oligonucleotides
1-16 of microchip II correspond to the normal and IVS I/2 G/T
allele. The mutated and corresponding normal bases are placed from
the 2.sup.nd to the 9.sup.th positions of the 10-mers from their
3'-end. The mutated bases are shown in lowercase bold letters and
corresponding oligonucleotide bases in the normal allele are
underscored. The oligonucleotide synthesis and the microchip
manufacturing were described by Yershov et al. (1996).
[0133] Microchip I was successively hybridized with RNA 75 and 133
nt long without fragmentation or after fragmentation (133fr, Table
5, probes 3a and 4a) and with 6 synthetic 19-mer
oligodeoxyribonucleotides corresponding to .beta.-thalassemia
mutations. The RNA and 19-mers were labeled with TMR except for RNA
probes 2a, 2b, and 6b, which were labeled with fluorescein (F1).
The melting curves (FIGS. 1A-B, FIG. 2) were measured
simultaneously for all microchip oligonucleotides at each
hybridization. These curves provided values of hybridization
intensities at the discrimination temperature, Td. R is the ratio
of the hybridization signal of a mismatched duplex (Im) to the
signal of the perfect duplex (Ip) estimated at Td in parallel for
all microchip oligonucleotides. R=Im/Ip. d.sub.19--synthetic
19-deoxymers were complementary to allele specific 10-mers
immobilized on the microchips.
[0134] Table 4 shows the effect of the position of the allelic base
within 10-mers on mutation detection. Microchip II contains two
sets of 10-mers corresponding to the normal and IVS I/2 T/G
alleles. The microchip was hybridized with the TMR-labeled normal
allele 19-mer and to an RNA 75 nt long. T.sub.0.1 is the
temperature at which the hybridization signals for a microchip
duplex drops to {fraction (1/10)} of its initial value at 0.degree.
C.-.DELTA.T.sub.0.1=T.sub.0.1 (a perfect duplex) minus T.sub.0.1
(the corresponding mismatched duplex.)
[0135] Fluorescently labeled RNA probes were prepared from a
fragment of the .beta.-globin gene from the first exon (Lawn et
al., 1980). PCR amplification of a 1.76-kb fragment of the human
.beta.-globin gene mapped from nucleotides -47 to +1714 (Lawn et
al., 1980) was carried out with _g genomic of DNA (Poncz et al.,
1982) and 50 pmol each of the forward primer:
5'GGAGCCAGGGCTGGGCATAAAAGT-3')(SEQ ID NO: 18) (-47.fwdarw.-23) and
the reverse primer 5'-ATTTTCCCAAGGTTTGAACTAGCTC-3'(S- EQ ID NO:
19)(+1689.fwdarw.+1714). (FIG. 7) The amplification was carried out
in a DNA thermal cycler (Gene Amp PCR System 2400, Perkin Elmer
Corporation in 100 .mu.l of a buffer containing 200 mM each of
dATP, dCTP, dGTP, dTTP, 2.5 MM M9Cl.sub.2, 2 units of Taq DNA
polymerase (BioMaster, Russia), 50 mM KCl, 10 mM Tris-Hcl, pH 9.0,
and 0.1% Triton X-100. The reaction conditions were 30 cycles, with
45 sec at 95.degree. C., 90 sec at 66.degree. C, and 120 sec at
72.degree. C. PCR product was purified from 2% low gel/ melting
temperature agarose gel (NuSieve agarose, FMC). The 159 bp and
102-bp DNA fragments were amplified with 10 ng of the 1.75 kb DNA
with three nested primers, two containing T7 promoter sequence and
a common reverse primer. The nested primers were T7-V2L-45.
(5'-GGAATTCCTAATACGACTCACTATAGGGACACCATGGTGCACCTGACTCC-3'(SEQ ID
NO: 5)-44.fwdarw.+66); T7-V2L-103
(5'-GGAATTCCTAATACGACTCACTATAGGGAGGT- GAACGTGGATGAAGTTGG -3'(SEQ ID
NO: 16);+102.fwdarw.-123); and 5'-TCTCCTTAAACCTGTCTTGTAACC-3' (SEQ
ID NO: 17) (common reverse; 153.fwdarw.+176). The amplification was
carried out in 25 cycles (15 sec at 95.degree. C., 30 sec at
62.degree. C., and 30 sec at 72.degree. C.). PCR products were
purified by QIAGEN QIAquick PCR Purification Kit. The PCR-amplified
159 or 102 bp DNA (4-5 .mu.g) containing T7 promoter was
transcribed with 400 units of T7 RNA polymerase (Promega) to
produce 133 and 75 nt long RNA in 80 .mu.l of buffer containing 300
mM HEPES, pH 7.6, 30 mM MgCl.sub.2, 16 _g of BSA, 40 mM DTT, 30
units of Rnasin (Promega) and 4 mM each of ATP, CTP, GTP, and UTP
for 3 h at 38.degree. C. Deproteinization of the reaction mixture
was carried out in 20 mM EGTA, pH 8.0, 2% SDS, and Proteinase K (10
mg/ml) for 15 min at 37.degree. C. The mixture was extracted first
with equal volumes of phenol and then with equal volumes of
chloroform, precipitated twice by one volume of isopropyl alcohol,
from 0.5 M LiCl0.sub.4 and dissolved on a Bio-Spin P6 column
(BioRad).
[0136] Fragmentation of 10-100 .mu.g of RNA to an average length of
20- to 40-mers was carried out in 50 .mu.l of 0.1 M KOH for 30 min.
at 40.degree. C. Then 5 .mu.l of 1M HEPES, pH 7.6, and 15 .mu.l of
1% HCO.sub.4 were added at 4.degree. C. The pellet of potassium
perchlorate was removed by centrifugation and RNA was precipitated
by 10 volumes of 2% LiClO.sub.4 in acetone. The RNA was washed
twice with acetone and dried for 20-30 min. at room temperature.
The fragmented RNA was dephosphorylated in 50 .mu.l of 20 mM
Tris-HCl, ph 8.0, 1 mM MgCl.sub.2, 1 mM ZnCl.sub.2, 10 units of
Rnasin, 5-7 units of calf intestine phosphatase (CIP) for 1 hour at
37.degree. C. RNA deproteinization and purification was carried out
as described herein.
[0137] For chemical fluorescence labeling of RNA the 3'-terminal
dephosphorylated nucleoside was oxidized in 20 .mu.l of 10 mM
sodium periodate for 20 min. at room temperature. RNA was
precipitated with acetone. A 10 molar excess of 10 mM TMR-hydrazine
in 10% acetonitrile was added to oxidized RNA fragments in 20 .mu.l
of 20 mM sodium acetate at pH 4.0.
[0138] The reaction mixture was incubated 30-40 min at 37.degree.
C., and the hydrazide bond between the RNA and dye was stabilized
by reduction with freshly prepared 1.5 .mu.l of 0.2 M NacNBH.sub.3
and incubated for 30 min. at room temperature. Then the mixture was
extracted four times with water saturated n-butanol and
precipitated with acetone. Alternatively, RNA was labeled by
incorporation of fluorescein-UTP during the transcription with
Ambion MEGAshortscript kit according to the manual.
[0139] The hybridization of fluorescently labeled RNA (1
pmol/.mu.l) with the microchips was carried out at 0.degree. C. for
18 h. In many cases, the intensities of the hybridization signals
at 0.degree. C. were similar for perfect and mismatched duplexes.
The perfect and mismatched duplexes as well as the duplexes having
various GC and AT contents displayed different stabilities and
therefore were tested at different temperatures.
[0140] Table 4 summarizes the results of hybridization of the
diagnostic microchips with 1) RNA probes derived from a number of
homozygous and heterozygous .beta.-thalassemia-patients; and 2)
with corresponding 19-mers. The table shows the Td for perfect
duplexes formed on each microchip oligonucleotide. The relative
intensities, R, of the hybridization signals for a different
microchip oligonucleotides in Table 3 are normalized to the signals
for a perfect duplex at the Td (estimated as 1.0). In most cases
the ratios for mismatched duplexes are less than 0.1 and close to
0. These values are low enough to allow unambiguous identification
of the homozygous and heterozygous mutations in patients at the Td
(when the hybridization signals from only perfect duplexes are
observed). The hybridization of homozygote RNA (Table 5, probes 1a,
2a, 2b, and 3a) with the microchip shows the distinctive formation
of a perfect duplex only with one immobilized oligonucleotide and
mismatched duplexes with all others. Two perfect duplexes were
unambiguously identified upon hybridization with a heterozygote RNA
(Table 5, probe 4a).
4TABLE 3 The sequence of the microchip allele specific 10-mers.
Position of # Allele mutated base Sequence Location MICROCHIP I 1
IVS (N) -- 5'-A TAC CAA CCT-gel +141 (SEQ ID NO: 20) 2 IVS I/1 G/A
8 5'-A TAC CAA tCT-gel +141 (SEQ ID NO: 21) 3 IVS I/1 G/T 8 5'-A
TAC CAA aCT-gel +141 (SEQ ID NO: 22) 4 IVS I/2 T/A 7 5'-A TAC Cat
CCT-gel +141 (SEQ ID NO: 23) 5 IVS I/2 T/C 7 5'-A TAC Cag CCT-gel
+141 (SEQ ID NO: 24) 6 IVS I/2 T/G 7 5'-A TAC Cac CCT-gel +141 (SEQ
ID NO: 25) 7 IVS I/5 G/A 4 5'-A TAt CAA CCT-gel +141 (SEQ ID NO:
26) 8 IVS I/5 G/C 4 5'-A TAg CAA CCT-gel +141 (SEQ ID NO: 27) 9 IVS
I/5 G/T 4 5'-A TAa CAA CCT-gel +141 (SEQ ID NO: 28) 10 IVS I/6 T/C
3 5'-A TgC CAA CCT-gel +141 (SEQ ID NO: 29) 11 CD 26 (N) -- 5'-G
GCC TCA CCA-gel +125 (SEQ ID NO: 30) 12 CD 26 G/A 6 5'-G GCC TtA
CCA-gel +125 (SEQ ID NO: 31) MICROCHIP II 1 IVS (N) 9 5'-TGA TAC
CAA C-gel +143 (SEQ ID NO: 32) 2 IVS I/2 T/G 9 5'-TGA TAC CAc C-gel
+143 (SEQ ID NO: 33) 3 IVS (N) 8 5'-GA TAC CAA CC-gel +142 (SEQ ID
NO: 34) 4 IVS I/2 T/G 8 5'-GA TAC CAc CC-gel +142 (SEQ ID NO: 35) 5
IVS (N) 7 5'-A TAC CAA CCT-gel +141 (SEQ ID NO: 36) 6 IVS I/2 T/G 7
5'-A TAC Cac CCT-gel +141 (SEQ ID NO: 37) 7 IVS (N) 6 5'-TAC CAA
CCT G-gel +140 (SEQ ID NO: 38) 8 IVS I/2 T/G 6 5'-TAC CAc CCT G-gel
+140 (SEQ ID NO: 39) 9 IVS (N) 5 5'-AC CAA CCT GC-gel +139 (SEQ ID
NO: 40) 10 IVS I/2 T/G 5 5'-AC CAc CCT GC-gel +139 (SEQ ID NO: 41)
11 IVS (N) 4 5'-C CAA CCT GCC-gel +138 (SEQ ID NO: 42) 12 IVS I/2
T/G 4 5'-C CAc CCT GCC-gel +138 (SEQ ID NO: 43) 13 IVS (N) 3 5'-CAA
CCT GCC-gel +137 (SEQ ID NO: 44) 14 IVS I/2 T/G 3 5'-CAc CCT GCC
C-gel +137 (SEQ ID NO: 45) 15 IVS (N) 2 5'-AA CCT GCC CA-gel +136
(SEQ ID NO: 46) 16 IVS I/2 T/G 2 5'-Ac CCT GCC CA-gel +136 (SEQ ID
NO: 47)
[0141]
5TABLE 4 The effect of the position of the allele base within
10-mers on mutation detection. 19-mer RNA Position T.sub.0.1 of
T.sub.0.1 of allele perfect T.sub.0.1 of (G-A) .DELTA.T.sub.0.1
perfect T.sub.0.1 of (G-A) .DELTA.T.sub.0.1 9 40 32 8 35 37 -2 8 47
32 15 49 38 11 7 42 30 12 44 41 3 6 47 28 19 49 41 8 5 52 38 14 50
42 8 4 54 39 15 54 44 10 3 55 46 9 59 54 5 2 52 46 6 58 53 5
[0142]
6TABLE 5 Identification of .beta.-thalassemia mutations by
hybridization with the microchip Immobillized 10-mer
Oligonucleotide IVS I/1 I/1 I/2 I/2 I/2 I/5 I/5 I/5 I/6 CD26 CD26
Hybridized Probe (N) G/A G/T T/A T/C T/G G/A G/G G/T T/C (N) G/A
Size R at Td = # Allele (nt) 42.degree. C. 39.degree. C.
38.5.degree. C. 42.degree. C. 48.degree. C. 45.5.degree. C.
37.degree. C. 44.5.degree. C. 40.degree. C. 50.degree. C.
54.5.degree. C. 49.degree. C. 1 a IVS(N) 75 1.00 0.04 0 0.2 0.05
0.07 0 0 0 0.04 1.0 -- b IVS(N) 19.sup.a 1.00 0.09 0.07 0.02 0.03
0.01 0.03 0.03 0.07 ND 0 0 2 a IVS I/2 F1 0.15 0 0 1.00 0.12 0.08 0
0 0 0 1.00 -- T/A 75 b IVS I/2 F1 0.03 0 0 1.00 0 0.30 0 0 0 0 1.00
0.19 TA 133 c IVS I/2 19.sup.a 0.01 0 0 1.00 0.07 0.03 0 0 0 0 0.01
0 TA 3 a IVS I/1 133.function.r 0.2 0.85 0 0.2 0 0.05 0 0 0 1.00
1.00 -- G/A b IVS I/1 19.sup.c 0.01 1.00 0.01 0 0 0.01 0 0 0 0 0 0
G/A 4 a IVS I/1 133.function.r 0.2 0.85 0 0.2 0 0.05 0 0 0 1.00
1.00 -- G/A & IVS I/6 T/C b IVS I/1 19.sup.c 0.01 1.00 0.01
0.01 0 0 0.01 0 0 0 0 0 G/A c IVS I/6 19.sup.a 0.1 0 0 0 0 0 0 0 0
1.0 0 0 T/C 5 a IVS I/5 19.sup.a 0 0 0 0 0 0 0.03 0.02 1.00 0 0 0
G/T b CD26 19.sup.c 0 0 0 0 0 0 0 0 0 0 1.00 0.03 (N) c CD26
19.sup.a 0.03 0 0 0 0 0 0 0 0 0 0.04 1.00 G/A 6 a IVS (N) 75 1.00
0.04 0 0.20 0.05 0.07 0 0 0 0.04 1.00 -- b IVS I/2 F1 0.03 0 0 1.00
0 0.30 0 0 0 0 1.00 -- T/A 133
[0143] The noticeable exceptions are oligonucleotides corresponding
to IVS I/2 T/A and IVS I/2 T/G mutations that show strong
mismatched signals upon hybridization with non-corresponding
samples of IVS (N) and IVS I/2 T/A RNA's, respectively (Table 5,
1a, 2b, 4a, 6a and 6b). The relative intensities of these
mismatched signals can be significantly decreased by choosing the
proper oligonucleotides for immobilization. It appears that the
diagnostic assays can be carried out with RNAs 75 nucleotides (nt)
long (Table 5, probes 1a, and 6a), and 133 nt long (probes 2a and
6b), as well as with 133 nt long RNA fragmented to pieces 20-40 nt
long (probes 3a and 4a). However, the intensities of the
hybridization signals after fragmentation are increased by about 5
times and the time of hybridization is decreased from several hours
to a tens of minutes.
[0144] The longer RNA probes diffuse more slowly into the gel and
can form stable secondary structures or aggregates. These factors
interfere with their hybridization with rather short immobilized
oligonucleotides. Thus, the fragmentation seems to be an essential
step in sample preparation, since it enhances and speeds the
hybridization.
[0145] In addition to the measuring of the melting curves, the
reliability of identification of mutations and base changes can be
enhanced by the use of a multicolor fluorescence microscope
(Yershov et al., 1996). For this purpose, the tested RNA is marked
by one fluorescence label and is hybridized with a microchip in the
presence of a normal allele sample labeled with a different dye.
The pattern and the ratio of hybridization measured with the two
dyes will be similar for all microchip oligonucleotides except for
those that correspond to different allele bases, i.e., mutations.
Table 4 shows the results of such an experiment. The patterns of
hybridization detected at two wavelengths are very similar.
[0146] As shown in Table 3, the immobilized 10-mers matching the
mutations IVS I-2 T/G, IVS I-2 T/C, and IVS I-2 T/A are hybridized
rather strongly with some RNA probes that correspond to other
alleles. Different structural factors in RNA could cause this
hybridization. The effect of these factors can be minimized by
placing a variable IVS I-2 base into different positions of the
10-mers. The results of such experiments are shown in Table 4.
Microchip II was successively hybridized with fragmented 75-nt-long
RNA or with a synthetic DNA 19-mer, both corresponding to, the
normal allele. Microchip II contained two similar sets of eight
overlapped immobilized 10-mers that are complementary either to a
normal allele or to IVS I-2 T/G allele. The allele specific bases A
for the first set and C for the second set are located in these
10-mers in all internal positions from the 2.sup.nd to the
9.sup.th. These bases form perfect A-T or mismatched A-G base
pairs, respectively. The stability of the perfect and mismatched
duplexes formed on the microchip is determined as T.sub.0.1 the
temperature at which the initial hybridization signal of the duplex
is decreased to one-tenth of the original intensity.
.DELTA.T.sub.0.1 corresponds to the difference in T.sub.0.1 between
the perfect and similar mismatched duplexes. A better
discrimination of the perfect and mismatched duplexes is reflected
in higher values of .DELTA.T.sub.0.1. The discrimination efficiency
(.DELTA.T) was lower for hybridized RNAs than for the 19-mers. The
discrimination was surprisingly low, .DELTA.T=-2.degree. and
3.degree. C., when the allelic bases were placed at the 9.sup.th or
7.sup.th position, respectively, of the immobilized
oligonucleotides. It appears that secondary structures and the
presence of similar sequences in other regions of the RNA causes
this lowering. These effects can be partly predicted from the
sequence of the region that is searched for mutations. However, it
is impossible to reach a high discrimination (.DELTA.T=8-11.degree.
C.) when allele bases are placed in other positions, for example
the 8, 6, 5, or 4 positions.
[0147] The hybridization of RNA transcripts of PCR-amplified DNA
with oligonucleotide microchips allows the reliable identification
of base changes and discrimination of homozygous and heterozygous
.beta.-thalassemia mutations in the genomic DNA of patients.
[0148] RNA transcribed from PCR-amplified DNA provides an easier
method for preparing a sufficient amount of labeled,
single-stranded samples than the use of DNA prepared by PCR
amplification. RNA can be fragmented and one fluorescent dye
molecule can be introduced per fragment.
Example 7
[0149] Use of a Customized Microchip Biosensor to Detect Gene
Expression
[0150] Gene expression is one of the central themes in modem
molecular biology. DNA from well studied genetical sources has
already been systematically sequenced. For these sequences
hybridization procedures are successfully used to estimate a level
of differential gene expression. The results of this estimation are
useful for understanding fundamental mechanisms of development
biology, embryology and treatment of genetic and infectious
diseases.
[0151] To determine whether oligonucleotide microchips are useful
to identify gene expression, microchip biosensor hybridization was
carried out with ssDNA fragments isolated from six different
genes:
[0152] 205 b fragment from glyceraldehyde 3-phosphate dehydrogenase
(G3PDH);
[0153] 281 b fragment from human transferrin receptor (HTR);
[0154] 224 b fragment from human .beta..sub.2-microglobulin
(B2M);
[0155] 545 b fragment from human interleukin-1 receptor (IL1R);
[0156] 188 b fragment from human NF-kB (p50);
[0157] 224 b fragment from human interferon .gamma. receptor
(IGR).
[0158] A customized microchip, containing immobilized 60 b
oligonucleotides, having at the 3'-terminal position
3-methyluridine residues, corresponding to five housekeeping genes
(G3PDH, HTR, B2M, IL1R and NF-kb(p50)) (CLONTECH catalog 94/95
"Tools for the Molecular Biologist", pp. 90-93) were produced for
hybridization experiments with complementary ssDNA fragments. Each
oligonucleotide was applied at two positions on the microchip in a
1:10 ratio of amount (0.3 pmol:0.03 pmol each). ssDNA fragments
complementary to immobilized oligonucleotides were synthesized by
asymmetric PCR amplification (using only one primer) with
fluorescently labeled nucleotide triphosphates (FUORscript T7,
Fluorescein-Labeling In vitro Transcription Kit). Moreover, the
PCR-primer bore a biotin tag that was utilized for following
isolation of synthesized ssDNA fragments with avidin carried on a
column (Sambrook et al. "Molecular Cloning" 2d edit., p. 12.14).
FIG. 8 demonstrates hybridization on the microchip. Intensity of
fluorescence in each spot depends on the amount of immobilized
oligonucleotide and on the length of the DNA fragment in the spot.
For hybridization, 10 .mu.l of Buffer A (50% formamide, 10% dextran
sulfate, 1% SDS, 50 mM sodium phosphate at pH 7.4, 750 mM sodium
chloride, 5 mM sodium EDTA) containing ssDNA with a concentration
of 0.5 pmol/.mu.l (approximately 0.05 .mu.g/.mu.l) was incubated
for about 6-12 h at room temperature, washed briefly with H.sub.2O
and analyzed with a fluorescent microscope. Before rehybridization
the microchip was treated in Buffer B (50% formamide, 1% Tween 20)
for 30 min. at 50.degree. C. to completely remove hybridized
ssDNA.
[0159] These results indicate that concentration of fluorescently
labeled ssDNA may be decreased up to 100 fold. Hybridization with
individual ssDNA fragments indicates high specificity of studied
oligonucleotides. There was no cross-hybridization detected between
different tested DNAs and immobilized oligonucleotides. None of the
oligonucleotides demonstrated a signal when hybridized with
non-specific DNA (e.g. probe IGR). This differentiates "expression"
of non-expressed genes from expression of housekeeping genes. Genes
that are not expressed in a particular cell or tissue, may actually
be picked up in conventional screening procedures as having a low
expression, while other genes being expressed in all cells
(housekeeping genes) will also be picked up as having low to
moderate expression. The housekeeping genes are actually being
expressed. In this example a difference in signal is detectable so
that low level expression could be unambiguously distinguished from
low level background.
[0160] The procedure detects expression of genes of high and middle
expression level. To determine low level gene expression selective
RT-PCR amplification is preferred.
Example 8
[0161] Use of a Customized Microchip Biosensor of the Present
Invention to Detect HLA Polymorphisms
[0162] A difficult problem of genotype recognition arises in
studying different haplotypes (alleles) of genes encoding Human
Leucocyte Antigens (HLA) in regions of histocompatibility genes.
The HLA locus (class I and class II genes) is responsible for
histocompatability of tissue transplantation. The need for allele
identification is encountered also in various medical and
biological tasks involving HLA class II genes. There are many
clinical data showing strong association between HLA genotype and
susceptibility to some disorders, for example some alleles
DQA1/DQB1 are clearly related to IDDM (Insulin--Dependent Diabetes
Mellitus), malaria, autoimmune diseases, such as rheumatoid
arthritis and pemphigus vulgaris--a skin disease which causes
severe blistering. The high level of polymorphism of HLA has been
shown to be useful for identification of individuals determining
the group of risk for some diseases. HLA typing is particularly
crucial for matching donors for transplants. It is also proposed
for infertility work-ups.
[0163] In this aspect, the present invention provides a method
which allows an array of immobilized 8-12 bp-long oligonucleotides
to form an oligonucleotide microchip thereby facilitating
identification of HLA DQA1 allies.
[0164] An algorithm has been designed and special computer programs
have been constructed which allow the analysis of the nucleotide
sequences of all alleles of various HLA subloci. Forming an
optimized set of oligonucleotides provides high reliability of
detection of homo- and heterozygotes for the HLA alleles.
[0165] A customized microchip, containing an array of eighteen
PAA--gel immobilized (1 pmol of each) short oligonucleotides has
been produced for hybridization with fluorescently labelled
complementary HLA DQA1 DNA or RNA probe for allele identification.
18 decamers were loaded on the chip in the following order, from
left to right:
7 TABLE 6 first (upper) row 1 2 3 4 g.sup.4-control oligo second: 5
6 7 8 g.sup.3-control oligo third: 9 10 11 12 13 4-th: 14 15 16 17
18
[0166] The sequence of the oligos used was as shown in FIG. 9.
[0167] The oligonucleotides immobilized on the microchip are
complementary to the sense strand of different alleles of DQA1 DNA
and some control oligos. A microchip with 20 oligonucleotides was
manufactured for partial identification of 15 different alleles in
the HLA DQA1 region. PCR was used to prepare 229 bp (starting from
condon 12 to condon 87) DNA fragments of the polymorphic second
exon of the DQA1 gene from human genomic DNA. Nested primers were
used 2DQAAMP--A:5'-a t ggt gta aac ttg tac cag t (SEQ ID NO: 73);
and 2DQAAMP--B:5'tt ggyt agc agc ggt aga gtt g (SEQ ID NO: 74).
Nested PCR primers were: T7-2DQAAMP-A and primer B. The first
primer containing the promoter for T7 RNA polymerase and PCR
product were used for in vitro transcription. RNA probes were
identical to the coding DNA strand. RNA was fragmented, labeled
with fluorescein and used for hybridization with the microchip.
Hybridization conditions were as follows:
[0168] overnight incubation at 5.degree. C. in 1M NaCl, 1 mM EDTA,
5 mM Na-phosphate, pH7.0, 1% Tween 20. The temperature was then
increased stepwise at 10.degree. C. intervals, and fluorescence
measurements were taken at each step. BUFFER WAS NOT CHANGED.
[0169] FIG. 10 shows the hybridization results and presents
schematically the HLA DQA1--chip for allele identification. In FIG.
10 three diagonally placed oligos (11-0101/0104 allele specific;
6-specific for 0101,01021,01022,0103,00104; 17--correspond to all
alleles, except 0502) gave a positive hybridization signal, and are
observed as three diagonally placed bright fluorescence spots. The
probes were identified as the 0101 or 0104 allele (both alleles are
identical in the second exon). All other oligos yielded much weaker
fluorescence signals compared with those described above, because
none of them contain sequences complementary to alleles 0101 and
0104. On the other hand any allele different from 0101 or 0104,
reveals another set of hybridization signals.
[0170] The brightest fluorescent squares on the chip were: Oligo# 4
which is 03011 or 0302 specific; oligo# 8 is Taq
polymerase-specific artifacts; oligo# 18--belong to alleles
0101-05011; oligo 11-0101,0104 allele specific; 17--corresponds to
all alleles, except 0502; g4 is a fluorescent control
oligo;#13--mismatch to #18. All other chip elements showed
significantly less intensive fluorescence. The genotype identified
by these probes has a 0101/0104-0302/03011 heterozygote.
Example 9
[0171] Use of a Customized Microchip Biosensor to Detect the Lyme
Disease Spirochetes
[0172] Bacteria belonging to the species Barretia burgdorferi and
related species of tick-borne spirochetes are capable of causing
human and veterinary disease. Nucleic acid probes are available to
detect bacteria causing Lyme disease. These bacteria cannot be
identified by standard microbiological methods, although
immunological tests are available.
[0173] Using the methods of the present invention, oligonucleotides
are prepared according to Weisburg (1995) and added to a
micromatrix designed for use in detecting Lyme disease in a
clinical sample.
Example 10
[0174] Use of a Customized Microchip Biosensor to Detect Salmonella
In Food Samples
[0175] Salmonella presence is detected most commonly by preparing
cultures according to standard microbiological laboratory
procedures, and testing the cultures for morphological and
biochemical characteristics. After about 48 hours after collection
of a sample testing begins and takes several days to complete.
[0176] However, RNA and DNA probes for Salmonella testing are
available. Using the methods of the present invention,
oligonucleotides are prepared according to Lane et al. (1996)
incorporated herein by reference and added to a microchip designed
for use in detecting Salmonella in food samples by distinguishing
rRNA of Salmonella from non-Salmonella.
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* * * * *