U.S. patent application number 11/719582 was filed with the patent office on 2012-09-13 for real-time pcr of targets on a micro-array.
This patent application is currently assigned to EPPENDORF ARRAY TECHNOLOGIES. Invention is credited to Isabelle Alexandre, Dieter Husar, Sylvain Margaine, Jose Remacle.
Application Number | 20120231962 11/719582 |
Document ID | / |
Family ID | 34927448 |
Filed Date | 2012-09-13 |
United States Patent
Application |
20120231962 |
Kind Code |
A9 |
Remacle; Jose ; et
al. |
September 13, 2012 |
REAL-TIME PCR OF TARGETS ON A MICRO-ARRAY
Abstract
The present invention relates to a method and apparatus for
monitoring on a micro-array a PCR amplification of a nucleotide
molecule being present in a solution. The method includes the steps
of: providing a support having fixed upon its surface a microarray
having at least a capture molecule being immobilized in
specifically localized areas of the support and a reaction chamber;
introducing a solution containing the nucleotide molecule into the
reaction chamber and reagents for nucleotide molecule amplification
and labelling; submitting the solution to at least 2 thermal cycles
having at least 2 and preferably 3 different temperature steps in
order to obtain labelled target nucleotide molecule by PCR
amplification; performing at least a measurement of the labelled
target nucleotide molecule in at least one thermal cycle by
incubating the labelled target nucleotide molecule under conditions
allowing a specific binding between the target nucleotide molecule
and its corresponding capture molecule and measuring the light
emission from the bound labelled target nucleotide molecule in
response to excitation light with the solution being present in the
chamber and containing the labelled target nucleotide molecule. The
surface of emission for a localized area is between about 0.1
.mu.m.sup.2 and about 75 mm.sup.2. The method further includes
processing the data obtained in at least one thermal cycle in order
to detect and/or quantify the amount of nucleotide molecule present
in the solution before the amplification.
Inventors: |
Remacle; Jose; (Malonne,
BE) ; Alexandre; Isabelle; (Namur, BE) ;
Margaine; Sylvain; (Namur, BE) ; Husar; Dieter;
(Hamburg, DE) |
Assignee: |
EPPENDORF ARRAY
TECHNOLOGIES
Namur
BE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20090156415 A1 |
June 18, 2009 |
|
|
Family ID: |
34927448 |
Appl. No.: |
11/719582 |
Filed: |
November 18, 2005 |
PCT Filed: |
November 18, 2005 |
PCT NO: |
PCT/EP05/12383 PCKC 00 |
371 Date: |
February 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10991087 |
Nov 18, 2004 |
|
|
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11719582 |
|
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Current U.S.
Class: |
506/9 ;
506/39 |
Current CPC
Class: |
C12Q 1/6837 20130101;
C12Q 1/6837 20130101; C12Q 2563/107 20130101 |
Class at
Publication: |
506/9 ;
506/39 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 60/12 20060101 C40B060/12 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 18, 2004 |
EP |
04027435.9 |
Claims
1. A method for monitoring on a micro-array a PCR amplification of
a nucleotide molecule being present in a solution comprising the
steps of: providing a support having fixed upon its surface a
micro-array comprising at least a capture molecule being
immobilized in specifically localized areas of said support and a
reaction chamber, introducing a solution containing said nucleotide
molecule into said reaction chamber and reagents for nucleotide
molecule amplification and labelling, submitting the solution to at
least 2 thermal cycles having at least 2 different temperature
steps in order to obtain labelled target nucleotide molecule by PCR
amplification, performing at least a measurement of the labelled
target nucleotide molecule in at least one thermal cycle in the
following way, incubating said labelled target nucleotide molecule
under conditions allowing a specific binding between said target
nucleotide molecule and its corresponding capture molecule,
measuring the light emission from the bound labelled target
nucleotide molecule in response to excitation light with said
solution being present in the chamber and containing the labelled
target nucleotide molecule, wherein the surface of emission for a
localized area is comprised between about 0.1 .mu.m and about 75
mm, and processing data obtained in at least one thermal cycle in
order to detect and/or quantify an amount of nucleotide molecule
present in the solution before the amplification.
2. The method of claim 1, wherein a measurement of the labelled
target nucleotide molecule is performed in at least 5 thermal
cycles.
3. The method of claim 1, wherein the light emission is measured at
a defined timing from the beginning of a temperature step.
4. The method of claim 1, wherein the light emission is measured at
within 5 min after the beginning of the annealing temperature
step.
5. The method of claim 1, wherein the light emission is measured at
the end of at least one of the 3 temperature steps used for the PCR
amplification.
6. The method of claim 1, wherein the light emission is measured at
the end of the PCR amplification.
7. The method of claim 1, wherein the data are processed in order
to obtain a signal value for each of the localized area.
8. The method of claim 1, wherein the data are processed in order
to obtain a signal value for each of the localized area and for the
local background.
9. The method of claim 8, wherein the data are further processed by
subtracting the background from the signal value for each of the
localized area.
10. The method of claim 1, wherein the quantification of the amount
of nucleotide molecule is performed by comparing the signal value
of the localized area with a fixed value.
11. The method of claim 1, wherein the quantification of the amount
of nucleotide molecule is performed by comparing the number of
thermal cycles necessary to reach a fixed value (CT) with the CT of
a reference nucleotide molecule.
12. The method of claim 11, wherein the reference nucleotide
molecule is amplified in the same solution and detected on the same
micro-array as the target nucleotide molecule.
13. The method of claim 1, wherein the quantification of the amount
of nucleotide molecule is performed by comparing the number of
thermal cycles necessary to reach a fixed value (CT) with a
standard curve wherein the CT are plotted against standard
concentrations.
14. The method of claim 1, wherein the reagents for nucleotide
molecule amplification comprise a primer pair, dNTPs, a
thermostable DNA polymerase and buffer.
15. The method of claim 1, wherein the reagents for nucleotide
molecule amplification comprise a primer and/or dNTP labelled with
a fluorescent dye.
16. The method of claim 1, wherein two fluorescent dyes are used in
the same solution.
17. The method of claim 1, wherein the solution composition is
adapted for performing the annealing of the primers on the
nucleotide molecule and the specific binding of the labelled target
molecule on the capture molecule during the same temperature
step.
18. The method of claim 1, wherein the capture molecules bound to
the labelled target nucleotide molecules are elongated during the
temperature step of elongation.
19. The method of claim 18, wherein the capture molecules elongated
are detected during the temperature step of denaturation.
20. The method of claim 1, wherein the localized area is comprised
between about 10 .mu.m.sup.2 and about 1 mm.sup.2.
21. The method of claim 1, wherein the micro-array comprises more
than 5 different capture molecules.
22. The method of claim 1, wherein between 1 and 4 nucleotide
molecules present in a solution are amplified and detected and/or
quantified in the same assay.
23. The method of claim 1 wherein between 20 and 1000 nucleotide
molecules present in a solution are amplified and detected and/or
quantified in the same assay.
24. The method of claim 1, wherein the support contains a substrate
on which are fixed the capture molecules.
25. The method of claim 1, wherein the support bears several
micro-arrays separated by physical boundaries.
26. The method of claim 25, wherein the support has a multi-well
plate or strip format.
27. The method of claim 26, wherein the multi-well plate is
submitted to a temperature gradient during the measurement of light
emission.
28. The method of claim 1, wherein an excitation light from a light
source is directed on the surface of the support.
29. The method of claim 1, wherein a thermal cycle is performed
within 10 min.
30. The method of claim 1, wherein 30 thermal cycles are performed
within 5 h.
31. The method of claim 1, wherein the capture molecule is a single
stranded polynucleotide containing a sequence able to specifically
bind the labelled target nucleotide molecule and a spacer of at
least 20 nucleotides.
32. An apparatus for monitoring on a micro-array a PCR
amplification of a nucleotide molecule being present in a solution
comprising: a support having fixed upon its surface a micro-array,
comprising at least one capture molecule being immobilized in
specifically localized areas of said support, which is in fluid
communication in a chamber with said nucleotide molecule and
reagents for nucleotide molecule amplification and labelling, a
thermal cycler for carrying out an automated PCR process, said
thermal cycler capable of alternately heating and cooling said
support for producing labelled target nucleotide molecule, an
excitation light source, a detector for measuring the
electromagnetic light emission from the bound labelled target
nucleotide molecule in response to said excitation light with said
solution being present in the chamber and containing the labelled
target nucleotide molecule wherein the surface of emission for a
localized area is comprised between about 0.1 .mu.m.sup.2 and about
75 mm.sup.2, wherein the different parts are integrated into the
same apparatus in order to read the light emission of the bound
labelled target nucleotide molecule during the PCR
amplification.
33. The apparatus of claim 32, further comprising: a storage system
for storing the data of the different measurements for at least 5
localized areas of the support at a defined timing of a thermal
cycle, a controller repeating the steps of excitation, detection
and storage at least one time in at least one thermal cycle for
each localized area of the micro-array, a program for processing
data obtained in at least one thermal cycle in order to detect
and/or quantify an amount of nucleotide molecule present in the
sample before the amplification.
34. The apparatus of claim 32, wherein the support contains a
substrate on which are fixed the capture molecules.
35. The apparatus of claim 32, wherein the micro-array comprises
more than 5 different capture molecules.
36. The apparatus of claim 32, wherein the heating and cooling is
performed at a ramping of 5.degree. C. per min.
37. The apparatus of claim 32, wherein the localized area is
comprised between about 10 .mu.m.sup.2 and about 1 mm.sup.2.
38. The apparatus of claim 32, further comprising: an optical
system for directing and focusing an excitation light from said
excitation light source directly on said support, wherein the
excitation light reaches the micro-array surface within an angle
comprised between 45 and 135.degree..
39. A diagnostic kit for monitoring on a micro-array a PCR
amplification of a nucleotide molecule being present in a solution
comprising: a support having fixed upon its surface a micro-array,
comprising at least one capture molecule being immobilized in
specifically localized areas of said support wherein the surface of
said support is maintained flat at temperature higher than
85.degree. C. and wherein said support have a low
self-fluorescence, a reaction chamber comprising 2 parts being in
fluid connection to each other comprising a fiat surface carrying
the micro-array.
40. The diagnostic kit of claim 39, further comprising: dNTPs a
thermostable DNA polymerase and buffer.
41. The diagnostic kit of claim 39, further comprising: dNTPs a
thermostable DNA polymerase, buffer and primers.
42. The diagnostic kit of claim 39, further comprising a nucleotide
molecule being used as an internal standard.
43. The diagnostic kit of claim 39, wherein the support and the
reaction chamber are part of a cartridge.
44. The method of claim 15, wherein the fluorescent dye is selected
from the group consisting of Cyanin 3, Cyanin 5 and Cyanin 7.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method and an apparatus
and diagnostic kit for monitoring a polymerase chain reaction (PCR)
for nucleic acid amplification over multiple thermal cycles on
capture molecules fixed on a micro-array. More particularly, the
invention allows the detection and quantification of nucleotide
molecule in a real time PCR amplification using micro-arrays. The
invention also comprises means and apparatus for performing the
method.
DESCRIPTION OF THE RELATED ART
[0002] The disclosed nucleotide molecule detection method offers
the advantages of speed, simplicity and multiplexing over prior
methods for detecting amplified nucleic acids. Nucleic acid
detection techniques in general are very useful in medical
diagnostic assays.
[0003] The sensitivity and specificity of nucleic acid detection
methods was greatly improved by the invention of the polymerase
chain reaction (PCR). PCR is a process for amplifying nucleic acids
and involves the use of two oligonucleotide primers, an agent for
polymerization, a target nucleic acid template, and successive
cycles of denaturation of nucleic acid and annealing and extension
of the primers to produce a large number of copies of a particular
nucleic acid segment. With this method, segments of single copy
genomic DNA can be amplified more than 10 million fold with very
high specificity and fidelity. Methods for detecting PCR products
are described in U.S. Pat. No. 4,683,195. Those methods require an
oligonucleotide probe capable of hybridizing with the amplified
target nucleic acid. These methods require separate steps of
amplification, capture, and detection and generally require several
hours to complete.
[0004] Due to the enormous amplification possible with the PCR
process, small levels of DNA carryover from samples with high DNA
content, positive control templates, or from previous
amplifications, can result in PCR product even in the absence of
purposefully added template DNA. Because the possibility of
introducing contaminating DNA to a sample will be increased as the
amount of handling steps required for sample preparation,
processing, and analysis is increased, it would be preferable to
minimize sample handling for their detection and quantification,
particularly after the amplification reaction is complete.
[0005] Methods for simultaneous amplification and detection of
target nucleic acids have been described in order to minimize the
problems of sample contamination. The U.S. Pat. No. 4,683,195 and
U.S. Pat. No. 6,171,785 involve the introduction of detectable DNA
binding agents (such as ethidium bromide) into the amplification
reaction, which agents produce a detectable signal that is enhanced
upon binding double-stranded DNA. An increased in fluorescence of
the PCR mixture indicates that amplification has occurred. The U.S.
Pat. No. 6,395,518 and U.S. Pat. No. 5,952,202 discloses an
oligonucleotide probe including a fluorescer molecule attached to a
first end of the oligonucleotide and a quencher molecule attached
to the opposite end of the oligonucleotide such that the fluorescer
is substantially unquenched whenever the oligonucleotide probe is
in a double-stranded state. A DNA polymerase having 5' to 3'
nuclease activity digests said probes during amplification to
separate the reporter dye from the quencher. An increased in
fluorescence of the PCR mixture indicates that amplification has
occurred. The U.S. Pat. No. 5,716,784 provides an alternative
method based on the use of two complementary probes, the first
analytical probe being labelled at its 5' terminus with an energy
transfer donor fluorophore, and the second detection probe being
labelled at its 3' terminus with an energy transfer acceptor
fluorophore. Measurement of oligonucleotide analytical probe
hybridized in solution to oligonucleotide detection probe measured
spectrophotometrically in solution by energy transfer measurement,
provides a measure of the amount of oligonucleotide analytical
probe used up in the amplification of the target nucleic acid
sequence and thus provides a measure of amount of target nucleic
acid sequence amplified in the PCR replication procedure. The U.S.
Pat. No. 5,928,907 describes an apparatus for monitoring the
formation of a nucleic acid amplification reaction product in real
time which uses a fiber optic focused in the volume of the
sample.
[0006] Although those methods are capable of monitoring in real
time the quantification of nucleic acids in an homogeneous PCR
hybridization system, they are limited to the quantification of one
target nucleic acid per fluorescent dye. The multiplexing is not
easy to implement due to the requirement of non overlapping
fluorescent dyes for measuring the increase in signal related to
the amplification of several target nucleic acids in the same
apparatus.
[0007] A problem underlying the present invention resides in
providing an improved method for monitoring a PCR in real-time in
heterogeneous system, obviating the shortcomings associated with
prior art methods. Specifically, the method should be simple to
carry out and cost effective.
[0008] The present invention aims to overcome most of these
limitations by proposing a simple and effective method and
apparatus for the simultaneous amplification of multiple target
molecules on a micro-array.
SUMMARY OF THE INVENTION
[0009] In order to realize the above-mentioned objectives, the
method for monitoring on a microarray a PCR amplification of a
nucleotide molecule being present in a solution comprises the steps
of: [0010] providing a support (15) having fixed upon its surface a
micro-array comprising at least a capture molecule (20) being
immobilized in specifically localized areas (21) of said support
and a reaction chamber (14), [0011] introducing a solution
containing said nucleotide molecule into said reaction chamber (14)
and reagents for nucleotide molecule amplification and labelling,
[0012] submitted the solution to at least 2 thermal cycles having
at least 2 and preferably 3 different temperature steps in order to
obtain labelled target nucleotide molecule (13) by PCR
amplification, [0013] performing at least a measurement of the
labelled target nucleotide molecule in at least one thermal cycle
in the following way, [0014] incubating said labelled target
nucleotide molecule (13) under conditions allowing a specific
binding between said target nucleotide molecule (13) and its
corresponding capture molecule (20), [0015] measuring the light
emission (7) from the bound labelled target nucleotide molecule in
response to excitation light (2) with said solution being present
in the chamber and containing the labelled target nucleotide
molecule (13), wherein the surface of emission for a localized area
is comprised between about 0.1 .mu.m.sup.2 and about 75 mm.sup.2,
and [0016] Processing the data obtained in at least one thermal
cycle in order to detect and/or quantify the amount of nucleotide
molecule present in the solution before the amplification.
[0017] The apparatus for monitoring on a micro-array a PCR
amplification of a nucleotide molecule being present in a solution
according to the present invention comprises: [0018] a support (15)
having fixed upon its surface a micro-array, comprising at least
one capture molecule (20) being immobilized in specifically
localized areas (21) of said support, which is in fluid
communication in a chamber with said nucleotide molecule and
reagents for nucleotide molecule amplification and labelling,
[0019] a thermal cycler for carrying out an automated PCR process,
said thermal cycler capable of alternately heating and cooling said
support for producing labelled target nucleotide molecule, [0020]
an excitation light source (1), [0021] a detector (10) for
measuring the electromagnetic light emission (7) from the bound
labelled target nucleotide molecule in response to said excitation
light with said solution being present in the chamber and
containing the labelled target nucleotide molecule wherein the
surface of emission for a localized area is comprised between about
0.1 .mu.m.sup.2 and about 75 mm.sup.2, [0022] wherein the different
parts are integrated into the same apparatus in order to read the
light emission of the bound labelled target nucleotide molecule
during the PCR amplification.
[0023] The apparatus further comprises: [0024] a storage system for
storing the data of the different measurements for at least 5
localized areas of the support at a defined timing of a thermal
cycle, [0025] a controller (11) repeating the steps of excitation,
detection and storage at least one time in at least one thermal
cycle for each localized area of the micro-array, [0026] a program
for processing the data obtained in at least one thermal cycle in
order to detect and/or quantify the amount of nucleotide molecule
present in the sample before the amplification.
[0027] The invention also comprises a diagnostic kit for monitoring
on a micro-array a PCR amplification of a nucleotide molecule being
present in a solution comprising: [0028] a support (15) having
fixed upon its surface a micro-array, comprising at least one
capture molecule (20) being immobilized in specifically localized
areas (21) of said support wherein the surface of said support is
maintained flat at temperature higher than 85.degree. C. and
wherein said support have a low self-fluorescence, [0029] a
reaction chamber comprising 2 or even better 3 parts being in fluid
connection to each other comprising a flat surface carrying the
micro-array.
DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1. General scheme of the integrated apparatus
comprising the support (15) a carrier (12), temperature regulating
device (16) and a temperature controlling device (17) and the
detector (10).
[0031] FIG. 2. Schematic description of the online detection of PCR
product on micro-array using labelled primer. PCR is performed in
the presence of a micro-array comprising different capture
molecules. Alternate steps of annealing, elongation and
denaturation during one cycle of reaction result in the
accumulation of labelled products which hybridize on their capture
molecule present on the micro-array but deshybrizes from their
specific capture molecules after each denaturation cycle.
[0032] FIG. 3. Schematic description of the online detection of PCR
product on micro-array using labelled dNTPs. PCR is performed in
the presence of a micro-array comprising different capture
molecules. Alternate steps of annealing, elongation and
denaturation during one cycle of reaction result in the
accumulation of labelled product which is partly integrated into
its specific capture molecule after each denaturation cycle and
detected.
[0033] FIG. 4. Results for the online detection of PCR
amplification on micro-array using labelled primer as schematically
represented in FIG. 2. PCR is performed on a GMO inserted sequence
in the presence of a micro-array comprising different bound capture
molecules, with one being specific of the amplified product (P35S)
and the other one (PAT1) not. Measurements are performed during the
annealing step of different thermal cycles on P35S and PAT1 capture
molecules.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0034] In the context of the present application and invention the
following definitions apply:
[0035] As used herein, "capture molecule" refers to a molecule, or
complex or combination thereof, that is capable of specifically
binding to one target molecule, or to a family of target molecules,
or to one or more member (s) of a plurality of target molecules, or
portion(s) thereof. The capture molecules are preferably nucleic
acids, which are either synthesized chemically in situ on the
surface of the support or laid down thereon. Nucleic acid binding
is achieved via base pairing between two polynucleotides, one being
the immobilized capture molecule and the other one the target to be
detected. Capture molecule also comprises derivative of the nucleic
acid such as PNA or LNA as long as they can bind specifically the
target polynucleotide molecule.
[0036] The term "single capture probe species" is a composition of
related polynucleotides for the detection of a given sequence by
base pairing hybridization or by molecular recognition between
polypeptides or proteins. Polynucleotides are synthesized either
chemically or enzymatically or purified from samples but the
synthesis or purification is not always perfect and the capture
molecule is contaminated by other related molecules like shorter
polynucleotides. The essential characteristic of one capture
species for the invention is that the overall species can be used
for capture of a given target nucleotide molecule.
[0037] The term "directly on the surface of the support" means that
the main part of the light beam is directed on the surface of the
support and excites itself the fluorescence molecules being present
on the surface.
[0038] The terms "nucleic acid, micro-array, probe, target nucleic
acid, bind substantially, hybridizing specifically to, background,
quantifying" are as described in the international patent
application WO97/27317, which is incorporated herein by way of
reference.
[0039] The term "nucleotide triphosphate" also called dNTP refers
to nucleotides present in either as DNA or RNA and thus includes
nucleotides, which incorporate adenine, cytosine, guanine, thymine
and uracil as bases, the sugar moieties being deoxyribose or
ribose. Other modified bases capable of base pairing with one of
the conventional bases adenine, cytosine, guanine, thymine and
uracil may be employed. Such modified bases include for example
8-azaguanine and hypoxanthine.
[0040] The term "nucleotide" as used herein refers to nucleosides
present in nucleic acids (either DNA or RNA) compared with the
bases of said nucleic acid, and includes nucleotides comprising
usual or modified bases as above described.
[0041] References to nucleotide(s), polynucleotide(s) and the like
include analogous species wherein the sugar-phosphate backbone is
modified and/or replaced, provided that its hybridization
properties are not destroyed. By way of example the backbone may be
replaced by an equivalent synthetic peptide, called Peptide Nucleic
Acid (PNA).
[0042] The term "polynucleotide" sequences that are complementary
to one or more genes or to the genome sequence described herein,
refers to polynucleotides that are capable of hybridizing under
stringent conditions to at least part of the nucleotide sequence of
said genes or genome or copy thereof. Polynucleotides also include
oligonucleotides being of more than 2 bases but below 100 bases
long which can be used under particular conditions. Such
hybridizable polynucleotides will typically exhibit at least about
75% sequence identity at the nucleotide level to said genes or
genome, preferably about 80% or 95% sequence identity or preferably
more than 95% nucleotide sequence identity to said genes or genome.
They are composed of either small sequences typically 15-30 base
long or longer ones being between 30 and 100 or even longer between
100 and 800 base long depending on the specificity and sensitivity
requirements for the assay.
[0043] The term "homology" is intended to mean the degree of
identity of one polynucleotide sequence to another polynucleotide
sequence. There may be complete homology (i.e. 100% identity)
between two or more polynucleotides. The degree of homology is
calculated after alignment of the sequence and may be determined by
any methods well known for a person skilled in the art.
[0044] "Micro-array" means a support on which multiple capture
molecules are immobilized in order to be able to bind to the given
specific target molecule. The micro-array is preferentially
composed of capture molecules present at specifically localized
areas on the surface or within the support or on the substrate
covering the support. A specifically localized area is the area of
the surface which contains bound capture molecules specific for a
determined target molecule. The specific localized area is either
known by the method of building the micro-array or is defined
during or after the detection. A spot is the area where specific
target molecules are fixed on their capture molecules and seen by
the detector. A spot is the area where specific target molecules
are fixed on their capture molecules and seen by the detector. In
one particular application of this invention, micro-arrays of
capture molecules are also provided on different supports as long
as the different supports contain specific capture molecules and
may be distinguished from each other in order to be able to
quantify the specific target molecules. This can be achieved by
using a mixture of beads having particular features and being able
to be recognized from each other in order to quantify the bound
molecules. One bead or a population of beads are then considered as
a spot having a capture molecule specific of one target
molecule.
[0045] The terms "background" or "background signal intensity"
refers to hybridization signals resulting from non-specific
binding, or other non specific interactions, between the labelled
target nucleic acids and components of the polynucleotide
micro-array (e.g. the polynucleotide probes, control probes, the
micro-array substrate, etc.). Background signals may also be
produced by intrinsic fluorescence of the micro-array components
themselves. A single background signal can be calculated for the
entire micro-array, or different background signals may be
calculated for each target nucleic acid. In a preferred embodiment,
the background is calculated individually for each spot, being the
level intensity of the signal on the surface surrounding the spot
and not bearing the specific capture molecule.
[0046] The nucleotide molecules of the invention are typically
detected by detecting one or more "labels" attached to the
nucleotide molecule. The labels may be incorporated by any of a
number of means well known to those of skill in the art, such as
detailed in WO 99/32660, which is incorporated herein by way of
reference. The label is detected directly preferably in
fluorescence.
[0047] The nucleotide molecule is intended to mean a polynucleotide
present in the biological material of interest and to be detected.
They are obtained either after extraction or purification of the
molecules of interest present in a sample being preferentially a
biological material. The term "biological material", includes
within its meaning organisms, organs, tissues, cells or biological
material produced by a cell culture.
[0048] Advantageously, the measurement of the target nucleotide
molecules is performed on a solid phase in the presence of labelled
amplified target molecules being present in the solution. The
method avoids removing the solution from the surface of the support
carrying a micro-array and avoids washing before the measurement.
The washing includes liquid handling of the solution containing
amplified target and possible contamination of further assays in
the laboratories.
[0049] Advantageously, the method of the invention does not require
the use different fluorescent dyes to quantify different nucleotide
molecules. One fluorescent dye is sufficient for the quantification
of multiple different nucleotide molecules because of their
specific binding by hybridization on capture molecules being
specific of each target nucleotide sequence and being localized in
distinct areas of the micro-array. For example, a nucleotide
molecule is amplified together with another nucleotide molecule
using the same or different primers and both amplicons are labelled
with the same fluorescent dye. The different amplicons are detected
and/or quantified on separated capture molecules without the need
of several fluorescent dyes as required in the real time solution
PCR.
[0050] Another advantage of the method is its great specificity. A
first specificity level is obtained through the annealing of the
primers and a second level of specificity is obtained by the
hybridization on the capture molecules. Such double specificity
increases very much the specificity of the final detection which is
often required for analysis in complex biological sample. Another
advantage is that primer dimers or non specific amplified product
formed during the PCR amplification will not generate signal on the
micro-array since there is no complementary capture molecules for
the primers nor for unspecific products.
[0051] The specificity can still be increased by the use of
different capture molecules for the same target nucleotide
molecule. Two or more capture molecules can be designed to bind the
same strand or one capture probe will bind the sense strand of the
amplified product and another capture molecule the antisense
strand.
[0052] Advantageously, the nucleotide molecules to be amplified are
homologous nucleotide sequences which are quantified on micro-array
during the PCR using consensus primers as described in WO0177372.
The same primers are used to amplify all the homologous sequences
possibly present in a sample. The amplicons which are labelled with
the same fluorescent dye are discriminated on different capture
molecules, each one targeting a different homologous sequence. So
with only one primer pair and one fluorescent dye, the assay is
rendered multiplex by the use of multiple capture probes present on
the micro-array. In one embodiment the number of sequences
amplified by the same primer pair is higher than 2 and even higher
than 5 and even higher than 20. The amplified targets are then
detected on the array.
[0053] In another embodiment, standards nucleotide sequences are
incorporated into the tested solution and the standards are
amplified with the same primers as the target nucleotide
sequences.
[0054] In still another embodiment, the
[0055] In the main embodiment, target and/or capture molecules are
polynucleotides. The capture molecules are attached preferably by
covalent link on the support or substrate present on the support.
In another embodiment, the capture molecules are adsorbed on the
support as long as they are not significantly released in solution
during the PCR cycles.
[0056] Deposition of the capture probe is preferentially done with
physical means such as pin or "pin and ring" touching the surface,
or by release of a micro-droplet of solution by methods such as
piezo or nanodispenser. Alternatively, in situ synthesis of capture
molecules is one of the invention embodiment with light spacial
resolution of the synthesis of oligonucleotides or polynucleotides
in known locations such as provided by U.S. Pat. No. 5,744,305 and
U.S. Pat. No. 6,346,413.
[0057] In another embodiment the nucleotide molecules are DNA
present in a biological sample. The DNA is extracted from the
sample and amplified by PCR and the amplicons are detected online
by their fixation on their specific capture molecules. In one
particular embodiment, the nucleotide molecules are homologous
nucleotide sequences which are detected and/or quantified online on
micro-array after amplification of genomic DNA by consensus primers
as described in WO0177372.
[0058] According to the invention, the solid support for the
micro-array is preferably selected from the group consisting of
glass, metallic supports, polymeric supports (preferably
thermo-resistant having low self-fluorescence) or any other support
used in the microchips (or micro-arrays) technology (preferably
activated glass bearing aldehyde or epoxide or acrylate groups),
said support comprising also specific coatings, markers or devices
(bar codes, electronic devices, etc.) for improving the assay.
[0059] In a preferred embodiment, the support (15) contains a
substrate on which are fixed the capture molecules.
[0060] If glass presents many advantages (like being inert and
having a low self-fluorescence), other supports like polymers, with
various chemically well-defined groups at their surface, allowing
the binding of the nucleotide sequences are useful. In another
preferred embodiment, the support bearing the capture molecules has
a 3 dimensional porous structure. Conventional glass slides have
less than 60% silicon dioxide on their surface. This inherently
limits the amount of chemical bonding available on the surface.
Porous material exhibits increased loading capacity of capture
molecules. Typical porous supports are gel pads, fused-fiber
matrix, fibrous polymer matrix. The array can be constructed
entirely of the porous material, or can comprise a layer of porous
material mounted on top of a flat surface such as glass, plastic,
or metal.
[0061] In another embodiment capture molecules are present on
different supports being preferentially beads with chemical or
physical characteristics for their identification with a specific
capture molecule.
[0062] In still another embodiment, the support bears several
micro-arrays separated by physical or chemical boundaries. Examples
for physical barriers are wells, e.g. the support being a 96, 384,
1536 multi-well plate, thus creating separated localized areas onto
which capture molecules may be spotted individually. 384-well and
1536-well plates are available from BD Falcon for cell based assays
(Merck Eurolab sa, Leuven, Belgium) or from Nunc A/S (Roskilde,
Denmark). 6144 format microtiter plates are available from Parallel
Synthesis Technologies Inc. (PSTI, Menlo Park, Calif., USA). The
multiwells are present as one plate or in strips. Other physical
barriers are tubes such as 96, 384, 1536 or even 6144 tubes deposit
at the surface of the support. Tubes are similar to the well
formats but do not have a plain bottom so that when deposit on the
surface of the support, they create localized areas isolated from
each other. An example for a chemical barrier is e.g. described in
DE 0019949735A1, where defined areas within a hydrophobic surface
are provided with hydrophilic anchors allowing the precise location
and confinement of capture molecules on a solid support.
[0063] In a preferred embodiment, the support bears several
micro-arrays separated by physical boundaries, preferably in a
multi-well plate or strip format. In another embodiment, the
multiwell plate is submitted to a temperature gradient during the
measurement of light emission (7).
[0064] In a preferred embodiment, the reaction chamber contains 2
or even better 3 parts being in fluid connection to each other
comprising a flat surface carrying the micro-array. The support is
preferably made of a plastic slide covered with a flow through
observation channel where the micro-array is build up. The
observation channel is terminated by one or two reservoirs
preferably located at both sides. One reservoir is preferably used
to introduce the solution and the other one to remove it. The
reservoirs are sealed by specific lids to avoid the evaporation of
the solution during thermal cycles. In a particular embodiment, the
solution is moved over the micro-array in order to increase the
speed of the binding reaction of the labelled target nucleotide
molecule on its capture molecule. This is obtained by rotating,
translating or moving up and down the reaction chamber during at
least the annealing step of the thermal cycle. In still another
embodiment, the height of the liquid on the surface having fixed
the micro-array is lower than 1 mm and preferably lower than 0.1 mm
and even more preferably lower than 0.02 mm.
[0065] In the preferred embodiment, the polynucleotides being used
as capture molecule are between 10 and 1000 nucleotide long and
preferably between 100 and 400 nucleotides long. For specific
binding of homologous sequences possibly present in the same
sample, the polynucleotide capture molecules contain a spacer
according to the patent WO0177372. Specific binding of homologous
sequences or SNP possibly present in the same sample, are obtained
using capture molecules having a specific part being between 10 and
30 nucleotides.
[0066] In the preferred embodiment, the polynucleotides being used
as capture molecules are present on the micro-array localized area
at a density superior to 10 fmoles, and preferably 100 fmoles per
cm.sup.2 surface of the solid support.
[0067] The micro-array according to this invention contains between
4 and 100000 spots per cm.sup.2 and preferably between 20 and 1000
spots per cm.sup.2, each spot being the localized area for one
capture molecule. Miniaturization allows performing one assay onto
a surface (usually circular spots of about 0.1 to about 1 mm
diameter). A low density array, containing 20 to 400 spots is
easily obtained with pins of 0.25 mm at low cost. Higher density of
spots going to 1,600 spots per cm2 can be obtained by reducing the
size of the spots for example to 0.15 mm. Method for obtaining
capture molecules of higher density have been described earlier as
in U.S. Pat. No. 5,445,934. Miniaturization of the spot size allows
obtaining a high number of data which can be obtained and analyzed
simultaneously, the possibility to perform replicates and the small
amount of biological sample necessary for the assay.
Miniaturization for detection on micro-arrays is preferably
associated with microfluidic substrate for separation, extraction
of nucleotide molecules from the cell extract.
[0068] In a preferred embodiment, the micro-array comprises more
than 5 different capture molecules (20), preferably more than 20
and even more than 50.
[0069] In a preferred embodiment, the localized area is comprised
between about 10 .mu.m.sup.2 and about 1 mm.sup.2 and preferably
between about 1 .mu.m.sup.2 and about 100 .mu.m.sup.2.
[0070] In one preferred embodiment, the capture molecules present
on the micro-array are complementary to at least one part of the
sequence of amplified target nucleotide sequence present in
solution. The capture molecules comprise a nucleotide sequence
which is able to specifically bind the amplified target nucleotide
sequence, said specific nucleotide sequence is also preferably
separated from the surface of the solid support by a spacer arm of
at least about 6.8 nm or 20 nucleotides in a double stranded form
which has no binding affinity for the amplified target molecule. In
a preferred embodiment, the capture molecule is a single stranded
polynucleotide containing a sequence able to specifically bind the
labelled target nucleotide molecule and a spacer of at least 20
nucleotides and better more than 90 nucleotides. The spacer part
can be either single or double stranded DNA.
[0071] In a preferred embodiment the probe sequence specific for
the target binding is comprised between 15 and 100 nucleotides and
more preferably between 15 and 35 nucleotides
[0072] Detectable labels suitable for use in the present invention
include any composition detectable by electromagnetic light
emission. In an embodiment, the target molecules are labelled with
a fluorescent dye. The fluorescent label can be incorporated into
the target by enzymatic or chemical reaction. Typical enzyme
reaction includes the incorporation of nucleotide analogues into
the target. Alternatively, primers labelled at their 5' end with a
fluorescent dye can be incorporated into the target. Fluorochromes
can also be incorporated into the targets by chemical reaction such
as the reaction of fluorescent dye bearing a N-hydroxysuccinimide
(NHS) group with amines groups of the targets. Useful fluorescent
dyes in the present invention include cyanine dyes (Cy3, Cy5, Cy7),
fluorescein, texas red, rhodamine, green fluorescent protein.
Preferably, the excitation wavelength for cyanin 3 is comprised
between 540 and 558 nm with a peak at 550 nm and the emission
wavelength is comprised between 562 and 580 nm with a peak at 570
nm.
[0073] Preferably, the excitation wavelength for cyanin 5 is
comprised between 639 and 659 nm with a peak at 649 nm and the
emission wavelength is comprised between 665 and 685 nm with a peak
at 670 nm. Preferably, the excitation wavelength for cyanin 7 is
comprised between 733 and 753 nm with a peak at 743 nm and the
emission wavelength is comprised between 757 and 777 nm with a peak
at 767 nm.
[0074] Patents teaching the use of such labels include U.S. Pat.
Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149; and 4,366,241. In a preferred embodiment, the
fluorescent dye is cyanin 3, cyanin 5 or cyanin 7.
[0075] Some fluorescent labels may be of particular interest, such
as nanocrystals particles having fluorescent properties. The most
common one are the Quantum dots (Han et al., Nature Biotechnology
19, 631-635, 2001). They are fluorescent and do not bleach with
time or with illumination. Their stability makes them particularly
suitable for the use in continuous reading as proposed in this
invention. Also, they contain metals which confer to these
particles specific properties so that other methods than the
fluorescence can be used to follow their attachment on the capture
probes. Thermal heating of these particles is one of the parameters
that may be followed with time. The fact that the metal absorbed
the energy of a light beams preferably a laser and induce a heating
of the particle has been used as a basis for the detection of low
density gold particle on a support and even single particles are
detected (Boyer et al Science, 297, 1160-2002). The method is
called Photothermal Interference contrast.
[0076] Another technology for the direct measurement of
nanoparticles is the Rayleigh Scattering. This method is based on
the use of a light beam adapted in order to obtain an oscillation
of the electrons in the metal particle so that an electromagnetic
radiation is obtain from the particle which can be detected.
(Stimpson et al., Proc. Natl. Acad. Sci. USA 100 (2003),
11350-11353) (real-time detection of DNA hybridization and melting
on oligonucleotide arrays by using optical wave guides) The method
is lacking sensitivity for the applications on biological
samples.
[0077] Alternatively, Raman scattering and the surface plasmon
resonance may be applied in the present invention, which technique
has been extensively used for the detection of antibody/antigen
binding but are also well suited for the multiparametric
measurement of the arrays and for the required sensitivity on
biological samples. (Thiel et al., Analytical Chemistry, 69 (1997),
4948-4956).
[0078] In another embodiment, quartz crystal microbalances may be
applied, which are now sensitive enough that they can measure
changes of mass lower than nanogram (cf. Caruso et al., Analytical
Chemistry 69 (1997), 2043-2049). This is one proposal for
micro-array detection in real-time.
[0079] Cantilevers are another option for the detection of DNA on
micro-arrays. (McKendry et al. Proc. Natl. Acad. Sci. USA, 99
(2002), 9783-9788).
[0080] Also, another technology is the electrical detection of the
nanoparticles which takes into account their metal properties. The
electrochemical detection was first applied but with low
sensitivity. The more advanced and sensitive method is the
detection by differential pulse voltametry (Ozsoz et al.,
Analytical Chemistry 75 (2003), 2181-2197).
[0081] The resistivity and the capacitance properties of the metal
are also one of the best properties to be detected on electronic
chips. The presence of a metal between two electrodes will induce a
change of resistivity and of capacitance. The detection of the DNA
or proteins is then observed when the capture molecules are present
on one of the electrode (Moreno-Hagelsieb et al Sensors and
Actuators B-Chemical, 98, 269-274, 2004). The capacitance assay of
the gold labelled DNA has been described by Guiducci et al. ESSDERC
2OO2. Since electronic chips can be made of several plots,
different targets may be detected on different plots and the change
in the resistivity or in the capacitance may be recorded. If the
methods have not yet been able to produce reliable and sensitive
detections as required by the biological samples, it is, however,
predicted that some of them will succeed to fulfil the requirements
for the realtime detection.
[0082] Another promising technology for measuring the binding of
the target molecules on capture molecule of the micro-array is the
chemical cartography based on optical process of non-linear
generation frequency spectroscopy (GFS) (L. Dreesen et al. Chem
Phys Chem, 5, 1719-1725, 2004). This technology allows the imaging
in real time of the vibrational properties of surfaces and
interfaces with a submicron spacial resolution. The measurement is
obtained by mixing at the surface of a substrate two laser beams,
one having a fixed frequency in the visible (green) and the other
having a variable frequency in infrared. The vibrational signature
at the interface is obtained by measuring the light emitted by the
sample in function of the frequency of the infrared laser beam.
This method allows to avoid labelling of the target in order to be
detected.
[0083] The original nucleotide molecule is not necessary labelled
before the amplification but lead to amplified labelled target
molecules during the amplification step.
[0084] The amplified nucleotide molecules are able to hybridize on
the capture molecules after a denaturation step. As the amplified
nucleotide molecules are double stranded, in theory they must
reassociate in solution much faster than to hybridise on capture
molecules fixed on a solid support where diffusion is low and the
specific binding sequence is short, thus reducing even more the
rate of reaction. Therefore, it was unexpected to observe a
significant signal increase on the capture molecules over multiple
thermal cycles after a short period of incubation time (FIG.
4).
[0085] In a particular embodiment the measurement is performed on
bound target labelled molecules while they reassociate in a double
stranded form in the solution during annealing and/or elongation of
the thermal cycle.
[0086] In a preferred embodiment, the reagents for nucleotide
molecule amplification comprise a primer pair, dNTPs, a
thermostable DNA polymerase and buffer.
[0087] In a particular embodiment, the assay is performed in a
continuous or semi-continuous way over the annealing and/or
elongation and/or denaturation step.
[0088] In a preferred embodiment, the reagents for nucleotide
molecule amplification comprise a primer and/or dNTP labelled with
a fluorescent dye, preferably Cyanin 3, Cyanin 5 or Cyanin 7.
[0089] In a specific embodiment, two or more fluorescent dyes are
used in the same solution. In an alternative embodiment, the
solution composition is adapted for performing the annealing of the
primers on the nucleotide molecule and the specific binding of the
labelled target molecule on the capture molecule during the same
temperature step.
[0090] In a preferred embodiment, the thermostable DNA polymerase
used for PCR on micro-array is the hot Master (Eppendorf, Hamburg,
Germany) which works at 62.degree. C. In a preferred embodiment the
steps of annealing, elongation and hybridization on the array are
performed at the same temperature which is comprised between 60 and
68.degree. C. Advantageously, the method of the invention is
compatible with most of the thermostable DNA polymerase available
on the market. It does not necessary require a 5' to 3' nuclease
activity as described in the U.S. Pat. No. 5,952,202.
[0091] In an embodiment, the solution contains 5' end labelled
oligonucleotides or primers which serve as anchors for the
polymerase to copy the target sequences to be detected on the
micro-array. FIG. 2 shows the detection of PCR product on
micro-array using labelled primer. Unexpectedly, during the
temperature step of annealing of a thermal cycle, the primers
hybridize with the nucleotide molecule in solution while the
amplified target molecule obtained in a previous thermal cycle
hybridize in the same time and in the same conditions, on a capture
molecule being immobilized in a specifically localized area of a
support. During the temperature step of elongation of a thermal
cycle, the primers hybridized to the nucleotide molecule are
elongated in solution. The capture molecules (20) bound to the
labelled target nucleotide molecules are possibly elongated but not
labelled. Alternate steps of annealing, elongation and denaturation
during one cycle of reaction result in the accumulation of labelled
products which hybridize on their capture molecule present on the
micro-array but deshybrizes from their specific capture molecules
after each denaturation cycle.
[0092] In a preferred embodiment, the labelled target nucleotide
molecules are specifically bound on their corresponding capture
molecules (20) preferably during the temperature step of annealing
and/or elongation.
[0093] In another embodiment, the solution contains labelled dNTP
which are incorporated by the polymerase into the target sequences
to be detected on the micro-array.
[0094] FIG. 3 shows the detection of PCR product on micro-array
using labelled dNTPs. During the temperature step of annealing of a
thermal cycle, the primers hybridize with the nucleotide molecule
in solution while the amplified target molecule obtained in a
previous thermal cycle hybridize in the same time and in the same
conditions, on a capture molecule being immobilized in a
specifically localized area of a support.
[0095] During the temperature step of elongation of a thermal
cycle, the primers hybridized to the nucleotide molecule are
elongated in solution while, the immobilized capture molecules (20)
having bound to the target nucleotide molecules are elongated and
labelled. Alternate steps of annealing, elongation and denaturation
during reaction cycles result in the accumulation of labelled
product being partly integrated into its specific capture molecule
and which can be detected during or at the end of each of the
denaturation step.
[0096] In an embodiment, some capture molecules are elongated by
the polymerase and some are in the same time hybridized with the
amplified products which accumulate in solution during the thermal
cycle. In one embodiment, the capture molecules elongated are
detected during the temperature step of denaturation. In another
embodiment, the capture molecules elongated and the labelled
nucleotide molecules bound on their capture molecule are both
detected during the temperature step of annealing and/or
elongation.
[0097] In a preferred embodiment, the hybridization is favoured
over the elongation by using capture probes which are not capable
of being elongated. In this case, capture molecules preferably
include a base terminator or long stretch of identical bases at
their 3' end such as polyA. Alternatively, the capture molecules
are immobilized on the support by their 3' end, the free 5' end
being not able to be elongated by the polymerase.
[0098] In another embodiment, the elongation is favoured over the
hybridization by performing PCR in the presence of one primer in
excess and a reduced amount of the other primer.
[0099] In another embodiment, at the end of the thermal cycles, an
annealing step of at least 10 min, and better at least 30 min and
even better at least 60 min is performed in order to increase the
signal of hybridization for nucleotide molecules present at a very
low concentration before the amplification.
[0100] In a preferred embodiment, a thermal cycle is performed
within 10 min and better within 6 min and even better within 3 min.
In an alternative embodiment, 30 thermal cycles are performed
within 5 h and better within 3 h and even better within 1.5 h.
[0101] Advantageously, the length of the amplified target
nucleotide molecules are selected as being of a limited length
preferably between 100 and 800 bases, preferably between 100 and
400 bases and more preferably between 100 and 200 bases. This
preferred requirement depends on the possibility to find primers to
amplify the required sequences possibly present in the sample. Too
long target may reallocate faster and adopt secondary structures
which can inhibit the fixation on the capture nucleotide
sequences.
[0102] The thermal cycler is preferably composed in its simplest
version of the following relevant components:
a thermocouple, a transmitter, a converter and a heater.
[0103] The thermocouple, sticks as close as possible of the
localized area of the micro-array to heat, measures the temperature
thought the transmitter. This temperature information is given to a
computer via the converter. Every 0.1 second, the software compares
the real temperature measured to the temperature set point
requested by the final user. If the measured temperature is higher
than the requested one, the heater is simply stopped (no active
cooling). If the measured temperature is lower than the set point,
the system continues the heating process. The thermal cycler is
preferably adapted to fit the support format being preferably a
microscopic slide of about 2.5.times.7.5 cm or a 96 wells
microtiter plate. The alternative heating and cooling is preferably
obtained using a peltier or pulsed air.
[0104] In a preferred embodiment, the thermal cycler is capable of
alternatively heating and cooling the support at a ramping of
5.degree. C. per min, preferably 10.degree. C. per min and better
30.degree. C. per min and ever better 40.degree. C. per min.
[0105] The method is particularly well fitted to control the light
excitation since the light is directed on the surface of the
support and the homogeneity of the excitation at each localized
area can be determined and corrected if necessary
[0106] In a preferred embodiment, the light beam is a laser beam
which is focused on the surface of the micro-array in order to
excite directly the fluorescent molecules. The laser beam is
preferably focused perpendicular to the surface of the array either
through the solution or through the support. The emitted light is
detected in the opposite direction of the excitation laser beam.
The emitted light is preferably detected as a confocal light and
measure after amplification by a photomultiplier. In the preferred
embodiment the surface of the microarray is scanned by the laser
beam in order to obtain a maximum light excitation of the bound
targets.
[0107] In a preferred embodiment, the excitation light (2) from a
light source (1) is directed on the surface of the support.
[0108] In a preferred embodiment, the signal associated with a
capture molecule on the micro-array is quantified. The preferred
method is the scanning of the array(s) with a scanner being
preferentially a laser confocal scanner for the detection of
fluorescent labelled targets. The resolution of the image is
comprised between 1 and 500 .mu.m and preferably between 5 and 50
.mu.m.
[0109] In a preferred embodiment, a measurement of the labelled
target nucleotide molecule is performed in at least 5, preferably
at least 10 thermal cycles and even preferably at least 20 thermal
cycles.
[0110] In a preferred embodiment, the light emission (7) is
measured at a defined timing from the beginning of a temperature
step, for example after 1 min of annealing.
[0111] In another preferred embodiment, the light emission (7) is
measured at within 5 min and even within 2 min and even better
within 1 min after the beginning of the annealing temperature step.
In an alternative embodiment, the light emission (7) is measured at
the end of at least one of the 3 temperature steps used for the PCR
amplification.
[0112] In still another embodiment, the light emission (7) is
measured at the end of the PCR amplification.
[0113] The micro-array is preferably scanned and each localized
area is subsequently measured. Preferably the scanning of the array
is performed within 1 min and better within 30 sec and even better
within 10 sec. Preferably the scan of each localized areas is
measured at the same precise moment of a temperature step when
reading is repeated over multiple thermal cycles, The fact that
each localized area is subsequently measured can be advantageously
used to monitor a kinetic of hybridization of a labelled target
nucleotide molecule on the same capture probe which has been
immobilized at different localized areas of the support and which
are scanned in a time dependant manner. Since the temperature is
maintained constant during the measurement, the target nucleotide
molecule continues to hybridize on their capture probe during the
scanning.
[0114] In a particular embodiment, the data on the quantification
of the amplified target molecules performed at different PCR cycles
are processed in order to quantify the amount of nucleotide
molecule present in the original solution before the amplification.
The amplification cycles lead to the doubling of the target
sequence in each cycle when the efficiency of the amplification is
maximal. Quantification of the original nucleotide concentration is
calculated from the extrapolation of the first cycle which gives a
detectable value or a value crossing a fixed threshold. The
concentration is then calculated from a reference curve or from the
data obtained on a standard molecule.
[0115] In a preferred embodiment, the data are processed in order
to obtain a signal value for each of the localized area. In another
embodiment, the data are processed in order to obtain a signal
value for each of the localized area and for the local background.
The data are further processed by subtracting the background from
the signal value for each of the localized area. In a preferred
embodiment, the quantification of the amount of nucleotide molecule
is performed by comparing the signal value of the localized area
with a fixed value.
[0116] In an alternative embodiment, the quantification of the
amount of nucleotide molecule is performed by comparing the number
of thermal cycles necessary to reach a fixed value (cycle threshold
or CT) with the CT of a reference nucleotide molecule. The
reference nucleotide molecule is preferably amplified in the same
solution and detected on the same micro-array as the target
nucleotide molecule.
[0117] In another embodiment, the quantification of the amount of
nucleotide molecule is performed by comparing the number of thermal
cycles necessary to reach a fixed value (CT) with a standard curve
wherein the CTs are plotted against standard concentrations.
[0118] In an embodiment, the micro-array is in contact with
reagents for carrying out the amplification of one or more
nucleotide sequences. In a preferred embodiment, between 1 and 4
nucleotide molecules and better between 1 and 20 nucleotide
molecules present in a solution are amplified and detected and/or
quantified in the same assay. In another embodiment, between 20 and
1000 nucleotide molecules present in a solution are amplified and
detected and/or quantified in the same assay.
[0119] The apparatus used in order to perform the method according
to the invention contains two different parts.
[0120] The first one contains the incubation system which provides
the conditions necessary for the binding reaction of the targets
onto their capture molecules. Preferably the first part contains a
temperature control system for regulating and controlling the
temperature during the binding reaction.
[0121] In a preferred embodiment, the temperature regulating device
is selected from the group consisting of a controlled peltier, a
micro-thin wire heating element laid in a pattern between optical
grade polyester sheets like Thermal-Clear.TM. transparent heaters
from Minco, or fluidic system circulating externally temperature
regulated fluid.
[0122] In a preferred embodiment, the temperature regulating device
is mounted on a carrier holding the support. The temperature
regulating device is preferably positioned between the carrier and
the support.
[0123] In another embodiment, the temperature regulating device is
mounted on the support and is not in contact with the carrier.
[0124] In a preferred embodiment, the incubation system provides
conditions so that the thickness of the solution being in contact
with the micro-array is constant above all the arrayed spots or
localized areas. The difference of thickness between two spots or
localized areas of the arrayed surface is preferably lower than 100
micrometers and even lower than 10 micrometers and even lower than
1 micrometer.
[0125] In another embodiment, the incubation system provides
conditions for the thickness of the solution being in contact with
the micro-array is changed between two measurements.
[0126] The first part of the apparatus also preferably contains a
mixing or agitation system for the liquid to be moved inside the
reaction chamber and increase the reaction rate. In a preferred
embodiment, the mixing is performed by movement of the liquid by
physical means such as pump, opening and closing valves,
electrostatic waves or piezoelectric vibrations.
[0127] The second part contains the detection system required to
detect the light emission from the target bound to their
corresponding capture molecules. A light source generates a beam of
light to excite the labeled targets on the support. In the
preferred embodiment, the detection part has to be settled in such
a way as to obtain the same detection efficiency on the overall
surface covered by the micro-array to be analyzed.
[0128] In a preferred embodiment, the excitation light is a laser
beam preferably having a wavelength of about 532 nm delivered at a
power of about 15 mW with a divergence that may be below 1.2 mrad.
In another embodiment, the detection system contains 2 or even 4
lasers.
[0129] In a preferred embodiment, the laser beam (2) generated by
the light source (1) is nearly collimated and nearly Gaussian. An
exchangeable excitation filter (4) is used to collect only the
wavelengths of interest. An additional filter wheel (3) is
preferably placed and used as an attenuation filter to precisely
regulate the laser power. This filter wheel is shaded differently
at variable know absorption levels. A lens (5) that may be
anti-reflection coated is used for focusing the laser beam on the
support (15). The distance between the light source, the lens and
the support is variable to allow focusing.
[0130] Thereafter, the light passes through a dichroic mirror or
beam splitter (6). This mirror pass light having a wavelength lower
than about 530 nm, but reflect light having a wavelength greater
than 560 nm. Consequently, the 532 nm light coming from the laser
is passed through the dichroic mirror to the support. The light
then passes through a reaction chamber (14) and the fluorescent
marked sample (13) and reaches the support (15), where bound
labeled target are excited and emit fluorescence at about 560
nm.
[0131] Emitted light (7) is then focused through a focusing lens
(9) to a photomultiplier tube (10) for detecting the number of
photons present therein.
[0132] In a specific embodiment, an additional emission filter (8)
that transmits light having a wavelength greater than about 550 nm
is added. Thus, photomultiplier tube (10) detects substantially
only fluoresced light. The Photomultiplier tube generates a pulse
for each photon detected. Each of these pulses is amplified and
converted to an electronic signal by photoelectric effect. A data
acquisition board or controller (11) then collects the resulting
signals. The controller includes a temperature controlling device
for controlling the temperature steps needed for PCR
amplification.
[0133] After data are collected from a region of the substrate, the
carrier (12) moves the support so that excitation light is directed
to a different region on the support (15). The process is repeated
until all regions on the substrate have been scanned. In another
embodiment the support is fixed and the light excitation beam is
moved from one part to the other on the surface of the support. In
still another embodiment, the overall micro-array is illuminated
and the light emission from each localized area is detected.
[0134] In one embodiment, the support itself is a carrier. In a
preferred embodiment, the data are stored and treated for
calculation of the amount or concentration of the different target
molecules in solution and in the original biological sample. Data
storage and data treatment are preferably performed using a
programmable computer which is integrated in the apparatus of the
invention. Data treatment can be performed at any time after data
storage.
[0135] In one embodiment, the support is moved relative to the
detection system during the reading. The support moves relative to
the excitation light to allow the reading of different regions of
the support. The excitation light may be fixed or moved in one
direction to scan the support.
[0136] In an alternative embodiment, the support is moved relative
to both the incubation and detection systems. During the
incubation, the support is in contact with the temperature control
system (incubation position). When a reading has to be effected,
the support is moved from the incubation system to the detection
system (reading position). During the reading, the support is
either moved relative to the excitation light or is fixed. After
the reading the support turns back to its initial position. One
advantage of moving the support relative to the incubation part
during the reading is to avoid deleterious effect of the heating
device on parts of the detection system.
[0137] In another embodiment, the two parts of the apparatus are
fixed and work together with no movement of the solid support
relative to the incubation and detection parts. A typical detector
used in this context is a CCD camera capable to take a picture of
the whole micro-array.
[0138] In a specific embodiment the apparatus is controlled by a
programmable computer which controls the parameters of the two
parts of the system. The scanner is comparable to a Genepix 4200A
scanner from Axon coupled with the scriptable Genepix 5.1 software
from Axon.
[0139] At STEP 1, the user is prompted to fill in the required
parameters, such as: resolution, voltage of the PMT, laser power,
number of scans, time between scan, scan area. Temperature of the
substrate is set separately on the heating system that can be a
peltier device mounted on the substrate.
Parameters of the System:
[0140] The resolution defines the pixel size. Generally, the pixel
size is chosen which results in more than 1 pixel per localized
area and preferably between 10 and 100. Setting a too high
resolution generates an overload of data while having a too low
pixel size generates low quality results. The PMT voltage
multiplies the detected signal. Increasing the laser power will
increase the photon count in each pixel.
[0141] The "number of scan" parameter corresponds to the number of
times the user wishes to scan the substrate while the "time between
scans" parameter controls the amount of time to wait before
commencing a subsequent scan. In this manner, the user may perform
a series of scans to follow the kinetics of the reaction.
[0142] Scan area parameter corresponds to the size of the substrate
to be tested. The temperature parameters control the temperature at
which detection is performed. Temperature may vary depending on the
type of polymers being tested. Preferably, testing is done at a
temperature that produces maximum binding affinity while minimizing
mismatches.
[0143] The system is then initialized: carrier is moved to home
position while laser power is checked. At STEP 2, first scan is
performed and the fluorescence emitted on the selected region
comprising the micro-array of the substrate is collected. The
JavaScript callback is launched when the scan is done (STEP 3). If
the number of scans to be done is not reached, then the program
waits for the delay asked by the user. Then the image is saved at
STEP 4, and if required a new scan is performed (STEP 5). The
JavaScript callback allows the loop to be continued. In STEP 6,
values are extracted from the data and in STEP 7; the calculation
and analysis are performed. For this purpose a grid which contains
the number of rows and columns of the micro-array to be measured is
positioned on the micro-array. The grid is composed of circles
which diameter in pixels correspond to the diameter of the spots to
be quantified. The diameter is depending on the resolution chosen
for the scanning. The means of the pixels intensity inside the
circle gives the spot signal. This signal is then calculated for
each time and plotted versus the incubation time. STEP 6 is
preferably performed by importing the scanned 16-bit images to the
software, `ImaGene4.0` (BioDiscovery, Los Angeles, Calif., USA),
which is used to quantify the signal intensities.
TABLE-US-00001 Algorithm <html> <head> <style
type="text/css"> @import url(GenePix_Style_Base.css);
</style> <title>Example Automation</title>
</head> <body marginheight="0" marginwidth="0"
topmargin="0" leftmargin="0"> <!-- HTML Layout portion -->
<p> <table width=600 border=0 cellspacing=0
cellpadding=5> <tr class="title"> <td> <p
class="heavy">Real-time scanning: allow scanning multiple time
the same sample at constant time intervals without any user
intervention </td> </tr> // STEP 1: USER PARAMETERS
<tr> <td class="underline instructions"> <p>PMT:
<input type=text size=2 name=setpmt value="700">
<p>Resolution: <input type=text size=2 name=setres
value="40"> .mu.m <p>Scan interval: <input type=text
size=2 name=interval value="120"> (s) <p>Scan numbers:
<input type=text size=2 name=snumber value="10"> <p>
<input type=checkbox size=5 name=saved value="10">Save images
? <p>Images directory: C:\Documents and
Settings\user\desktop\<input type=text size=20 name=ipath
value=""> </td> </tr> <tr> <td
class="underline instructions"> <input type="button"
value="Prescan" onclick="GenePix.PreviewScan( )"> <input
type="button" value="Start scanning" onclick="startscan( )">
</td> </tr> </tr> </table> <!--
Scripting portion --> <script language=vbscript> //
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++++++++++++++++++++++++++++++++++++++++++++ Option
Explicit Dim GenePix Dim Scanner dim i // PMT VALUE dim j //
RESOLUTION (.mu.m) dim k // SCAN INTERVAL (s) dim n // NUMBER OF
SCANS dim c // COUNTER dim s // IMAGES PATH dim t1 // TIMER // This
procedure is launched by pressing on the start scan button
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++++++++++++++++++++++++++++++++++++++++++++ sub
startscan( ) // STEP 2 c=0 Set GenePix = window.external //
declares scanner object Set Scanner = GenePix.Scanner
GenePix.DiscardImages( ) // clears the display call
InitializeCallbacks( ) // defines the Javascript callbacks
i=cint(setpmt.value) // sets the PMT value j=cint(setres.value) //
sets the resolution value k=cint(interval.value) // sets the time
interval between scans n=cint(snumber.value) // sets the number of
scans s=cstr(ipath.value) // sets the path of the images
Scanner.PixelSize=j Scanner.PMT(0)=i t1=timer( ) // sets the time 0
GenePix.DataScan // starts the first scan end sub // Saves the
image and launches a new scan
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++++++++++++++++++++++++++++++++++++++++++++ Function
ScanDone( ) // STEP 4 if saved.checked=true then // saves the image
GenePix.SaveImages "C:\" & s & "\RT-"&
cstr(formatnumber(timer( )-t1-k,0)) &" s.tif", "",
&h008000; end if GenePix.DiscardImages( ) // reinitializes the
display c=c+1 // counts the number of scans if c<n then //
STEP5: and if necessary, GenePix.DataScan // launches a new scan
end if End Function </script> <script
language="JavaScript"> // This function is called after a scan
is done
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++++++++++++++++++++++++++++++++++++++++++++ function
waitjs( ) // STEP3 { if c<n then // if more scans have to be
done, setTimeout("ScanDone( )",k*1000); // pauses the program
during the time else // interval and calls the ScanDone function
End if } // Javascript callback: defines which function has to be
run after which event
+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
++++++++++++++++++++++++++++++++++++++++++++++++++++++ function
InitializeCallbacks( ) { GenePix.OnScanDone = function ( ) {
waitjs( ); } } </script> </body> </html>
[0144] FIG. 1 represents one embodiment of the invention in which
parts of the two processes are present in the same compartment. The
two processes are performed in the integrated system as long as the
technical parts (necessary for having the specifications) are
compatible with each other. The light source (1) is directed on the
surface of the support (15) opposite to the surface in contact with
the thermostatized carrier (12). The controller (11) includes a
temperature controlling device.
[0145] In preferred embodiment, the excitation light (2) reaches
the micro-array surface within an angle comprised between
45.degree. and 135.degree., preferably between 60.degree. and
120.degree., even more preferably between 80.degree. and
100.degree.. The light excitation is a direct excitation of the
labelled target and do not use the internal reflection of the light
such as provided by the evanescent waves.
[0146] In a preferred embodiment, the apparatus contains a
substrate on which are fixed the capture molecules. In a preferred
embodiment the support of the apparatus is thermostable and the
surface is maintained flat at a temperature higher than 85.degree.
C. and even higher than 94.degree. C. The support also presents a
low self-fluorescence in order to be compatible fluorescence
measurement. Preferably, the micro-array contained in the apparatus
comprises more than 5 different capture molecules (20), preferably
more than 20 and even more than 50.
[0147] In a preferred embodiment, the heating and cooling of the
thermal cycler is performed at a ramping of 5.degree. C. and better
30.degree. C. per min. In a preferred embodiment, the localized
area comprising the capture molecule is comprised between about 10
.mu.m.sup.2 and about 1 mm.sup.2 and preferably between about 1
.mu.m.sup.2 and about 100 .mu.m.sup.2.
[0148] The apparatus further comprises an optical system for
directing and focusing an excitation light (2) from said excitation
light source (1) directly on said support, wherein the excitation
light reaches the micro-array surface within an angle comprised
between 45 and 135.degree..
[0149] In a specific embodiment a diagnostic kit is provided for
monitoring on a micro-array a PCR amplification of a nucleotide
molecule being present in a solution. The kit includes a cartridge
comprising: a support (15) having fixed upon its surface a
micro-array, comprising at least one capture molecule (20) being
immobilized in specifically localized areas (21) of said support
wherein the surface of said support is maintained flat at
temperature higher than 85.degree. C. and wherein said support have
a low self-fluorescence, and a reaction chamber comprising 2 or
even better 3 parts being in fluid connection to each other
comprising a flat surface carrying the micro-array.
[0150] The diagnostic kit according to the invention also better
comprises dNTPs, a thermostable DNA polymerase, buffer and
optionally primers and/or a nucleotide molecule being used as an
internal standard.
[0151] One example of the present invention applicable to the
determination of the measurement of amplified targets from an
original nucleotide molecule is presented here after.
EXAMPLE 1
Monitoring PCR Amplification on Micro-Array
Capture Nucleotide Sequence Immobilisation
[0152] The Diaglass slides (Eppendorf, Hamburg, Germany) are
functionalized for the presence of aldehydes according to the
method described in patent application WO02/18288. The protocol
described in this patent application was followed for the grafting
of aminated DNA to aldehyde derivatised glass. The aminated capture
nucleotide sequences were spotted from solutions at concentrations
of 3 .mu.M. The capture nucleotide sequences were printed onto
microscopic glass slides with a home made robotic device using 250
.mu.m diameter pins. The spots have 400 .mu.m in diameter and the
volume dispensed is about 0.5 nl. Slides were dried at room
temperature and stored at 4.degree. C. until used.
[0153] The capture probes used in this experiment have the
following sequences:
TABLE-US-00002 TP35S (SEQ ID NO:23): 5'Amine-
GTCATCCCTTACGTCAGTGGAGATAT -3' TGUT (PCR control) (SEQ ID NO:24):
5'Amine- GGGACTGGCTGCTATTGGGCGAA -3' TPAT1 (SEQ ID NO:25): 5'Amine-
CTGTGTATCCCAAAGCCTCATGCaa -3'
[0154] Each capture probe comprises a spacer of 95 base long at its
5' end which has the following sequence:
TABLE-US-00003 ATAAAAAAGTGGGTCTTAGAAATAAATTTCGAAGTGCAATAATTATTAT
TCACAACATTTCGATTTTTGCAACTACTTCAGTTCACTCCAAATTA.
PCR and Hybridization
[0155] PCR is designed for the amplification of the 35 promoter
element of DNA sample of a genetically modified organism (GMO) Bt11
from reference flour ERM-BF412f.
[0156] The primers used in this experiment have the following
sequences:
TABLE-US-00004 OP35SF (SEQ ID NO:26): 5'- Cy3-
CGTCTTCAAAGCAAGTGGATTG -3' OP35SR (SEQ ID NO:27): 5'-
TCTTGCGAAGGATAGTGGGATT -3'
[0157] The amplified product has part of one of its strand sequence
specific of capture molecules P35S (SEQ ID NO: 23).
[0158] A mix for PCR reaction is prepared as follows: for a final
volume of 100 .mu.l, we mix 10 .mu.l of PCR eppendorf buffer, 10
.mu.l of dNTP mix (each of dNTP at a final concentration of 200
.mu.M), 1 .mu.l of 20 .mu.M primer OP35SF-Cy3 labelled at 5' end
and 1 .mu.l of 20 .mu.M primer OP35SR, 2 .mu.l of Eppendorf Taq DNA
polymerase, 10 .mu.l of NaCl 600 mM, 55 .mu.l of water and 10 .mu.l
of 20 ng/.mu.l of DNA sample extracted from reference flour
ERM-BF412f.
[0159] 25 .mu.l of this PCR mix solution is loaded on the
micro-array framed by an hybridization chamber, of 9.times.9 mm
sealed with a smooth plastic coverslip (Grace Biolabs).
[0160] On the backside of the slide, we fix a special thermocouple
which is temperature controlled. The complete heating process test
bench is composed of the following relevant components:
"thermocouple": RS-COMPONENT no. 219-4321 Self adhesive
thermocouple Type K-Nickel Chromium/Nickel Aluminium, [0161]
"transmitter": RS-COMPONENT no. 363-0222 Transmitter temperature
thermocouple 4-20 mA, [0162] "converter": NATIONAL INSTRUMENTS
779026-01 USB-6009 48 Ksamples./sec DAQ multifonctions 14 bits for
USB, [0163] "heater": MINCO Heating thermofoil flexible heater:
Kapton 0.75''.times.0.75'' HK 5578 R 18.3 L12F.
[0164] The thermocouple, sticks as close as possible of the spot to
heat, measures the temperature thought the transmitter. This
temperature information is given to a computer via the converter.
Every 0.1 second, the software compares the temperature measured to
the temperature set point requested by the final user and the
controller adjusted the heating in order to provide the requested
temperature.
[0165] The slide is then entered upside down into the Axon scanner
(4100 personal) where it remains during the whole experiment.
Scanned is performed with the channel Cy3 at a gain of 500 with a
resolution of 10 micrometer.
[0166] The heating cover is then heated to 95.degree. C.
(denaturation) for 1 min then going to 56.degree. C. (annealing)
for 2 min and then for 1 min at 72.degree. C. (elongation). The
same cycle was repeated 39 times. The fluorescent light emission is
determined by scanning the micro-array surface starting 1 min after
the beginning of the annealing step (at 56.degree. C.) of the
cycles 6, 10, 14, 18, 22, 24, 26, 28, 30, 32, 33, 34, 35, 37, 38
and 40. The scanner uses as excitation light a laser which was
focussed on the surface of the support. The emission light is
detected and amplified by a photomultiplier. After image
acquisition, the scanned 16-bit images were imported to the
software, `Genepix 5" (Axon, Union city, Calif., USA) which was
used to quantify the signal intensities. The signal was quantified
on two capture probes P35S (SEQ ID NO: 23) and pat1 (SEQ ID NO: 25)
present in six replicates on the array. The local background was
subtracted and signal minus background is plotted against the
number of cycles. The arrays also contained capture probes for
negative hybridization control and positive detection control
labeled with Cy3 present in quadruplicate on the array.
[0167] Result of the real-time PCR on micro-array is presented in
FIG. 4. The result shows the appearance of a signal on the specific
capture probe P35S at cycle 22. The signal continues to increase
regularly until cycle 40. There is no signal observed on capture
probe pat1.
* * * * *