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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

• This invention relates to a method of rapid preparation of labeled and unlabeled target polynucleotides suitable for gene expression analysis.
• targets are generated in a single, multiple-compartment format container rather than in a set of single tubes, and both the cDNA and biotin-labeled cRNA are purified using filter plates suitable for such a multiple-compartment format container.
• the invention also is easily adaptable for automation, thereby cutting the time and expense even more.
• the invention disclosed herein describes a method of generating substantially pure cDNA or cRNA in a multiple-compartment container, comprising dispensing at least one total or poly A+ RNA sample into the multiple-compartment container, synthesizing cDNA using the RNA as a template, and transferring the synthesized cDNA to a multiple-compartment filter unit.
• the substantially pure cDNA is then transcribed in vitro and the reaction mixture transferred to a multiple-compartment filter unit to obtain substantially pure cRNA.
• This invention is directed to the generation and purification, in a multiple- compartment format, of labeled and unlabeled polynucleotides, such as biotinylated cRNA, which are suitable as targets for gene expression analysis.
• labeled and unlabeled polynucleotides such as biotinylated cRNA
• a large number of samples can be processed in less time than a single sample using current methods.
• one operator can generate up to 96 biotinylated targets in two days' time, whereas known prior art would allow only 25 to 30 samples to be processed in the same time period.
• sample manipulation via pipetting, etc. is minimized, thereby reducing the probability of operator-induced errors and variations.
• All samples undergo cDNA synthesis and, when desired, in vitro transcription using common reagent cocktails, thereby increasing product uniformity and reproducibility.
• the use of appropriate filters for purification improves the consistency and purity of the samples even further.
• the present invention also permits the reduction of the reaction volume of the in vitro transcription step by 50%, leading to a significant cost reduction.
• the present invention also allows the amplification of cDNA or RNA by using sub-microgram amounts of the starting RNA samples.
• the first step of the method of this invention requires dispensing at least one RNA sample into at least one compartment of a multiple-compartment container.
• a plurality of RNA samples are dispensed into individual compartments of the multiple-compartment container.
• the multiple-compartment container has 96 compartments or wells.
• Other exemplary multiple-compartment containers include 384 and 1536 multiple-compartment containers.
• the starting total or poly A+ RNA where total RNA includes all species of RNA and polyA+ RNA includes any RNA with a polyA+ tail, may be prepared by a method known in the art.
• the amount of the starting materials can vary, and advantageously may be less than what is currently thought to be necessary for the preparation of the end product using prior art methods.
• the amount of sample RNA dispensed into each individual compartment is in the range of about 0.5 to about 10 ⁇ g, and more preferably is an amount of about 5 ⁇ g.
• cDNA is then prepared from the RNA using techniques that are well known to those skilled in the art. For example, sample RNA in each compartment may be subjected to synthesis of first copy strand of cDNA, using reverse transcriptase and an oligo dT primer that does or does not incorporate the sequence of the T7 RNA polymerase promoter. After the first strand is synthesized, the second complementary strand is synthesized using T4 polymerase to produce cDNA. The synthesized product cDNA is then transferred to a multiple-compartment filter unit with a filter membrane that retains the product cDNA but does not bind such cDNA while allowing smaller molecules to pass through, thus forming substantially pure cDNA.
• the filter membrane is a standard cast membrane that works on the principle of size exclusion such that it retains double stranded DNA that is longer than approximately 130-150 nucleotides. Nucleotide triphosphates and oligonucleotide primers pass readily through the membrane.
• the multiple-compartment filter unit is a Millipore MultiScreen®-PCR Filter Plate, available from the Millipore Corporation, Bedford, Massachusetts.
• the resulting purified product cDNA is then collected by adding an appropriate buffer to the multiple-compartment filter unit containing the unbound product cDNA, gently shaking the multiple-compartment filter unit to resuspend the cDNA in the buffer, and then recovering the buffer containing the pure product cDNA.
• the buffer is 10 niM TRIS buffer.
• the resulting substantially pure product is suitable for use in microarray screening and assays.
• detection labels can be incorporated during this process.
• detection labels can be incorporated in the cDNA during the synthesis of the first strand.
• a non-radioactive label such as biotin or fluorescein can be incorporated by adding a labeled nucleotide in the synthesis reaction.
• isotopically labeled nucleotide either non-radioactive or radioactive, may be incorporated during the synthesis of the first strand.
• the resulting cDNA will be easily detectable by the virtue of such labels.
• a chemically reactive group such as an allyl amine can be incorporated into the cDNA by adding an amino allyl-dNTP to the synthesis reaction. After cDNA synthesis, the DNA is modified with a labeling molecule that is reactive with the amino group.
• the substantially pure cDNA may be used to form substantially pure cRNA. More specifically, the cDNA synthesized and made substantially pure as described above may then be transcribed with an RNA polymerase, such as T7 RNA polymerase. The synthesized product cRNA is then transferred to a multiple-compartment filter unit, such as Millipore MultiScreen®- PCR Filter Plate, to form substantially pure cRNA. This product is suitable for use in microarray screening and assays.
• the substantially pure cRNA may be labeled for detection.
• detection labels can be incorporated during the in vitro transcription.
• a non-radioactive label such as biotin or fluorescein can be incorporated by adding a labeled nucleotide in the in vitro transcription reaction.
• isotopically labeled nucleotide either non-radioactive or radioactive, may be incorporated during the in vitro transcription. The resulting substantially pure cRNA will be easily detectable by the virtue of such labels.
• detection labels can be incorporated directly into double stranded DNA and employed in microarray hybridization reactions that lead to genetic analysis and resequencing results.
• genomic DNA can be cut with a restriction enzyme that leads to fragments 200 - 1000 bases in length. These fragments are end-modified with an adapter and then subjected to PCR amplification.
• the amplified DNA is partially digested with Dnase, end-labeled with a dd-NTP and terminal transferase.
• the labeled DNA is then hybridized to a genetic analysis array that can detect, for example, single nucleotide polymorphisims (SNPs).
• SNPs single nucleotide polymorphisims
• DNA can be partially fragmented with DNase and then 3' end labeled with a labeled didioxy nucleoside triphosphate and terminal transferase.
• the partially fragmented, labeled DNA is then passed through a multiple-compartment filter unit to form a substantially pure DNA.
• Millipore MultiScreen®-PCR Filter Plate #MANU03010
• Millipore MultiScreen® Resist Vacuum Manifold #MAVM0960R
• 8-channel pipettors with 5-50 ⁇ l and 50- 300 ⁇ l capacities
• Beckman Modular Reservoir-quarter module either #372788 or #372790
• Vortex mixer with plate adapter
• MicrosealTM 'A' film MJ Research
• Polypropylene microtiter plate 96-well format or 48-well format, such as MJ Research #MAP-9601 (96-well) or #MAP-4801 (48-well); Nuclease-free H 2 O (Ambion #9938); V-bottom assay plate (Corning #9793); UV plate in a 96-well flat- bottom format (Corning #3536); Thermal cycler with a vortexer, accommodating 96- well format plates ; Tape sheets (Qiagen #19570 or comparable).
• RNA samples were thawed at 65°C for 5 minutes.
• Step 2 First strand synthesis:
• a first strand cocktail was prepared by mixing the following amounts for each reaction: 5X 1 st strand buffer (such as Gibco #18057-018), 4.0 ⁇ l; lOOmM DTT, 2.0 ⁇ l; lOmM dNTPs, 1.0 ⁇ l; Rnase Inhibitor such as Rnase outTM (Gibco), 1.0 ⁇ l; reverse transcriptase such as Superscript IT RT (Gibco), 1.0 ⁇ l;
• the cocktail was dispensed into a Beckman quarter-module reagent reservoir.
• the microtiter plate prepared as in Step 1 was kept in the thermal cycler. The plate was unsealed and the film was disposed of, afterwards 9 ⁇ l cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 5-50 ⁇ l, and mixed. 8. The plate was resealed with fresh "A' film and was incubated in the thermal cycler at 50°C for 1 hour.
• Step 3 Second strand synthesis:
• a second strand cocktail was prepared by mixing the following amounts of materials for each reaction: DEPC H 2 O, 83.5 ⁇ l; 5X 2 nd strand buffer such as Gibco # 10812-014, 30.0 ⁇ l; lOmM dNTPs, 3.0 ⁇ l; Bio-11 CTPs such as Enzo #43818 7.5 ⁇ l E. coli DNA Ligase, 1.0 ⁇ l; E. coli DNA Polymerase, 4.0 ⁇ l; Rnase H, 1.0 ⁇ l. (Total volume 130 ⁇ l). Enough cocktail was prepared for one more than the total number of reactions .
• the cocktail was dispensed into a Beckman quarter-module reagent reservoir.
• the plate was resealed and was incubated in the thermal cycler at 16°C for 2 hours.
• the plate was resealed and was incubated in the thermal cycler at 16°C for 5 minutes.
• the plate was removed from the thermal cycler and was placed immediately on ice.
• Step 4 cDNA Purification: 16. Using an 8-channel pipettor, 150 ⁇ l nuclease-free H 2 O was transferred for each reaction to be purified to a MultiScreen®-PCR plate.
• the manifold was connected to house vacuum and the samples were aspirated at 15" Hg for 20 minutes for each well to completely dry.
• the plate was placed on a vortex mixer with a plate adapter and was vortexed to resuspend the samples.
• the samples were diluted 1 to 20 and quantified.
• the samples were transferred from the plate to properly labeled 1.5 ml snap-cap tubes for storage at - 80°C.
• the cDNA samples were also transferred to a multiple- compartment container to serve as the templates for the in vitro transcription for amplification reaction.
• SPRI refers to a cDNA purification method based on the binding of DNA or RNA to carboxylate-modified paramagnetic micro-particles.
• the cRNAs resulting from the cDNAs prepared by the method of the present invention compared to a test tube - SPRI method or a multiple-compartment plate - SPRI method, yielded gene expression results that were characterized by higher average frequency values (measure of signal strength) and the detection of more genes (Table 2).
• Multiple-compartment filter unit 1 12.8 5518 2 12.0 5099
• Example 2 High-throughput protocol for generating Affymetrix® GeneChip ⁇ Targets (biotinylated cRNA) in 96-well format.
• Millipore MultiScreen®-PCR Filter Plate #MANU03010
• Millipore MultiScreen® Resist Vacuum Manifold #MAVM0960R
• 8-channel pipettors 5-50 ⁇ l and 50 - 300 ⁇ l capacities
• Beckman Modular Reservoir-quarter module either #372788 or #372790
• Vortex mixer with plate adapter
• MicrosealTM 'A' film MJ Research #MSA-5001
• Polypropylene microtiter plate 96-well format or 48-well format
• Nuclease-free H 2 O Ambion #9938
• V-bottom assay plate Coming #9793
• UV plate in a 96-well flat-bottom format Coming #3536
• Thermal cycler with a vortexer accommodating 96-well format plates
• Tape sheets Quality of 96-well format plates
• Tape sheets Qiagen #19570 or comparable
• RNA samples were thawed at 65°C for 5 minutes.
• Reagents were dispensed into polypropylene microtiter plate in the following quantities: total RNA, 5 ⁇ g; T7/T24 primer (High-quality, purified, 10 pmol/ ⁇ l), 2 ⁇ l; BAC Pool IX, 2.0 ⁇ l, DEPC H 2 O, sufficient to make the total volume to 11.0 ⁇ l.
• the plate was sealed with MicrosealTM 'A' film.
• Step 2 First strand synthesis:
• a first strand cocktail was prepared by mixing the following amounts for each reaction: 5X 1 st strand buffer such as Gibco #18057-018, 4.0 ⁇ l; lOOmM DTT, 2.0 ⁇ l; lOmM dNTPs, 1.0 ⁇ l; Rnase Inhibitor such as Rnase-outTM, 1.0 ⁇ l; reverse transcriptase such as Superscript II RT, 1.0 ⁇ l; (total 9.0 ⁇ l). Enough cocktail was prepared for five more reactions than the number of reactions planned. 6. The cocktail was dispensed into a Beckman quarter-module reagent reservoir. 7. The microtiter plate prepared as in Step 1 was kept in the thermal cycler. The plate was unsealed and the film was disposed of, and 9 ⁇ l cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 5-50 ⁇ l, and mix.
• 5X 1 st strand buffer such as Gibco #18057-018, 4.0
• microtiter plate was resealed with fresh "A' film and was incubated in the thermal cycler at 50° C for 1 hour.
• Step 3 Second strand synthesis:
• a second strand cocktail was prepared by mixing the following amounts of materials for each reaction: DEPC H 2 O, 91.0 ⁇ l; 5X 2 nd strand buffer Gibco #
• the cocktail was dispensed into a Beckman quarter-module reagent reservoir. 11. With the microtiter plate in thermal cycler, the plate was unsealed, 130 ⁇ l second strand cocktail was carefully aliquoted to each sample with an 8- channel pipettor, 50-300 ⁇ l, and mixed. 12. The plate was unsealed and was incubated in a thermal cycler at 16°C for 2 hours. 13. With the microtiter plate in the thermal cycler, the temperature was dropped to 4°C, the plate was unsealed, 2 ⁇ l T4 DNA polymerase was aliquoted to each sample with an 8-channel pipettor, 5-50 ⁇ l, and mixed. 14. The plate was resealed and was incubated in a thermal cycler at 16°C for 5 minutes.
• microtiter plate was removed from the thermal cycler and was placed immediately on ice.
• MultiScreen®-PCR plate was placed onto MultiScreen® Resist Vacuum Manifold.
• Step 5 hi vitro transcription for amplification:
• An TVT cocktail was prepared by mixing the following volumes for each reaction: DEPC H 2 O, 16.2 ⁇ l; 1 OX TVT buffer such as Ambion #8150G, 6 ⁇ l; r ⁇ TP mix #5, 6 ⁇ l; biotinylated UTP such as Bio-11 UTP, 2.4 ⁇ l; biotinylated CTP such as Bio-11 CTP, 2.4 ⁇ l; Rnase hihibitor, 2 ⁇ l; lOOmM DTT, 3 ⁇ l; T7 R ⁇ A Polymerase, 1 ⁇ l. (Total volume 40 ⁇ l.) Enough cocktail was prepared for one more than total number of reactions. 29.
• the IVT cocktail was dispensed into a Beckman quarter-module reagent reservoir. 30. 40 ⁇ l TVT cocktail was carefully aliquoted to each well of a polypropylene microtiter plate containing 20 ⁇ l cleaned cD ⁇ A product with an 8-channel pipettor, 5-50 ⁇ l, and mixed. 31. The microtiter plate was sealed with MicrosealTM 'A' film.
• the plate was incubated in a thermal cycler at 37°C for 16 hours.
• microtiter plate containing IVT reaction product was removed from the thermal cycler. The plate was placed on ice if not purifying immediately. 34. 120 ⁇ l ⁇ uclease-free H 2 O was added to each sample with an 8-channel pipettor and mixed. 35. The samples were transferred to a MultiScreen®-PCR plate.
• MultiScreen ⁇ -PCR plate was placed on a MultiScreen® Resist Vacuum Manifold. 38. The vacuum was set to 15" Hg and the plate was aspirated for 20 minutes.
• the vacuum was increased to 25" Hg (or maximum house vacuum if it is below 25" Hg) and the plate was aspirated for 10 minutes or until wells were dry.
• the plate was placed on a vortexer with a plate adapter, and the plate was vortexed to resuspend the samples.
• the plate was covered and was placed on ice.
• the samples were diluted 1 to 20 and quantified. The samples were then transferred from plate to properly labeled 1.5 ⁇ l snap-cap tubes for storage at
• Multiple-compartment filter unit 1 18.42 6415

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