Patent Application
20110015085
1. Field of Endeavor
The present invention relates to DNA libraries and more particularly to repetitive sequence-free DNA Libraries.
2. State of Technology
U.S. Pat. No. 6,841,347 for in vivo construction of DNA libraries issued Jan. 11, 2005 to Anntonis Aervos provides the following state of technology information, “A cDNA library is a collection of cloned DNA molecules propagated in an appropriate host. It is usually derived from the mRNA population of a particular cell, tissue or organ by reverse transcription, cloned into a vector molecule and propagated in an appropriate host cell. cDNA libraries are useful in numerous applications. cDNA libraries can be used to isolate and identify cell-specific expressed sequences. A cDNA clone isolated from a library can be sequenced and translated (e.g., by computer programs) to derive the primary amino acid sequence of the encoded protein or can be used as a labeled probe to investigate gene expression in vivo. cDNA libraries can also be used in a two-hybrid assay to screen a large number of candidate proteins and identify those which interact with a particular target protein. In this approach, cDNAs are incorporated into activation domain vectors to provide random proteins fused to an activation domain of a known transcription factor. Vectors encoding the target protein fused to the DNA binding domain of the transcription factor, and the library of activation domain hybrids are cotransformed into a reporter strain. Interaction of the target protein moiety of a target protein DNA binding domain fusion protein with a protein encoded by cDNA brings the DNA binding domain into proximity with the activation domain fused to the cDNA encoded protein. The resulting transcription identifies a positive clone. Once a positive clone has been identified, the gene corresponding to the interacting protein can be isolated and analyzed.”
Features and advantages of the present invention will become apparent from the following description. Applicants are providing this description, which includes drawings and examples of specific embodiments, to give a broad representation of the invention. Various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description and by practice of the invention. The scope of the invention is not intended to be limited to the particular forms disclosed and the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.
DNA libraries are used daily, in research laboratories and hospitals, as probes to locate abnormalities in chromosomes. Many birth defects such as Down's Syndrome and diseases like cancer are detected using fluorescently-labeled DNA probes made from these libraries. These probes can be made specific to particular chromosomes, or even to regions within chromosomes. However, to use these probes requires the co-hybridization of unlabeled DNA to block the repetitive elements of DNA in the probes. Without this addition, the probe is non-specific and will bind to every chromosome in the cell. The ability to make libraries that are free of these repetitive elements, and thus do not require blocking DNA to be added to the reactions, would represent a significant savings in cost for a research laboratory.
The present invention provides a system for making such libraries that holds for any species of animal. The present invention provides a method of creating a repetitive sequence-free DNA library. The method comprises the steps of providing a DNA library, providing an amplification mixture from the DNA library, and adding a repetitive sequence fraction DNA to the amplification mixture to produce the repetitive sequence-free DNA library. The present invention also provides a method of creating a whole chromosome painting probe. The method comprises the steps of providing a DNA library, providing an amplification mixture from the DNA library, adding a repetitive sequence fraction DNA to the amplification mixture to produce the repetitive sequence-free DNA library, and labeling the repetitive sequence-free DNA library to produce the whole chromosome painting probe. The present invention also provides a method of in-situ hybridization. The method comprises the steps of providing a DNA library, providing an amplification mixture from the DNA library, adding a repetitive sequence fraction DNA to the amplification mixture to produce the repetitive sequence-free DNA library, labeling the repetitive sequence-free DNA library to produce the whole chromosome painting probe, and using the painting probe in in-situ hybridization.
The present invention provides a simple method for producing significant volumes of chromosome-specific painting probes. The entire process can be done in less than one day and yields probes with high specificity without the use of additional competitor DNA. The present invention has use in research and in hospitals. Cytogenetic analysis is an important diagnostic tool in prenatal care, as well as oncology. These laboratories are set up to require processes that are as simple and foolproof as possible. To remove an element of the in situ hybridization process, as well as decreasing the cost of the probes, will represent a significant improvement over existing technology.
The invention is susceptible to modifications and alternative forms. Specific embodiments are shown by way of example. It is to be understood that the invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.
The accompanying drawings, which are incorporated into and constitute a part of the specification, illustrate specific embodiments of the invention and, together with the general description of the invention given above, and the detailed description of the specific embodiments, serve to explain the principles of the invention.
Referring to the drawings, to the following detailed description, and to incorporated materials, detailed information about the invention is provided including the description of specific embodiments. The detailed description serves to explain the principles of the invention. The invention is susceptible to modifications and alternative forms. The invention is not limited to the particular forms disclosed. The invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the claims.
Modern cytogenetic techniques including fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), Multiplex-FISH (M-FIST and Spectral karyotyping (SKY), have become extensively used techniques in both diagnostic and research laboratories. The probes used in these techniques contain both unique and repetitive sequences, which bind to target DNA. The repetitive sequences are suppressed from binding to the target by the addition of competitive blocking DNA, usually Cot-1 DNA. This process requires large amounts of Cot-1 DNA, often 20-50-fold excesses, that is expensive when purchased commercially.
Recent publications have described a method for removing repetitive sequences using affinity chromatography producing PCR-amplifiable, chromosome-specific painting probes. This method solves the problem of having to use high levels of expensive Cot-1 DNA, but is difficult and time consuming. It requires multiple PCR amplifications and affinity chromatography purifications for many of the probes. Furthermore, the initial outlay for materials can be rather expensive.
Applicants have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequences, greatly decreasing PCR efficiency selectively across these blocked regions. A second labeling reaction is then done and this product is resuspended in FISH hybridization mixture without further addition of blocking DNA. The hybridization produces little if any non-specific binding on any other chromosomes. Applicants have been able to successfully use this procedure with both human and rat chromosome probes. This technique should be applicable in producing probes for CGH, M-FISH and SKY, as well as reducing the presence of repetitive DNA in genomic libraries.
The present invention provides a simple method for producing significant volumes of chromosome-specific painting probes. The entire process can be done in less than one day and yields probes with high specificity without the use of additional competitor DNA. Additional details of the present invention are described in the article “Polymerase Chain Reaction-based Suppression of Repetitive Sequences in Whole Chromosome Painting Probes for FISH” by Lawrence C. Dugan, Melissa S. Pattee, Jennifer Williams, Mike Eklund, J. Karen Sorensen, Joel S. Bedford and Allen T. Christian, in Chromosome Research, 13 (1), p 27-32 (2005). The article and all figures, data, and information “Polymerase Chain Reaction-based Suppression of Repetitive Sequences in Whole Chromosome Painting Probes for FISH” by Lawrence C. Dugan, Melissa S. Pattee, Jennifer Williams, Mike Eklund, J. Karen Sorensen, Joel S. Bedford and Allen T. Christian, in Chromosome Research, 13 (1), p 27-32 (2005) is incorporated herein by reference. A copy of the article is enclosed in a Prior Art statement accompanying this application.
Referring now to the drawings,
Referring to
Referring now to
Referring to the drawings,
Referring to
Referring now to
Standard Library Amplification and Labeling by PCR
A 15 l reaction volume was prepared using 200 ng chromosome specific library, 1.5 μl Thermosequenase reaction buffer (USB, Cleveland, Ohio), 1.5 μl 10×dNTP solution (Roche Molecular, Indianapolis, Ind.) (80 μM final concentration) 0.6 μl 100 μM Telenius primer (5′-CCGACTCGAGNNNNNNATGTGG-3′) (MacroMolecular Resources, Fort Collins, Colo.), 6 U Thermosequenase polymerase (USB, Cleveland, Ohio) and distilled, diionized H2O. The libraries were amplified using a MWesearch PT-100 thermocycler (MJResearch, Watertown, Mass.). The reaction profile used was as follows; 95° C. for 5 min. followed by 15 cycles of 94° C. for 1 min., 60° C. for 1 min. and 72° C. for 3 min. This was followed by 5 min. at 72° C. and a hold at 4° C. until tubes were removed. Products were then purified using Qiagen's Qiaquick PCR purification kit and 3 μl were run on a 1.5% agarose gel against size and concentration standards for 60 minutes at 100V. Products should be in the range of 300-800 by in size and a concentration of 100-200 ng/μl. 3 μl of the product was diluted 1:10 in 10 mM Tris-HCl, pH 8.5. A labeling step was then performed in a 50 μl volume containing 1 μl of diluted chromosome library (˜10-20 ng) from the above reaction, 5 μl Thermosequenase reaction buffer (USB, Cleveland, Ohio), 5 μl of 10×dNTP solution (Roche Molecular, Indianapolis, Ind.) (80 μM final concentration), 2 μlof 100 μM Telenius primer, 0.5 μl AmpliTaq LD polymerase, 2 μl of digoxigenin-11-dUTP and distilled, deionized H2O. The same reaction profile was used as above. This product was not further purified.
Repetitive-Sequence Depletion by PCR using Cot-1 DNA
The standard PCR amplification procedure was used with the addition of 1 mg of species-specific Cot-1 DNA in the amplification reaction. The result of this protocol is a DNA library that does not require that any blocking DNA be used in subsequent in situ hybridization experiments.
Applicants' invention provides a simple method to produce virtually unlimited quantities of Cot-1 depleted whole chromosome-specific painting probes (WCPs). The entire process can be done in less than one day and yields probes with high specificity without the use of additional competitor DNA. Human chromosome X and rat chromosome 1 specific probes were prepared by microdissection of normal metaphase chromosomes. The microdissected chromosomes were then amplified using the degenerate oligonucleotide-primed PCR protocol (DOP-PCR). This protocol uses a single primer containing a degenerate 6-nucleotide sequence and an initial ramping step to randomly incorporate the primer into target DNA. Once incorporated, target DNA can be further amplified with this same single primer. WCPs produced in this manner are complex enough to provide continuous coverage of target chromosomes. The WCPs produced here were tested against traditional hybridization cocktails on normal human fibroblast and rat lymphocyte metaphase spreads.
Materials and Methods
Chromosome libraries—Chromosome X was kindly provided by Dr. Maria Muhlmann-Diaz, formerly of Colorado State University and was produced using traditional microdissection techniques. Rat chromosome 1 library (RNO1) was also prepared using standard microdissection techniques. Briefly, metaphase spreads are dropped onto glass coverslips and air-dried. Chromosomes are identified under phase-contrast, transmitted light on an inverted microscope, often with the aid of G-banding. A glass needle attached to a micromanipulator is then used to scrape desired chromosomes from the coverslip, one at a time. The chromosome DNA is then transferred to a PCR tube by breaking off the tip of the needle containing the DNA in the tube. Multiple copies of a single chromosome are usually collected in the same tube. Collection of multiple copies of the same chromosome improves complexity and coverage of the probe on target chromosomes. The tubes were then centrifuged and loaded with PCR reaction solution. The PCR reaction solution contains 1.5 μl Thermosequenase reaction buffer, 1.5 μl 10×dNTP solution, (200 mM dATP, dTTP, dCTP, dGTP), 0.6 μl of 100 μM DOP primer (5′-CCGACTCGAGNNNNNNATGTGG-3′), 6 U Thermosequenase polymerase and distilled, deionized H2O to a final volume of 15 μl. The microdissected chromosomes were amplified using an MJResearch PT-100 thermocycler. The temperature-time reaction cycling profile used was as follows; 95° C. for 10 min, 8 cycles at 94° C. for 1 min, 30° C. for 5 min, and a ramp of 0.1 ° C/s to 65° C. for 5 min, followed by 12 cycles of 94° C. for 1 min, 56° C. for 1 min and 72° C. for 5 min. This was followed by 5 min at 72° C. and a hold at 4° C. until tubes were removed.
The standard amplification procedure described above was used with the inclusion of 1 μg human Cot-1 DNA for human Chromosome X and 1 μg rat Cot-1 DNA in the amplification reaction. Prior to use, the Cot-1 DNA was ethanol precipitated and resuspended at 1 μg/μl in 10 mM Tris-HCl, pH 8.5, to remove EDTA, which inhibits PCR amplification.
Metaphase Chromosome Preparation
Human metaphase spreads were prepared by growing human BJ1 cells (ATCC) to confluence in DMEM/F12 supplemented with 10% fetal bovine serum (FBS). The cells were then subcultured at a 1:5 dilution and incubated for 30-36 hours. Mitotic cells were collected and dropped on slides as previously described.
Rat metaphase spreads were prepared from blood cultures obtained by cardiac puncture of 8-12 week old, male Sprague-Dawley rats following the procedure in with minor modifications. Animals were housed in LLNL Animal Care Facility. Fresh blood was washed 2× with 5 ml RPMI 1640 media containing 10 U/ml Heparin (Sigma) and incubated in RPMI-1640 supplemented with 10% FBS (Sigma), 3 μg/ml Concanavalin A (Sigma), 100 μg/ml Lipopolysaccharide (Sigma), 1% L-glutamine (Gibco) and Antibiotic/antimycotic (Gibco). Cultures were incubated for 62 h at 37° C. in a humidified incubator containing 5% CO2. Colcemid (Gibco) was then added at a final concentration of 0.1 μg/ml for 4 h. Mitotic cells were then collected and dropped on slides as previously described.
Fluorescence in situ hybridization
A 4 μl volume of Alexa Fluor-488 labeled library containing 200 ng/ul was diluted with or without 4 μg Cot-1 DNA in a final volume of 15 μl containing 50% formamide, 2×SSC and 10% Dextran sulfate. The probe cocktail was then denatured for 10 min. at 84° C. and incubated at 37° C. for 45-60 min. Target slides were prepared by dehydration in an ethanol series consisting of 2 min. washes in 70%, 85% and 100% ethanol at room temperature. Slides were air-dried and denatured for 2-3 min. in 70% formamide, 30% 2×SSC 72° C. This was followed by a second dehydration series. The denaturations were timed so as to be completed simultaneously. The probe cocktail was then placed on the target slide and covered with a 22×22 mm coverslip. The coverslip was sealed with rubber cement and the slide was placed in a sealed slide box and incubated for 1-2 days at 37° C.
After incubation, the rubber cement and coverslip were carefully removed and the slide was rinsed 2× in 50% formamide, 2×SSC at 45° C. for 5 min per rinse. Slides were then rinsed 2× in 2×SSC at 45° C. for 5 min per rinse. This was followed by rinsing 2× in room temperature 1×PN buffer for 3 min per rinse. Finally, 10 μl of antifade solution containing 2.5 ng/μl DAPI counterstain was placed on the target area and covered with a coverslip.
Image Capture and Analysis
Slides were imaged using a Zeiss Axioskop microscope equipped with epifluorescence and standard DAPI/FITC/Texas red excitation filters and a triple bandpass DAPI/FITC/Texas red filter set. Images were captured using a Photometrics' SenSys CCD camera and Applied Imaging's Quips image analysis software.
Results and Discussion
As expected, the unblocked presence of labeled, repetitive sequences common to all chromosomes results in the more or less uniform painting of all chromosomes. FIG. 1B in the article, “Polymerase Chain Reaction-based Suppression of Repetitive Sequences in Whole Chromosome Painting Probes for FISH” by Lawrence C. Dugan, Melissa S. Pattee, Jennifer Williams, Mike Eklund, J. Karen Sorensen, Joel S. Bedford and Allen T. Christian, in Chromosome Research, 13 (1), p 27-32 (2005) shows results of hybridization of the chromosome X library amplified under standard conditions, without Cot-1 DNA in the PCR reaction, but in this case hybridized in the usual way with the addition of a 20-fold excess of unlabeled, blocking Cot-1 DNA during probe hybridization on the slide. The presence of the unlabelled Cot-1 DNA competitively blocks painting of all but the X chromosome.
This method can be expanded for use in other non-human mammalian species as shown in
The addition of a 10-fold excess of Cot-1 DNA to a PCR amplification reaction involving chromosome-specific libraries blocks, or at least drastically reduces the PCR efficiency of the amplification of the highly repetitive sequences in the library while allowing for the unimpeded amplification of unique sequences. It is thought that the presence of the Cot-1 DNA in the PCR reaction binds competitively with the DOP-primer to repetitive elements during the annealing step, minimizing the amplification of these repetitive elements. As shown in
Applicants have presented a method for removing repetitive sequences from chromosome-specific libraries that is quick, inexpensive and produces results equaling traditional FISH methods. Applicants have estimated that for each initial 15 PCR-blocking reaction, Applicants can produce >1000×10 μl hybridizations without additional Cot-1 DNA. At 20 μl Cot-1/hybridization and a cost of ˜$100/500 μl Cot-1DNA, this comes to a savings of >$5000.
Using this technique, a large volume of a chromosome specific DNA library can be generated in a single day. This method should be applicable to any process where the need exists for high quality libraries of unique or low-copy DNA sequences.
While the invention may be susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and have been described in detail herein. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the following appended claims.
This application is a Divisional of application Ser. No. 11/299,347 filed Dec. 8, 2005, entitled “Repetitive Sequence-Free DNA Libraries”. This application claims the benefit of U.S. Provisional Patent Application No. 60/637,367, filed Dec. 16, 2004, and titled “Repetitive Sequence-Free DNA Libraries”, which are incorporated herein by this reference.
The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC for the operation of Lawrence Livermore National Laboratory.
| Number | Date | Country | |
|---|---|---|---|
| 60637367 | Dec 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11299347 | Dec 2005 | US |
| Child | 12788165 | US |
