JP2003534551A - Method and computer software product for transcription annotation - Google Patents

Abstract

  (57) [Summary] Methods and computer software products are provided for transcript annotation. In one embodiment of the invention, regions of the genome where the intensity of hybridization of all probes is above a threshold (usually the level of non-specific hybridization). This region can be identified by aligning the probes to the genome and walking across the genome to find a region where all successive probes have an intensity above a threshold level.

Description

Detailed Description of the Invention

(Related Application) This application is related to USSN 09 / 641,081 filed on August 16, 2000.
USSN 60 / 205,432, filed May 19, 2000; and 2;
Claimed priority of USSN 60 / 206,866, filed May 24, 000, which are incorporated by reference in their entirety for all purposes.

[0002]   (Field of the invention)   The present invention relates to genetic analysis and bioinformatics.

BACKGROUND OF THE INVENTION Many biological functions are mediated by alterations in the copy number of gene DNA, through alterations in the level of transcription of a particular gene (eg, initiation control, RNA precursors). Preparation,
By controlling the expression levels of various genes, either by (such as by RNA processing) or through changes in protein synthesis. For example, control of cell cycle and cell differentiation, and disease are characterized by alterations in the transcriptional levels of groups of genes.

Complete genomic sequences of more than 20 bacterial genomes are already publicly available.
However, little is known about the transcribed region of the genome. Methods for discovering transcribed regions and their functional significance are needed.

SUMMARY OF THE INVENTION The present invention provides methods and computer program products for identifying transcripts. This method and computer program product has applications in genetic research, drug discovery, diagnostics and pharmacogenetics.

In some embodiments, a nucleic acid probe for a region of the genome is hybridized with a biological sample derived from a species bearing that genome. Hybridization signals are analyzed to determine potential transcripts.
Regions of the genome where the intensity of hybridization of all probes is above a threshold (usually the level of non-specific hybridization) are identified. This region is identified by aligning the probe to the genome; walking all over the genome to find regions where all consecutive probes have intensities above a threshold. In some embodiments, the threshold can be the strength of the corresponding mismatch probe.

The identified region potentially corresponds to one transcript. In some embodiments, the intensity of probe hybridization in each of the identified regions is further analyzed to detect changes in size. For example, 4
In the identified region covered by 0 probe, the intensities of the first 20 probes have the same intensity, and the last 20 probes also have the same intensity; and the intensities of the first 20 probes Is twice the intensity of the last 20 probes, this region may contain at least two additional transcribed regions: the first and the second 20.
, The second is covered by the last 20 probes.

In some embodiments, probe intensity is used to identify the location of transcripts with respect to computer annotation. If multiple probes have the same intensity, it is more likely to be one transcript.

In some other embodiments, the strength of the probe in the non-coding region is used to identify previously unidentified transcripts. If multiple probes have the same intensity, it is more likely to be one transcript.

In some further embodiments, probe strength at the 5 ′ and 3 ′ ends of the coding region is used to identify potential control sequences, termination sequences or regions with unknown function.

In some further embodiments, multiple contiguous transcripts containing coding and non-coding regions with the same probe strength are likely one operon.

In another aspect of the invention, a computer software product for transcription annotations is provided. In some embodiments, the computer software product has computer program code for entering probe intensity, computer program code for identifying genomic regions where the intensity of the probe exceeds a threshold, and regions of the same probe intensity. Includes code to compare the intensities of the probes in the genomic region to Each of these regions can be designated as a potential transcript. These codes may be stored on any computer readable medium, including CD-ROM or hard drive.

Detailed Description of the Preferred Embodiments A detailed reference will now be made to the preferred embodiments of the present invention. Although the present invention is described in connection with the preferred embodiments, they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention.

As will be appreciated by one of skill in the art, the present invention may be embodied as a method, data processing system or program product. Accordingly, the present invention may take the form of a data analysis system, method, analysis software, or the like. Software written in accordance with the present invention may be a memory, hard drive, DVD ROM or CD.
It is stored in some form of computer readable medium, such as a ROM, or is transmitted over a network and executed by a processor.

FIG. 1 illustrates a computer system that may be used to execute the software of an embodiment of the invention. FIG. 1 shows a computer system 1 comprising a display 3, a screen 5, a cabinet 7, a keyboard 9 and a mouse 11. Mouse 11 may have one or more buttons for interacting with the graphic user interface. The cabinet 7 is preferably a CD-RO
M or DVD-ROM drive 13, saves system memory and hard drive (eg, FIG. 2), which stores software programs incorporating computer code for implementing the present invention, data for use with the present invention, etc. And can be used for searching. Although CD15 is shown as an exemplary computer-readable medium, other computer-readable storage media (floppy disk, tape, flash memory, system memory, and hard drive) may be utilized. Further, a data signal embodied in a carrier wave (eg, in a network including the Internet) can be a computer-readable storage medium.

FIG. 2 shows a system block diagram of a computer system 1 used to execute the software of an embodiment of the present invention. As shown in Figure 1,
The computer system 1 includes a monitor 3, a keyboard 9, and a mouse 1.
1 is provided. Computer system 1 further includes central processing unit 51, system memory 53, fixed storage 55 (eg, hard drive), removable storage 57 (eg, CD-ROM), display adapter 59, sound card 61, speaker 63, and network. A subsystem such as the interface 65 is provided. Other computer systems suitable for use with the present invention may include additional subsystems or fewer subsystems. For example, another computer system may include more than one processor 51 or cache memory. A computer system suitable for use with the present invention may also be incorporated into the measuring device. This embedded system
For example, it may control, for example, the operation of the GeneChip® Probe Array Scanner as well as the execution of the computer code of the present invention.

In one aspect of the invention, methods, computer software products are provided for annotating genomic transcripts according to transcription patterns (transcript format and quantification). The method involves detection of transcripts and analysis of patterns of transcription.

I. Transcription Detection A. Nucleic Acid Samples Transcription patterns (transcript morphology and quantification) can be determined by examining samples containing transcripts. In some preferred embodiments, a biological sample is obtained from cells of the species of interest (the species whose genome is annotated, eg, E. coli, yeast, dog, or human) and the nucleic acid sample is prepared. To do.

Those skilled in the art will appreciate that it is desirable to have a nucleic acid sample that contains a target nucleic acid sequence that reflects the transcript of the cell of interest. Thus, a suitable nucleic acid sample may contain the transcript of interest. However, a suitable nucleic acid sample may contain nucleic acids from the transcript of interest. As used herein, transcript-derived nucleic acid refers to a nucleic acid to which the mRNA transcript or its successor ultimately serves as a template for nucleic acid synthesis. Therefore, the cDNA reverse transcribed from the transcript, this c
RNA transcribed from DNA, DNA amplified from this cDNA, RNA transcribed from this amplified DNA, etc. are all derived from the transcript, and the detection of such derived products can be detected in a sample. The presence and / or abundance ratio of the original transcript is indicated. Thus, suitable samples include, but are not limited to: a transcript of a gene, a cDNA reverse transcribed from this transcript,
CRNA transcribed from this cDNA, DNA amplified from this gene, RNA transcribed from the amplified DNA, etc. Transcripts, as used herein, include, but are not limited to: pre-mRNA early transcripts, transcription processing intermediates, mature RNAs and degradation products.

In one embodiment, such a sample is a homogenate of cells or tissues or other biological samples. Preferably such sample is a total RNA preparation of a biological sample. More preferably, in some embodiments, such nucleic acid sample is a total mRN isolated from the biological sample.
It is A. Those skilled in the art will appreciate that the total mRNA prepared using most methods will
It is understood to include not only RNA, but also RNA processing intermediates and early pre-mRNA transcripts. For example, total mRNA purified using a poly (T) column
Comprises an RNA molecule with a poly (A) tail. These poly A + RNA molecules can be mature mRNA, RNA processing intermediates, early transcripts, or degradation intermediates.

The biological sample can be any biological tissue or fluid or cell sample. Representative samples include, but are not limited to, sputum, blood, blood cells (eg, white blood cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells derived therefrom. . The biological sample also
It may include sections of tissue such as frozen sections obtained for histological purposes.

Another representative source of biological samples is cell cultures, which can manipulate gene expression status to explore correlations between genes.

Those skilled in the art will appreciate that it is desirable to inhibit or destroy RNase present in the homogenate, after which the homogenate can be used for hybridization. Methods of inhibiting or destroying nucleases are well known in the art. In some preferred embodiments, cells or tissues are homogenized in the presence of chaotropic agents to inhibit nucleases. In some preferred embodiments, RNase is inhibited or destroyed by heat treatment followed by proteolytic enzyme treatment.

Methods of isolating total RNA are well known to those of skill in the art. For example, methods for isolating and separating nucleic acids are described in detail below: Laboratory Technology.
ues in Biochemistry and Molecular Bi
Chapter 3: Hybridization With Nucleic
Acid Probes, Part I, Theory and Nucle
ic Acid Preparation, P.I. Edited by Tijssen, Elsev
ier, N.M. Y. (1993) and Laboratory Technology.
es in Biochemistry and Molecular Bio
Chapter 3 of the Logic: Hybridization With Nucleic
Acid Probes, Part I, Theory and Nuclei
c Acid Preparation, P.C. Edited by Tijssen, Elsevi
er, N.N. Y. (1993).

In a preferred embodiment, total RNA is isolated from a given sample using, for example, the acid guanidinium-phenol-chloroform extraction method, and poly.
A ⁺ mRNA is isolated by using oligo dT column chromatography or (dT) n magnetic beads (eg Sambrook et al., Mole.
color Cloning: A Laboratory Manual (2nd
Edition), Volumes 1-3, Cold Spring Harbor Laborato
ry (1989), or Current Protocol in Mole
circular Biology, F.M. Edited by Ausubel et al., Green Publ
Ishing and Wiley-Interscience, New Yo
rk, 1987).

In one preferred embodiment, the total RNA is RNeasy Total RN.
It is isolated from mammalian cells using the A isolation kit (QIAGEN). When mammalian tissue is used as a source of RNA, TRIzol Reagent (
Commercially available reagents such as GIBCOL Life Technologies) are used. A second cleaning after ethanol precipitation of TRIzol extraction with the RNeasy total RNA isolation kit may be beneficial.

Schmitt et al. (1990) Nucleic Acid Res. , 18:
3091-3092 describes the warm phenol protocol for total RN of yeast cells.
Useful for isolating A.

High quality mRNA can be obtained, for example, by first isolating total RNA and then Oligo.
It can be obtained by isolating mRNA from total RNA using the tex mRNA kit (QIAGEN).

Total RNA from prokaryotes (eg, E. coli. Cells) is available from Epicent
MasterP from re Technologies (Madison, WI)
ure complete DNA / RNA purification kit protocol.

Frequently, it is desirable to hybridize after amplifying the nucleic acid sample. Those of skill in the art will be familiar with using methods that maintain or control the relative frequency of amplified nucleic acids to achieve quantitative amplification when quantitative results are desired, whatever the amplification method used. Understand that you must turn on.

Methods of “quantitative” amplification are well known to those of skill in the art. For example, quantitative PCR involves co-amplifying known amounts of control sequences with the same primers. This provides an internal standard that can be used to calibrate the PCR reaction. The high density array can then include probes specific to an internal standard for quantification of amplified nucleic acids.

Other suitable amplification methods include, but are not limited to, the polymerase chain reaction (PCR) (Innis et al., PCR Protocols. Augui).
de to Methods and Application, Academ
ic Press, Inc. San Diego, (1990)), ligase chain reaction (LCR) (Wu and Wallace. Genomics, 4: 560.
(1989), Landegren et al., Science, 241: 1077 (1.
988) and Barringer et al., Gene, 89: 117 (1990)), transcription amplification.
on) (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:
1173 (1989)), and self-sustaining sequence replication (self-sustai).
Ned sequence replication) (Guatelli et al. P)
roc. Natl. Acad. Sci. USA, 87: 1874 (1990)).
.

Cell lysates or tissue homogenates often contain numerous inhibitors of polymerase activity. Therefore, RT-PCR typically first incorporates a step of isolating total RNA or mRNA for subsequent use as an amplification template. 1-tube mRNA capture method (one tube mRNA captu
re-method) for rapid RT-PCR (B
A poly (A) + RNA sample suitable for Oehringer Mannheim can be prepared. The captured mRNA is then transferred to the reverse transcription mixture (reverse t
It can be directly subjected to RT-PCR by adding a transcription mixture and then a PCR mixture (PCR mix).

In a particularly preferred embodiment, the sample mRNA is reverse transcribed with a reverse transcriptase and a primer consisting of oligo dT and a sequence encoding the phage T7 promoter to provide a single-stranded DNA template. The second DNA strand is polymerized using a DNA polymerase with or without a primer. (See US patent application Ser. No. 09 / 102,167 and US provisional patent application Ser. No. 60 / 172,340, both incorporated herein by reference for all purposes). After synthesis of double-stranded cDNA, T7 RNA polymerase was added and RN
Transcribe A from the cDNA template. Successive rounds of transcription from each single cDNA template yields amplified RNA. Methods of in vitro polymerization are well known to those of skill in the art. (Eg Sambrook, supra), and this particular method is described by Van Gelder et al., Proc. Natl. Acad. Sci.
USA, 87: 1663-1667, 1990. Furthermore, E
Berwine et al., Proc. Natl. Acad. Sci. USA, 89: 3
010-3014 achieves> 10 ⁶ -fold amplification of the original starting material using two rounds of amplification via transcription in vitro, which results in expression of expression even when the biological sample is limited. Provides a protocol that allows monitoring. 1
In one preferred embodiment, the in vitro transcription reaction can be combined with labeling of the resulting cRNA with biotin using the Bioarray High Yield RNA Transcription Labeling Kit (Enzo P / N900182).

The resulting cRNA can be fragmented prior to hybridization.
One preferred method for fragmentation uses Rnase-free RNA fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate). About 20 μg of cRNA is 8 μ
Mix with L fragmentation buffer. Water without Rnase is added to bring the volume to 40 μL. This mixture is incubated at 94 ° C for 35 minutes and cooled in ice.

It will be appreciated by those skilled in the art that the direct transcription method described above provides an antisense (aRNA) pool. When antisense RNA is used as the target nucleic acid, the oligonucleotide probes provided in the array are chosen to be complementary to a subsequence of the antisense nucleic acid. In contrast, if the target nucleic acid pool is a pool of sense nucleic acids, then the oligonucleotide probe is selected to be complementary to a subsequence of the sense nucleic acid. Finally, if the nucleic acid pool is double-stranded, the probe can be a probe of either sense, such that the target nucleic acid contains both the sense and antisense strands.

The above protocol includes methods for generating pools of either sense or antisense nucleic acids. In fact, one approach may be used to produce either the sense or antisense nucleic acids as desired. For example, the cDNA may be a vector (eg, Stratagene's p Bluscr
was cloned in the direction of ipt II KS (+) phagemid, so that the cDNA is flanked by the T3 and T7 promoters.
In vitro transcription with T3 polymerase produces one sense of RNA, where the sense depends on the orientation of the insert, whereas in vitro transcription with T7 polymerase produces RNA with the opposite sense. . Other suitable cloning systems include phage lambda vectors designed for Cre-loxP plasmid subcloning (eg, Palazzolo et al., Gene, 88: 2).
5-36, 1990).

The biological sample should contain nucleic acids that reflect at least some levels of the transcript present in the cell, tissue or organ of interest. In some embodiments, biological samples can be prepared from cells, tissues or organs in a particular condition. For example, if the dog is pregnant, total RNA is prepared from the pituitary gland of the dog. In another example, the sample is E. coli after treating the cells with IPTG. E. coli cells. Since certain genes are only expressed under certain conditions, biological samples induced under various conditions may be needed to observe all transcripts. In some examples, transcriptional annotation may be specific to a particular physiological, pharmacological or toxicological condition. For example, certain regions of the gene are transcribed only under certain physiological conditions. Transcriptional annotations obtained from a particular physiological condition using a biological sample may not be applicable to other physiological conditions.

B. Nucleic Acid Probe Assay Design One preferred method for transcript detection uses a high density oligonucleotide probe array. High density oligonucleotide probe arrays and their uses for transcription detection are described, for example, in US Pat. Nos. 5,800,992 and 6,0.
40,193 and 5,831,070.

Those skilled in the art will appreciate that a vast number of array designs are suitable for practicing the present invention. High density arrays typically include multiple probes that specifically hybridize to sequences of interest, including potential and putative transcripts. Moreover, in a preferred embodiment, the array comprises one or more control probes.

The high density array chip contains test probes. The probe ranges from about 5 to about 45 or about 5 to about 500 nucleotides in length, more preferably about 10 to about 40 nucleotides in length, and most preferably about 15 to about 40 nucleotides in length. It can be an oligonucleotide. In another particularly preferred embodiment, the probe is 20 or 25 nucleotides in length. In another preferred embodiment, the test probe is a double-stranded or single-stranded DNA sequence. DNA sequences are isolated or cloned from natural sources, or amplified from natural sources using natural nucleic acids as templates. These probes have a sequence that is complementary to a particular subsequence of the gene, which is designed for these probes to detect the expression of this gene. Thus, the test probe may specifically hybridize to the target nucleic acid (the target nucleic acid for the test probe to detect).

In addition to a test probe that binds to the target nucleic acid of interest, the high density array can include multiple control probes. Control probes are divided into three categories, which are referred to herein as 1) normalized controls; 2
) Expression level control; and 3) mismatch control. These controls are designed to contain at least one base different from the base of the target nucleic acid. A normalization control is an oligonucleotide or other nucleic acid probe that is complementary to a labeled reference oligonucleotide or other nucleic acid sequence that is added to a nucleic acid sample. The signals obtained from the normalization control after hybridization are hybridization conditions, label strength,
It provides controls for "reading" effects, and variations in other factors where the signal of complete hybridization can vary between arrays. In a preferred embodiment, the signal read from all other probes in the array (eg fluorescence intensity) is divided by the signal from the control probe (eg fluorescence intensity), thereby normalizing the measurement.

In fact, any probe can serve as a normalization probe. However, it is recognized that hybridization effects vary with base composition and probe length. Preferred normalized probes are chosen to reflect the average length of the other probes present in the array, although they can be chosen to cover a range of lengths. Normalized controls may also be selected to reflect the (average) base composition of the other probes in the array, but in a preferred embodiment only one or several normalized probes are used and these probes Are selected such that they hybridize well (ie, do not contain secondary structure) and do not match any target-specific probe.

Expression level controls are probes that specifically hybridize to genes that are constitutively expressed in the biological sample. Indeed, any constitutively expressed gene provides a suitable target for expression level control.
Representative expression level control probes include, but are not limited to, β-actin gene, transferrin receptor gene, GAPDH gene, and the like, and have a sequence complementary to a partial sequence of a constitutively expressed “housekeeping gene”. Have.

Mismatch controls can also be provided for probes to target genes, expression level controls, or normalization controls. Mismatch controls are oligonucleotide probes or other nucleic acid probes designed to match their corresponding test, target, or control probe except for the presence of one or more mismatched bases. A mismatched base is a base selected so that it is not complementary to the corresponding base in the target sequence (otherwise the probe hybridizes specifically). The test probe or control probe is expected to hybridize to its target sequence under one or more mismatches under appropriate hybridization conditions (eg, stringent conditions), while the mismatch probe will hybridize. Not selected (or hybridized to a significantly lesser extent). Preferred mismatch probes contain a central mismatch. Thus, for example, where the probe is a 20-mer, the corresponding mismatch probe is identical except for a single base mismatch at any of positions 6-14 (eg, replacing G, C, or T with A). Has a sequence (central mismatch).

Mismatch probes thus provide controls for non-specific binding or cross-hybridization to nucleic acids in the sample other than the target to which the probe is directed.

The difference in intensity of (I (PM) -I (MM)) between the perfectly matched and mismatched probes provides a good measure of the concentration of hybridized material.

The high density array may also include sample preparation / amplification control probes.
These are probes that are complementary to partial sequences of selected control genes. Because they usually do not occur in the nucleic acid of the particular biological sample being assayed. Suitable sample preparation / amplification control probes include, for example, a probe for a bacterial gene (eg, Bio B) when the sample of interest is a biologic of eukaryotic origin.

The RNA sample is then, prior to processing, a known amount of nucleic acid directed against a control probe for sample preparation / amplification. Quantitation of control probe hybridization for sample preparation / amplification then provides a measure of the alteration in large amounts of nucleic acid caused by processing steps (eg, PCR, reverse transcription, in vitro transcription, etc.).

In a preferred embodiment, oligonucleotide probes in a high density array are selected to specifically target nucleic acid targets directed with minimal non-specific binding or cross-hybridization under the particular hybridization conditions used. Be combined. The high-density arrays of the invention can include over 1,000,000 different probes, making it possible to provide any probe of a characteristic length that binds to a particular nucleic acid sequence. Thus, for example, a high density array can be used for IL-2 mRN
It may include any possible 20-mer sequence complementary to A.

However, there may be 20-mer subsequences that are not unique to IL-2 mRNA. Probes directed to these subsequences are expected to cross-hybridize by the appearance of their complementary sequences in other regions of the sample genome.
Similarly, other probes may simply not hybridize efficiently under hybridization conditions (due to, for example, secondary structure, or interaction with substrates or other probes). Therefore, in a preferred embodiment, probes exhibiting such poor specificity or hybridization efficiency are identified and either in the high density array itself (eg, during array production) or during post-hybridization data analysis. Can be included in

Probes as short as 15, 20 or 25 nucleotides are sufficient to hybridize to a subsequence of a gene, and for most genes a set of probes that perform well over a wide range of target nucleic acid concentrates. Exists. In a preferred embodiment, it is desirable to select a preferred or "optimal" subset of probes for each gene before synthesizing the high density array.

In one aspect of the invention, for transcriptional annotation, the probe array can include probes selected to direct regions of interest in the genome. This probe
The entire region of interest or a selected region of the region of interest may be tiled. Figure 3 shows one such probe array design. Multiple probes are selected to tile the gene sequence of interest without duplication. The method and strategy of tiling is discussed in considerable detail in published PCT Application No. 95/11995, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. To be done. However, in some embodiments overlapping probes may be preferred.

C. Formation of Nucleic Acid Probe Arrays Methods for forming high density arrays of oligonucleotides, peptides and other polymer sequences with a minimal number of synthetic steps are described in, for example, 5,143,854, 5,25.
2,743,5,384,261,5,405,783,5,424,186,
5,429,807, 5,445,946, 5,510,270, 5,677,
195, 5,571, 639, 6,040, 138, all incorporated herein by reference for all purposes. Oligonucleotide analog arrays can be synthesized on a solid substrate by a variety of methods including, but not limited to, photodirected chemical coupling, and mechanically directed coupling. (See Pirrung et al., US Pat. No. 5,143,854). (PCT Application No. WO 90/15070, and disclose methods of forming vast arrays of peptides, oligonucleotides and other molecules using, for example, photodirected synthetic techniques, Fodor et al., PCT Publication No. WO 92 / 100
92 and WO 93/09668 and US Pat. No. 5,677,195). (Fodor et al., Science, 251, 767-77, 1
See also 991). These steps for the synthesis of polymer arrays are
This is referred to as the VLSIPS ^™ procedure. Using the VLSIPS ^™ approach, one heterogeneous array of polymers is converted to a different heterogeneous array via simultaneous coupling at many reaction sites. (See US Pat. Nos. 5,384,261 and 5,677,195).

US Pat. No. 5,143,854 and PCT Patent Publication No. WO 90 /
As described in 15070 and 92/10092, the development of VLSIPS ^™ technology is considered to be a state of the art in the field of combinatorial synthesis and screening of combinatorial libraries.

Briefly, the photo-directed combinatorial synthesis of oligonucleotide arrays on glass surfaces proceeds using automated phosphoramidite chemistry and chip making techniques. In one particular implementation, the glass surface is derivatized with a silane reagent containing functional groups (eg, hydroxyl or amine groups blocked by photolabile protecting groups). Photolysis via a photolithographic mask was selectively used to expose the functional groups, which then readily reacted with the incoming 5'photoprotected nucleoside phosphoramidite. This phosphoramidite reacts only with these irradiated sites (and is therefore exposed by removal of the photolabile blocking group). Therefore, phosphoramidites are added only to those regions that were selectively exposed from the previous step. These steps are repeated until the desired array of arrays is synthesized on the solid surface. Combinatorial synthesis of different oligonucleotide analogs at different positions on the array is determined by the pattern of irradiation during synthesis and the order of addition of coupling reagents.

When oligonucleotide analogues with a polyamide backbone are used in the VLSIPS ^™ procedure, it is generally inadequate to use phosphoramidite chemistry to carry out the synthetic steps. This is because the monomers do not bond to each other via a phosphate bond. Instead, peptide synthesis methods are substituted. (See, eg, Pirrung et al., US Pat. No. 5,143,854).

Peptide nucleic acids are described, for example, in Biosearch, Inc. (Bedford,
MA), which contain a polyamide backbone and the bases found in naturally occurring nucleotides. Peptide nucleic acids can bind nucleic acids with high specificity and are considered “oligonucleotide analogs” for the purposes of this disclosure.

In addition to the methods described above, a further method that can be used to make an array of oligonucleotides on one substrate is described in PCT Publication No. WO93 / 09668. In the method disclosed in that application, the reagents are delivered to the substrate by either: (1) by flowing within defined channels in defined areas or (2) on defined areas. By "spotting" or (3) through the use of photoresist. However, other approaches and combinations of concentration and flow can be used. In each case, certain activated areas of the substrate are mechanically separated from other areas when the monomer solution is delivered to the various reaction sites.

Representative “flow channel” methods applied to the compounds and libraries of the invention can be generally described below. A variety of polymer sequences form flow channels on the surface of a substrate through which suitable reagents flow or into which suitable reagents are placed to form a substrate or solid support. Are combined in the selected area of. For example, assume that monomer "A" binds to a substrate in a selected area of the first group. If necessary, all or part of the surface of the substrate in all or part of the selected area is, for example, by flowing the appropriate reagent through all or some of the channels, or It is activated for binding by washing the entire substrate with. After placing the channel block on the surface of the substrate,
A reagent with monomer A flows or is placed there through all or some of the channels. This channel provides the fluid in contact with the first selected area, thereby allowing the monomer A to be directly or indirectly (via the spacer) on the substrate in the first selected area. Join).

Thereafter, the monomer B is attached to the second selected region, some of which are
It may be included between the first selected regions. The second selected area is via translation, rotation or displacement of the channel block on the surface of the substrate; via opening or closing of the selected valve; or deposition of a layer of chemical or photoresist. Is in fluid contacting the second flow channel via. If necessary,
The steps are performed to activate at least the second region. Thereafter, the monomer B is flowed through or placed in the second flow channel, the monomer B
To the second selected position. In this particular example, the resulting sequence bound to the substrate at this stage of processing is, for example, A, B or AB. This process is repeated to form a huge array of arrays of desired length at known locations on the substrate.

After the substrate is activated, monomer A can be flowed through some of the channels, monomer B can be flowed through the other channels, and monomer C can be flowed through another channel. Can be flowed through, and so on. In this manner, many or all of the reaction regions are reacted with the monomer before the channel block is removed or the substrate is washed and / or reactivated. The number of wash and activation steps can be minimized by using some or all of the available reaction zones simultaneously.

One of ordinary skill in the art will recognize that there are alternative methods of forming channels or otherwise protecting portions of the surface of the substrate. For example, according to some embodiments, a protective coating (eg, a hydrophilic or hydrophobic coating (depending on the nature of the solvent)), sometimes in combination with a substance that promotes wetting by the reaction solution in other regions, Used on the part of the substrate to be protected. In this manner, the flowing solution is further prevented from passing outside these desired channels.

High density nucleic acid arrays can be made by attaching moieties, pre-synthesized nucleic acids or naturally occurring nucleic acids. Synthetic or natural nucleic acids can be used for light-directed targeting and oligonucleic acids as disclosed in US Pat. No. 5,040,138 and its patent application, previously incorporated by reference for all purposes. It is attached to a specific location on the substrate by nucleotide-directed targeting. Nucleic acids can also be directed to specific locations in much the same way as flow channel methods. For example, nucleic acid A may be delivered to and associated with a suitably activated reaction zone of the first group. Nucleic acid B is then delivered to and reacts with the activated reaction zones of the second group. The nucleic acid is attached to the selected area. Another embodiment uses a dispenser that is removed region by region to attach nucleic acids to specific spots. Typical dispensers include micropipettes or capillary pins for delivering nucleic acids to a substrate, and robotic systems for controlling the position of the micropipette with respect to the substrate. In other embodiments, the dispenser is a tube,
Including manifolds, pipettes or arrays of capillary pins, etc., so that
Various reagents can be delivered to the reaction zone simultaneously.

(D. Hybridization of Nucleic Acid Sample to Probe Array) Nucleic acid hybridization is performed under the condition that the probe and its complementary target can form a stable hybrid duplex through complementary base pairs. It simply comprises the step of contacting with the target nucleic acid. Nucleic acids that do not form hybrid duplexes are then washed away, leaving hybridized nucleic acids that are typically detected through detection of the attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under low stringency conditions (eg, low temperature and / or high salt), hybrid duplexes (eg, DNA: DNA, RNA: RNA or RNA :) even if the annealed sequences are not perfectly complementary. DNA) is formed. Therefore, the specificity of hybridization is reduced at lower stringency. Conversely, at high stringency (eg, higher temperature or lower salt) successful hybridization requires slight mismatches.

One of ordinary skill in the art will appreciate that hybridization conditions may be selected to provide any degree of stringency. In a preferred embodiment, in this case the hybridization is performed with low stringency (6 × SSPE-T (37C) (0.005% Triton) to ensure hybridization.
X-100)) and then subsequent washes to remove mismatched hybrid duplexes at high stringency (eg 1 × SSPE-T ().
37C)). Sequential washes will be of increasing high stringency (eg, 0.25 ×) until the desired level of hybridization specificity is obtained.
SSPE-T (until reduced to 37 C to 50 C). Stringency can also be increased by the addition of agents such as formamide. Hybridization specificity can be assessed by comparison of hybridization to a test probe with hybridization to various controls that may be present (eg, expression level control, normalization control, mismatch control, etc.).

In general, there is a relationship between hybridization specificity (stringency) and signal strength. Thus, in a preferred embodiment, washes can be performed at the highest stringency that produces consistent results and provides a signal intensity greater than about 10% of the background intensity. Thus, in a preferred embodiment, the hybridized array may be washed with successive higher stringency solutions and read between each wash. Analysis of the data set thus generated reveals the above wash stringency with which the hybridization pattern is not significantly altered and which provides the appropriate signal for the particular oligonucleotide probe of interest.

In a preferred embodiment, the background signal is a detergent (eg C-TAB) during hybridization to reduce non-specific binding.
Or it is reduced by the use of blocking agents (eg sperm DNA, cot-1 DNA, etc.). In a particularly preferred embodiment, this hybridization is performed in the presence of about 0.5 mg / ml DNA (eg herring sperm DNA). The use of blocking agents in hybridization is well known to those of skill in the art (see, eg, P. Tijssen, supra, Chapter 8).

The stability of duplexes formed between RNA or DNA is generally determined in solution by R
The order is NA: RNA> RNA: DNA> DNA: DNA. Long probes
Mismatch discrimination with better duplex stability with the target but less than shorter probes (mismatch discrimination is the observed hybridization signal ratio between a perfectly matched probe and a probe with one base mismatch). Only). Shorter probes (e.g. 8-mer) discriminate mismatches very well, but have poor overall duplex stability.

Altering the thermal stability (T _m ) of a duplex formed between a target and a probe, for example using known oligonucleotide analogs, can improve duplex stability and mismatches. Allows optimization of identification. One useful aspect of altering the T _m is adenine - results from the fact that it has a cytosine (G-C) lower than the duplex T _m - thymine (A-T) duplexes guanine. This is partly due to the fact that AT duplexes have two hydrogen bonds per base pair, while GC duplexes have three hydrogen bonds per base pair. In heterogeneous oligonucleotide arrays where there is a heterogeneous distribution of bases, it is generally not possible to optimize the hybridization for each oligonucleotide probe at the same time. Thus, in some embodiments it may be desirable to selectively destabilize the heavy chain at GC and / or increase the stability of the AT duplex. This is, for example, G
By substituting hypoxanthine for the guanine residue in the probe of the array forming the -C duplex, or by replacing the adenine residue in the probe forming the AT duplex with 2,6 diaminopurine Or by using tetramethyl ammonium chloride (TMAC1) salt in place of NaCl.

The altered double-stranded stability confirmed using the oligonucleotide analog probe follows, for example, the fluorescence signal intensity of the oligonucleotide analog array hybridized with the target oligonucleotide over time. Can be confirmed. This data allows optimization of specific hybridization conditions (eg at room temperature) (for subsequent simplified diagnostic applications).

Another method of assessing altered duplex stability is by following the signal intensity generated during hybridization over time. DNA target and D
Previous experiments using NA chips showed that the signal intensity increased over time and that the more stable duplex produced faster and higher signal intensity than the less stable duplex. These signals reach a plateau or "saturation" after a certain time due to all the binding sites being occupied. These data allow optimization of hybridization and determination of the best conditions at a particular temperature.

Methods for optimizing hybridization conditions are well known to those of skill in the art (eg, Laboratory Technologies in Biochemis.
try and Molecular Biology, Volume 24: Hybri
dization With Nucleic Acid Probes, P.M.
Tijssen, Elsevier eds. Y. , 1993).

E. Signal Detection In a preferred embodiment, hybridized nucleic acid is detected by detecting one or more labels bound to the sample nucleic acid. The label can be incorporated by any of numerous means well known to those of skill in the art. However, in a preferred embodiment, the label is co-incorporated during the amplification step in the preparation of the sample nucleic acid. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide labeled amplification products. In a preferred embodiment, labeled nucleotides as described above (e.g.,
Transcription amplification with fluorescein-labeled UTP and / or CTP) incorporates a label into the transcribed nucleic acid. Alternatively, cDNA is synthesized using an RNA sample as a template and cRNA is synthesized using in vitro transcription (IVT) using cDNA as a template. Biotin labeling was performed using IVT reaction (Enzo Bi
oarray high yield labeling kit).

Alternatively, the label may be labeled from the original nucleic acid sample (eg, mRNA, polyAmRN).
A, cDNA, etc.) or directly to the amplification product after amplification is complete. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example, nick translation, or kinase treatment of nucleic acids, followed by labeling of sample nucleic acid
For example, end labeling (for example, using labeled RNA) by binding (ligation) of a nucleic acid linker to be linked to a fluorophore is mentioned.

Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. . Labels useful in the present invention include biotin for staining with a labeled streptavidin conjugate, magnetic beads (eg, Dynabea).
ds ^™ ), fluorescent dyes (eg, fluorescein, Texas Red (texas
red), rhodamine, green fluorescent protein, etc.), radiolabel (eg, ³ H)
, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P), enzymes (eg horseradish peroxidase, alkaline phosphatase and others commonly used in ELISA),
And colloidal gold or colored glass or plastic beads (
Colorimetric labels such as polystyrene, polypropylene, latex, etc.). Patents teaching the use of such labels include US Pat.
No. 17,837; No. 3,850,752; No. 3,939,350; No. 3,996,345; No. 4,277,437; No. 4,275,149; And No. 4,366,241.

Means for detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters and fluorescent markers can be detected using photodetectors to detect emitted light. Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and the colorimetric label is the colored label. Is detected by simply visualizing. One particularly preferred method uses colloidal gold which can be detected by measuring scattered light.

The label may be added to the target (sample) nucleic acid before or after hybridization. So-called "direct labels" are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, so-called "indirect labels" are attached to the hybrid duplex after hybridization. Often, the indirect label is attached to the binding moiety attached to the target nucleic acid prior to hybridization. Thus, for example, the target nucleic acid can be biotinylated prior to hybridization. After hybridization, the avidin-conjugated fluorophore binds to the biotin-bearing hybrid duplex, providing a label that is easily detected. For a detailed review of methods for labeling nucleic acids and detecting labeled hybridized nucleic acids. (Laborato
ry Technologies in Biochemistry and Mo
Lecture Biology, Volume 24: Hybridization W
it Nucleic Acid Probes, P.I. Edited by Tijssen, E
Isevier, N.M. Y. , 1993).

Fluorescent labels are preferred and are easily added during the in vitro transcription reaction. In a preferred embodiment, fluorescein-labeled UTP and CTP are incorporated into the RNA produced during the in vitro transcription reaction as described above.

Labeled targets (samples) hybridized to the probes of the high density array
Means of detecting nucleic acids are known to those of skill in the art. Thus, for example, if a colorimetric label is used, simple visualization of the label is sufficient. When radiolabeled probes are used, detection of radiation (eg, with photographic film or solid state detectors) is sufficient.

However, in a preferred embodiment, the target nucleic acid is labeled with a fluorescent label and the localization of this label on the probe array is achieved by fluorescence microscopy. The hybridized array is excited by a light source with excitation light of a specific fluorescent label, and the fluorescence at the emission wavelength generated is detected. In a particularly preferred embodiment, the excitation light source is a laser suitable for exciting fluorescent labels.

A confocal microscope can be automated with a computer controlled stage to automatically scan through a high density array. Similarly, the microscope may be equipped with a photo-converter (photomultiplier, solid-state array, CCD camera, etc.), which is connected to an automated data collection system and is equipped with a high The fluorescent signal generated by the hybridization is automatically recorded. Such an automated system is described in US Pat. No. 5,143,854,
PCT application 20 92/10092 and US application serial number 08/195
, 889 (filed Feb. 10, 1994). The use of laser irradiation coupled with an automated confocal microscope for signal detection allows detection at resolutions above about 100 μm, more preferably above 50 μm, and most preferably above 25 μm.

Those skilled in the art will appreciate that the methods for assessing hybridization results will vary with the nature of the particular probe nucleic acid used and the controls provided. In the simplest embodiment, a simple quantification of fluorescence intensity for each probe is determined. It measures the signal intensity of the probe at each position on the high density array (indicating a different probe) (eg, if the label is a fluorescent label, detection of the amount of fluorescence (intensity) is at each position on the array). Generated by a fixed excitation irradiation in). Comparing the absolute intensity of an array hybridized to nucleic acid from a "test" sample with the intensity produced by a "control" sample is a measure of the relative expression of nucleic acid hybridized to each of its probes. I will provide a.

However, one skilled in the art will understand that the hybridization signal will vary with the strength of hybridization efficiency, the amount of label on the sample nucleic acid, and the amount of the particular nucleic acid in the sample. Representative nucleic acids present at very low levels (eg <1 pM) give very weak signals. At somewhat lower levels of concentration, this signal becomes virtually indistinguishable from the background.
In assessing hybridization data, threshold intensity values can be selected below the signals that do not count as essentially indistinguishable from the background.

(F. Transcriptional annotation
)) In some embodiments, a nucleic acid probe designed to be complementary to a region of the genome hybridizes to a nucleic acid sample from a species having that genome. Hybridization signals are analyzed to determine potential transcripts. Certain regions of the genome in which the hybridization intensities of all probes are above a threshold (usually the level of non-specific hybridization) are identified. This region can be identified by aligning the probe to the genome; walking through the genome to find regions where all consecutive probes have intensities above the threshold.
In some embodiments, the threshold may be the intensity of the corresponding probe designed to include the mismatch.

This region was identified as potentially matching one or more transcripts. In some embodiments, the hybridization intensities of the probe and each of the identified regions are further analyzed to detect changes in size. For example, in the identified region covered by 40 probes, if the intensities of the first 20 probes have similar intensities, the remaining 20 probes also have similar intensities; And if the intensity of the first 20 probes is twice that of the remaining 20 probes, this region may contain at least two separately transcribed regions:
The first region is covered by the first 20 probes and the second region is covered by the remaining 20 probes.

In some embodiments, probe intensity is used to identify the location of the transcript with respect to computer annotation. Multiple probes with similar intensities offer a high probability of being a single transcript.

In some other embodiments, probe strength in the non-coding region is used to identify previously unidentified transcripts. Multiple probes with similar intensities give a high probability of being a transcript.

In some further embodiments, probe strength at the 5 ′ and 3 ′ ends of the coding region is used to identify potential regulatory regions, termination sequences, or regions with unknown function. .

In some further embodiments, multiple contiguous transcripts (including coding and non-coding regions) with similar probe strengths provide a high likelihood of being an operon.

In another aspect of the invention, a computer software product for transcription annotation is provided. In some embodiments, the computer software product comprises a computer program code for entering probe intensity, a computer program code for identifying a genomic region, where the intensity of the probe is above a threshold, and A code is included to compare the intensity of the probe to the genomic region to identify regions of similar probe intensity.
Each region can be designated as a potential transcript. The code may be stored on any computer readable medium, including CD-ROM or hard drive.

[0092]   (II. Example)   This embodiment is based on E. 3 shows transcriptional annotation of several operons of E. coli.

A) RNA Isolation and cDNA Synthesis Single colony E. coli K-12 (MG1655) with 5 ml of Lur
Inoculated into ia-Bertini (LB) broth and grown overnight at 37 ° C. The next day, 20 ml of LB broth was inoculated with 0.2 ml of overnight culture and incubated at 37 ° C. with constant ventilation to an optical density (OD600) of 0.8.
Were grown in. IPTG was added 30 minutes before harvesting the bacteria to induce the lac operon.

Total RNA was collected from Epicentre Technologies (Madiso).
n, WI) was used to isolate from these cells using a MasterPure complete DNA / RNA purification kit. Cultures were briefly centrifuged at 5000 xg, resuspended in lysis buffer and RNA isolation proceeded according to the manufacturer's protocol. The isolated RNA was resuspended in diethyl pyrocarbonate (DEPC) treated water and
Quantification based on absorbance at nm and stored in aliquots at -20 ° C until further use.

CDNA was synthesized using full length total RNA. 9 mg of total RNA was reverse transcribed using random hexamers as primers. RNA to RNAse H and R
It was removed using a cocktail of NAseA. After purification of the cDNA (50% yield), a partial DNAseI digestion was performed. The cDNA fragment was labeled with Biotin.
-Biotin labeling with terminal transferase using ddATP as substrate. 2 μg of labeled cDNA was hybridized to the probe array. 7
0 ng of full length cDNA was used for operon testing by RT-PCR.
Standard PCR was performed with different combinations of forward and reverse primers from each gene. The positions of the primers are shown in the figure. A template made without reverse transcriptase was used as a control.

A. Identification of Transcription Initiation and Transcript Elongation FIG. 4 shows the genomic region containing the genes metA, aceB, aceA and aceK (top box). These genes have been previously described by Blattner et al. R. Blattner et al., 1997. The complete Gen
ome sequence of Escherichia coli K-1
2. Science: 277; 1453-1462. The vertical bar below the schematic showing the genomic region shows the intensity of the probe. The scale is provided on the left. The intensity of this probe is 100 in the region between metA and aceB.
It varies around ˜10,000, indicating that the gene metA is in a transcript different from that of the gene aceB. Gene metA and gene ac
To confirm that eB is not transcribed as a unit, RT-PCR
Carry out. When the gene metA and the gene aceB are transcribed as a unit, the product of RT-PCR is a 1.5 kb fragment. As shown in the lower part of FIG. 4, no putative 1.5 kb fragment was found, which is consistent with the finding that the genes metA and aceB are not transcribed as a unit.

The probe that tiles the intergenic region between metA and aceB was also hybridized with similar strength to the probe that tiles metA and aceB, respectively, which contained several genes. Interregions are also shown to be transcribed. Therefore, this transcript is 5 of the Blattner gene.
May extend beyond'or 3 '.

Similarly, in FIG. 5, a transcript extending in the intergenic region between the yadQ gene and the yadR gene was detected.

FIG. 6 shows that at least the genes ribF, ileS, lspA, slpA and l.
3 shows the identification of an operon containing ytB. The strength of the probe targeting the intergenic region between ribF and ileS was similar to the strength of the probe targeting the ribF gene and ileS gene. Similarly, a probe targeting the intergenic region between the genes lspA and sipA had similar strength to the probe targeting this gene. This result indicates that 5 adjacent genes can be transcribed as one operon. To confirm this result, RT-PCR was performed on the putative product over these intergenic regions. At the bottom of Figure 6, RT
-Shows the results of PCR experiments. A transcript (0.94 kb fragment) containing an intergenic region between the ribF and ileS genes was identified. A transcript (0.94 kb and 1.84 kb fragment) containing an intergenic region between lspA and sipA was also found. It was confirmed that these transcripts transcribed the intergenic region as shown by probe array experiments.

Similarly, FIGS. 7-11 show identification of additional operons using oligonucleotide probe arrays. Some results were confirmed by RT-PCR experiments as described for the operon shown in Figure 6.

FIG. 12 shows the genomic region containing tnaC, tnaA and tnaB. tn
The upstream region of aA is the transcriptional regulatory region (tnaL), which contains a small translated regulatory protein tnaC.

FIG. 13 shows the identification of the transcribed region (indicated by the arrow). This region was not previously known as the transcribed region.

CONCLUSION The present invention provides a greatly improved method for transcriptional annotation. It is understood that the above description is intended to be illustrative and not limiting. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description. For purposes of illustration, the present invention has been described primarily with respect to the use of high density oligonucleotide arrays, although other nucleic acid arrays, other methods of measuring transcription levels and gene expression to monitor protein levels may be used. Are easily recognized by those skilled in the art.

The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[Brief description of drawings]

FIG. 1 illustrates one example of a computer system that may be used to execute the software of an embodiment of the present invention.

FIG. 2 shows a system block diagram of the computer system of FIG.

[Figure 3]   FIG. 3 shows one design of expression arrays useful in embodiments of the invention.

[Figure 4]   Figure 4 shows initiation of transcription and identification of transcription elongation.

[Figure 5]   FIG. 5 shows transcription elongation.

[Figure 6]   FIG. 6 shows the identification of operons.

[Figure 7]   FIG. 7 shows the identification of operons.

[Figure 8]   FIG. 8 shows the identification of operons.

[Figure 9]   FIG. 9 shows the identification of operons.

[Figure 10]   FIG. 10 shows the identification of operons.

FIG. 11   FIG. 11 shows the identification of operons.

[Fig. 12]   FIG. 12 shows the identification of control elements.

[Fig. 13]   FIG. 13 shows the identification of previously unknown transcripts.

─────────────────────────────────────────────────── ─── Continued front page    (31) Priority claim number 09 / 641,081 (32) Priority date August 16, 2000 (August 16, 2000) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CO, CR, CU, CZ, DE , DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, I S, JP, KE, KG, KP, KR, KZ, LC, LK , LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, P T, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Zingeras, Thomas             United States California 92024,               Encinitas, Crest drive             1541 F-term (reference) 4B063 QA01 QA18 QQ42 QQ52 QR08                       QR42 QR55 QR62 QR82 QS25                       QS34 QX02

Claims (14)

[Claims] 1. A method for identifying potentially transcribed regions of a genome, the method comprising: hybridizing a plurality of nucleic acid probes with a nucleic acid sample, wherein the nucleic acid sample is Including a transcript from the genome, wherein the probe targets a region of the genome; and the transcribed region is above the threshold for hybridization of all consecutive probes targeting the region. Identifying a region of the genome that is
Including the method. 2. The method of claim 1, wherein the probe is an oligonucleotide. 3. The method of claim 2, wherein the oligonucleotide is immobilized on a substrate. 4. The method of claim 1, wherein the threshold is non-specific binding. 5. The method of claim 4, wherein the non-specific binding is measured using a probe designed to contain at least one mismatched base. 6. The method of claim 1, wherein the hybridization of the probe targeting a partial region is similar and the partial region is designated as the transcribed region. A method comprising identifying a region. 7. The method of claim 6, wherein the genome is of prokaryotic origin. 8. The method of claim 7, wherein the transcribed region is an operon. 9. The method of claim 8, wherein the prokaryote is a bacterium. 10. A computer software product, comprising: a) computer program code for receiving a plurality of hybridization intensities, each of said intensities being among a plurality of probes to a nucleic acid sample. Computer program code reflecting one hybridization and wherein the probe targets the genome; b) a computer program code for identifying a region of the genome, wherein the intensity of the probe for the region is above a threshold value. A computer software product comprising: a computer program code; and c) a computer-readable medium for storing the code. 11. The computer software product of claim 10, further comprising computer program code identifying a range of the region, wherein the intensities of the probe for the range are similar. Computer software product. 12. The computer software product of claim 11, wherein the difference in the intensity of the probe with respect to the range is within a factor of two. 13. The computer software product of claim 12, wherein the difference is within 150%. 14. The computer software product of claim 11, further comprising computer program code for indicating the range as a transcribed range.