CA2509512A1 - Novel high throughput method of generating and purifying labeled crna targets for gene expression analysis - Google Patents
Novel high throughput method of generating and purifying labeled crna targets for gene expression analysis Download PDFInfo
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- CA2509512A1 CA2509512A1 CA002509512A CA2509512A CA2509512A1 CA 2509512 A1 CA2509512 A1 CA 2509512A1 CA 002509512 A CA002509512 A CA 002509512A CA 2509512 A CA2509512 A CA 2509512A CA 2509512 A1 CA2509512 A1 CA 2509512A1
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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
Abstract
A method of generating substantially pure polynucleotides in a multiple-compartment container using a multiple-compartment purification filter is described. In particular, a method of generating cDNA or cRNA labeled with biotin for the ease of detection is described.
Description
TITLE
1~1~T~~~~ HIGH THROIJGHFZJT T~~°~°H~I~ ~F G~I~TE~AT~I~~TG
AI~'~Tl~
PIJRIF~II~TG I~A~EIJEl~ eRII~TTA T GETS F~R GE1~I~ E~PI~~I~T~T
AI~AIJ~~IS
EACI~GROUND OF THE INVENTION
Field of the Invention This invention relates to a method of rapid preparation of labeled and unlabeled target polynucleotides suitable for gene expression analysis.
Related Eacl~growd Art The analysis of genes and gene expression has become more widely applicable aald more in demand as the complete human genome sequence is now available and identities of expressed genes are elucidated. One important analysis is detecting which of the multitude of genes are expressed in any given cell. This is traditionally done by northern blots, nuclease protection assays, or differential display gel electrophoresis.
More recently, array-based methods have been developed to improve the accuracy and speed of such analysis. D.J. Locl~hart et al., Nature Biotechnol., 14, 1675 (1996).
However, even with the recent improvements, the previously available methods are time consuming and require a fairly large amount of samples. An investigator using a microanay screening or assay still must generate the samples to test with such techniques, and this sample preparation is often a most time consuming and a very expensive endeavor. The present invention greatly reduces the amount of time and materials necessary, and at the same time improves on the accuracy, reproducibility and unformity of the test results. Advantageously, 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.
SUMMARY OF INVENTI~N
The invention disclosed herein describes a method of generating substantially pure cI~NA or cRNA in a multiple-compaz-tment container, comprising dispensing at least 2 0 one total or poly A+ RNA sample into the multiple-compartment container, synthesising cDNA using the RNA as a template, and transferring the synthesised cI~NA to a multiple-compartment filter unit. 5~lhen preparing cRNA, the substantially pure cI~NA is then transcribed in vitro and the reaction mixture traa~sferred to a multiple-compartment filter unit to obtain substantially pure cRNA.
1~1~T~~~~ HIGH THROIJGHFZJT T~~°~°H~I~ ~F G~I~TE~AT~I~~TG
AI~'~Tl~
PIJRIF~II~TG I~A~EIJEl~ eRII~TTA T GETS F~R GE1~I~ E~PI~~I~T~T
AI~AIJ~~IS
EACI~GROUND OF THE INVENTION
Field of the Invention This invention relates to a method of rapid preparation of labeled and unlabeled target polynucleotides suitable for gene expression analysis.
Related Eacl~growd Art The analysis of genes and gene expression has become more widely applicable aald more in demand as the complete human genome sequence is now available and identities of expressed genes are elucidated. One important analysis is detecting which of the multitude of genes are expressed in any given cell. This is traditionally done by northern blots, nuclease protection assays, or differential display gel electrophoresis.
More recently, array-based methods have been developed to improve the accuracy and speed of such analysis. D.J. Locl~hart et al., Nature Biotechnol., 14, 1675 (1996).
However, even with the recent improvements, the previously available methods are time consuming and require a fairly large amount of samples. An investigator using a microanay screening or assay still must generate the samples to test with such techniques, and this sample preparation is often a most time consuming and a very expensive endeavor. The present invention greatly reduces the amount of time and materials necessary, and at the same time improves on the accuracy, reproducibility and unformity of the test results. Advantageously, 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.
SUMMARY OF INVENTI~N
The invention disclosed herein describes a method of generating substantially pure cI~NA or cRNA in a multiple-compaz-tment container, comprising dispensing at least 2 0 one total or poly A+ RNA sample into the multiple-compartment container, synthesising cDNA using the RNA as a template, and transferring the synthesised cI~NA to a multiple-compartment filter unit. 5~lhen preparing cRNA, the substantially pure cI~NA is then transcribed in vitro and the reaction mixture traa~sferred to a multiple-compartment filter unit to obtain substantially pure cRNA.
DETAILED DESCRIPTION OF THE INVENTION
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.
There are many advantages and benefits of this invention over current methods.
In particular, a large number of samples can be processed in less time than a single sample using current methods. For example, one operator can generate up to 96 biotinylated targets in two days' time, whereas lmown prior art would allow only 25 to 30 samples to be processed in the same time period. Moreover, 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 cocl~tails, 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. In addition, the present invention also allows the amplification of cDNA or RNA by using sub-microgram amounts of the starting 2 0 RNA samples.
The first step of the method of this invention reduires dispensing at least one RNA
sample into at least one compartment of a multiple-compartment contain er.
Preferably, a plurality of RNA samples are dispensed into individual compartments of 2 5 the multiple-compartment container. Most preferably, the multiple-compartment container has 96 compartments or wells. Other exemplary multiple-compartment containers include 3S4 and 1536 multiple-compartment containers.
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.
There are many advantages and benefits of this invention over current methods.
In particular, a large number of samples can be processed in less time than a single sample using current methods. For example, one operator can generate up to 96 biotinylated targets in two days' time, whereas lmown prior art would allow only 25 to 30 samples to be processed in the same time period. Moreover, 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 cocl~tails, 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. In addition, the present invention also allows the amplification of cDNA or RNA by using sub-microgram amounts of the starting 2 0 RNA samples.
The first step of the method of this invention reduires dispensing at least one RNA
sample into at least one compartment of a multiple-compartment contain er.
Preferably, a plurality of RNA samples are dispensed into individual compartments of 2 5 the multiple-compartment container. Most preferably, the multiple-compartment container has 96 compartments or wells. Other exemplary multiple-compartment containers include 3S4 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 lcnown 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 at-t methods. Preferably, the amount of sample RNA
dispensed into each individual compartment is in the range of about 0.5 to about 10 fig, and more preferably is an amount of about 5 ~,g.
cDNA is then prepared from the RNA using techniques that are well pnown to those slcilled 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
pol~nnerase promoter. After the first strand is synthesized, the second complementary strand is synthesized using T4 polymerise 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. Any multiple-compartment filter unit with a filter membrane that meets these requirements may be employed in this invention. Generally, the filter membrane is a standard cast ~ 0 membrane that worps 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.
Preferably, the multiple-compartment filter unit is a Ie~illipore I~ultiScreen~-PCR
Filter Plate, available fiom the l~Iillipore Corporation, Bedford, IVIassachusetts. The 2 5 resulting purified product cDNA is then collected by adding an appropriate buffer to the multiple-compartment filter unit containing the unbound product cDNA, gently shaping the multiple-compaWent filter unit to resuspend the cDNA in the buffer, and then recovering the buffer containing the pure product cDNA. Preferably, the buffer is _5_ mM TRIS buffer. The resulting substantially pure product is suitable for use in microarray screening and assays.
In a preferred embodiment of this invention, detection labels can be incorporated 5 during tlus process. For example, detection labels can be incorporated in the cDNA
during the synthesis of the first strand. Specifically, a non-radioactive label such as biotin or fluorescein can be incorporated by adding a labeled nucleotide in the synthesis reaction. Alternatively, isotopically labeled nucleotide, either non-radioactive or radioactive, may be incorporated during the synthesis of the first strand.
The 10 resulting cDNA will be easily detectable by the virtue of such labels.
Lil~ewise, a chemically reactive group such as an allyl amine can be incorporated into the cI)NA.
by adding an amino allyl-dNTP to the synthesis reaction. After cI~NA
synthesis, the FJ1VA is modified with a labeling molecule that is reactive with the amino group.
In yet another embodiment of this invention, 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 MultiScreen0-~ 0 PAR Filter Plate, to fonn substantially pure cRNA. This product is suitable for use in microarray screening and assays.
IIZ a preferred embodiment, the substantially pure cRNA may be labeled for detection.
For example, detection labels can be incorporated during the in vitro transcription.
2 5 Specifically, a non-radioactive label such as biotin or fluorescein can be incorporated by adding a labeled nucleotide in the ir~ vitro transcription reaction.
Alternatively, 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.
In another embodiment of this invention, detection labels can be incorporated directly into double stranded DNA and employed in microarray hybridization reactions that lead to genetic analysis and resequencing results. For genetic analysis, 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 allay that can detect, for example, single nucleotide polymorphisims (SNPs).
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.
Example 1:
High throu~ht~ut protocol for ~enerating~ labeled cDNA in a 96-well format.
The following equipment and plasticware was used in the methods of this invention:
Millipore MultiScreen~-PCR Filter Plate (#I~AlAN~T03010); Millipore I~IultiScreenc~
Desist Vacuum Manifold (#MAVM0960R); 8-channel pipettors with 5-50 pal and 50-300 ~1 capacities; Eeclnnan Modular Reservoir-quarter module (either #372788 or 2 5 #372790); Vortex mixer with plate adapter; MicrosealT~ 'A' film (MJ
Research #MSA-5001); 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 HZO
(Ambion #9938); V-bottom assay plate (Corning #9793); UV plate in a 96-well flat-_7_ bottom format (Corning #3536); Thermal cycler with a vortexer, accommodating well format plates ; Tape sheets (Qiagen #19570 or comparable).
Stet? 1: AnnealingLof Primer:
1. RNA samples were thawed at 65°C for 5 minutes.
2. The following reagents were dispensed into polypropylene microtiter plates in the following quantities: total RNA, 5 pig; T7/T24. primer (High-quality, purified, 10 pmol/~,1), 2 ~1; EAC Pool (1X), 2.0 ~1;1~EPC HZ~, sufficient to melee the total volume to 11.0 ~1. The microtiter plates were sealed with MicrosealTM 'A9 film.
3. The samples were incubated in a thermal cycler at 70°C for 10 minutes.
4. The temperature was dropped to 50°C.
Steb 2: First strand synthesis:
5. A first strand cocl~tail was prepared by mixing the following amounts for each reaction: SX 1St strand buffer (such as Gibco #18057-018), 4.0 ~,1; 100m1VI
1?TT, 2.0 ~,1; lOml~I dNTPs, 1.0 ~1; Rnase Inhibitor such as Rnase outT~
~ 0 (Gibco), 1.0 ~,1; reverse transcriptase such as Superscript II RT (Gibco), 1.0 ~1;
(total 9.0 pal). Enough cocktail was prepared for five more reactions than the number of reactions planned.
6. The cocktail was dispensed into a Eeckman quarter-module reagent reservoir.
and polyA+ RNA includes any RNA with a polyA+ tail, may be prepared by a method lcnown 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 at-t methods. Preferably, the amount of sample RNA
dispensed into each individual compartment is in the range of about 0.5 to about 10 fig, and more preferably is an amount of about 5 ~,g.
cDNA is then prepared from the RNA using techniques that are well pnown to those slcilled 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
pol~nnerase promoter. After the first strand is synthesized, the second complementary strand is synthesized using T4 polymerise 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. Any multiple-compartment filter unit with a filter membrane that meets these requirements may be employed in this invention. Generally, the filter membrane is a standard cast ~ 0 membrane that worps 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.
Preferably, the multiple-compartment filter unit is a Ie~illipore I~ultiScreen~-PCR
Filter Plate, available fiom the l~Iillipore Corporation, Bedford, IVIassachusetts. The 2 5 resulting purified product cDNA is then collected by adding an appropriate buffer to the multiple-compartment filter unit containing the unbound product cDNA, gently shaping the multiple-compaWent filter unit to resuspend the cDNA in the buffer, and then recovering the buffer containing the pure product cDNA. Preferably, the buffer is _5_ mM TRIS buffer. The resulting substantially pure product is suitable for use in microarray screening and assays.
In a preferred embodiment of this invention, detection labels can be incorporated 5 during tlus process. For example, detection labels can be incorporated in the cDNA
during the synthesis of the first strand. Specifically, a non-radioactive label such as biotin or fluorescein can be incorporated by adding a labeled nucleotide in the synthesis reaction. Alternatively, isotopically labeled nucleotide, either non-radioactive or radioactive, may be incorporated during the synthesis of the first strand.
The 10 resulting cDNA will be easily detectable by the virtue of such labels.
Lil~ewise, a chemically reactive group such as an allyl amine can be incorporated into the cI)NA.
by adding an amino allyl-dNTP to the synthesis reaction. After cI~NA
synthesis, the FJ1VA is modified with a labeling molecule that is reactive with the amino group.
In yet another embodiment of this invention, 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 MultiScreen0-~ 0 PAR Filter Plate, to fonn substantially pure cRNA. This product is suitable for use in microarray screening and assays.
IIZ a preferred embodiment, the substantially pure cRNA may be labeled for detection.
For example, detection labels can be incorporated during the in vitro transcription.
2 5 Specifically, a non-radioactive label such as biotin or fluorescein can be incorporated by adding a labeled nucleotide in the ir~ vitro transcription reaction.
Alternatively, 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.
In another embodiment of this invention, detection labels can be incorporated directly into double stranded DNA and employed in microarray hybridization reactions that lead to genetic analysis and resequencing results. For genetic analysis, 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 allay that can detect, for example, single nucleotide polymorphisims (SNPs).
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.
Example 1:
High throu~ht~ut protocol for ~enerating~ labeled cDNA in a 96-well format.
The following equipment and plasticware was used in the methods of this invention:
Millipore MultiScreen~-PCR Filter Plate (#I~AlAN~T03010); Millipore I~IultiScreenc~
Desist Vacuum Manifold (#MAVM0960R); 8-channel pipettors with 5-50 pal and 50-300 ~1 capacities; Eeclnnan Modular Reservoir-quarter module (either #372788 or 2 5 #372790); Vortex mixer with plate adapter; MicrosealT~ 'A' film (MJ
Research #MSA-5001); 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 HZO
(Ambion #9938); V-bottom assay plate (Corning #9793); UV plate in a 96-well flat-_7_ bottom format (Corning #3536); Thermal cycler with a vortexer, accommodating well format plates ; Tape sheets (Qiagen #19570 or comparable).
Stet? 1: AnnealingLof Primer:
1. RNA samples were thawed at 65°C for 5 minutes.
2. The following reagents were dispensed into polypropylene microtiter plates in the following quantities: total RNA, 5 pig; T7/T24. primer (High-quality, purified, 10 pmol/~,1), 2 ~1; EAC Pool (1X), 2.0 ~1;1~EPC HZ~, sufficient to melee the total volume to 11.0 ~1. The microtiter plates were sealed with MicrosealTM 'A9 film.
3. The samples were incubated in a thermal cycler at 70°C for 10 minutes.
4. The temperature was dropped to 50°C.
Steb 2: First strand synthesis:
5. A first strand cocl~tail was prepared by mixing the following amounts for each reaction: SX 1St strand buffer (such as Gibco #18057-018), 4.0 ~,1; 100m1VI
1?TT, 2.0 ~,1; lOml~I dNTPs, 1.0 ~1; Rnase Inhibitor such as Rnase outT~
~ 0 (Gibco), 1.0 ~,1; reverse transcriptase such as Superscript II RT (Gibco), 1.0 ~1;
(total 9.0 pal). Enough cocktail was prepared for five more reactions than the number of reactions planned.
6. The cocktail was dispensed into a Eeckman quarter-module reagent reservoir.
7. The microtiter plate prepared as in Step 1 was kept in the thermal cycler.
The 2 5 plate was unsealed and the film was disposed of, afterwards 9 ~.1 cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 5-50 ~,1, and mixed.
_g-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:
The 2 5 plate was unsealed and the film was disposed of, afterwards 9 ~.1 cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 5-50 ~,1, and mixed.
_g-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:
9. ' A second strand cocktail was prepared by mixing the following amounts of materials for each reaction: I~EPC HZ~, 83.5 ~l; 5~ 2nd strand buffer such as Caibco # 10812-014, 30.0 pal; lOmM dIVTPs, 3.0 ~,1; Eio-11 CTPs such as En~o #43 818 7.5 ~,1, E. coli DICTA I,igase, 1.0 ~ 1; E. coli DIVA Polymerase, 4.0 ~,1;
IW ase H, 1.0 ~,1. (Total volume 130 ~,1). Enough cocktail was prepared for one more than the total number of reactions.
IW ase H, 1.0 ~,1. (Total volume 130 ~,1). Enough cocktail was prepared for one more than the total number of reactions.
10. The cocktail was dispensed into a Beckman quarter-module reagent reservoir.
11. With the plate in the thermal cycler, the plate was unsealed, 130 ~1 cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 50-300 ~l, ' and mixed.
12. The plate was resealed and was incubated in the thermal cycler at 16°C for 2 hours.
13. With the plate in the thermal cycler: the temperature was dropped to 4°C, the plate was unsealed, 2 ~1 T4 DIVA polymerase was aliquoted to each sample 2 0 well with 8-cha~ugel pipettor, 5-50 ~l, ~a2d mixed.
14. The plate was resealed snd was incubated in the thermal cycler at 16°C for 5 mutes.
15. The plate was removed from the thermal cycler and was placed immediately on ice.
Step 4: cI~IVA Purification:
Step 4: cI~IVA Purification:
16. Using an 8-channel pipettor, 150 ~,l nuclease-free H20 was transferred for each reaction to be purified to a MultiScreen~-PCR plate.
17. Using an 8-channel pipettor, the entire cDNA reaction was transferred to the MultiScreen~-PCR plate.
18. The MultiScreen~-PCR plate was placed onto a MultiScreen~ Resist Vacuum Manifold.
19. The manifold was connected to house vacuum and the samples were aspirated at 15" Hg for 20 minutes for each well to completely dry.
20. The vacuum was completely released and the plate was removed from the manifold.
21. The bottom of the plate was blotted dry on I~imwipe~.
22. 25 ~1 lOmM TRIS buffer was aliquoted to each well with an 8-channel pipettor.
23. The plate was placed on a vortex mixer with a plate adapter and was vortexed to resuspend the samples.
24. 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.
In some instances, the cl~NA samples were also transferred to a multiple-compartment container to serve as the templates for the in vitro transcription 2 0 for amplification reaction.
Results:
Using the method described herein for the isolation of substantially pure cl~NA , 2 5 resulting cRNA yields were consistently in the range of 20 ~,g per reaction. In contrast, the SPRI-based cDNA purification executed in a multiple-compartment plate gave yields of approximately 13 to 22 ~,g with a wide variation in yield (Table 1).
SPRI
refers to a cDNA purification method based on the binding of I~NA or RNA to carboxylate-modified paramagnetic micro-particles. Furthermore, 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).
Table 1: Purifaeation I~Ieth~ds F~11~w~ing eDIVr~ Synthesis Influence the field and variability of field as Reflected in cltl~TA
Yield,Standard Standard Sample Pr~eess Replicatesug DeviationA260/A280Deviation SW120-1 Multiple-compartment8 12.94 2.34 1.88 0.039 plate/SPRI
SW120-1 Multiple-compartment4. 21.9 3.01 1.99 0.014 filter unit SW120-1 Test tubes/SPRI2 17.65 0.19 1.8 0.043 SW120-2 Multiple-compartment 4 21.99 11.45 1.77 0.39 plate/SPRI
SW120-2 Multiple-compartment 4 19.82 1.08 1.97 0.023 filter unit Table 2: Expression Results f~r vari~us eDhTA Pnrifieation I~lethods Pr~eess Replicate 1'Iumber Present Average Frequency falls 2 5 Multiple-compartment1 4.7 4413 plate/SPRI
2 3.8 4438 Multiple-compartment 1 12.8 5518 filter unit 2 12.0 5099 Test tubes/SPRI 1 6.1 5013 2 9.9 4828 3 0 Example 2:
High throughput protocol for generating, Aff~rnetrix~ GeneChip~ Targets ~biotinylated cRNA) in 96-well format.
The following equipment and plasticware was used: Millipore MultiScreen~-PCR
Filter Plate (#MANLJ03010); Millipore MultiScreen~ Resist Vacuum Manifold (#MAVM0960R); 8-channel pipettors, 5-50 ~,1 and 50 - 300 ~l capacities;
Beckman Modular Reservoir-quarter module (either #372788 or #372790); Voutex mixer with plate adapter; MicrosealT~ 'A' film (MJ Research #MSA-5001); Polypropylene microtiter plate, 96-well format or 48-well format; Nuclease-free HZ~ (Ambion #9938); V-bottom assay plate (Coming #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) Step 1: Annealing of Primer:
1. The RNA samples were thawed at 65°C for 5 minutes.
2. 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 ~,1; BAC Pool 1X, 2.0 ~.1, DEPC HZO, sufficient to make the total 2 0 volmne to 11.0 pal. The plate was sealed with MicrosealT~ 'A' film.
3. The samples were incubated in thermal cycler at 70°C for 10 minutes.
4.. The temperature was dropped to 50°C.
Sten 2: First strand synthesis:
5. A first strand cocktail was prepared by mixing the following amounts for each reaction: SX 1St strand buffer such as Gibco #18057-018, 4.0 ~,1; 100mM DTT, 2.0 ~1; lOmM dNTPs, 1.0 ~,1; Rnase Inlubitor such as Rnase-outTM, 1.0 ~1;
reverse transcriptase such as Superscript II RT, 1.0 ~,1; (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 Beclanan 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 ~.1 cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 5-50 ~1, and mix.
8. The 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:
9. A second strand cocktail was prepared by mixing the following amounts of materials for each reaction: DEPC Hz~, 91.0 ~1; SX 2nd strand buffer Gibco #
10812-014, 30.0 ~l; lOmM dNTPs, 3.0 ~.1; E. coli DNA Ligase, 1.0 ~1; E. coli DNA Polymerase, 4.0 ~.1; Rnase H, 1.0 ~,1. (Total volmne 130 ~.1). Enough cocktail was prepared for one more than the total number of reactions.
10. The cocktail was dispensed into a Beclcman quarter-module reagent reservoir.
2 0 11. With the microtiter plate in thermal cycler, the plate was unsealed, 130 ~1 second strand cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 50-300 ~1, and mixed.
12. The plate was unsealed and was incubated in a thermal cycler at 16°C for 2 hours.
2 5 13. With the microtiter plate in the thermal cycler, the temperature was dropped to 4°C, the plate was unsealed, 2 pal T4 DNA polymerise was aliquoted to each sample with an 8-channel pipettor, 5-50 ~1, and mixed.
14. The plate was resealed and was incubated in a thermal cycler at 16°C for 5 minutes.
15. The microtiter plate was removed from the thermal cycler and was placed immediately on ice.
Step 4: cDNA Purification:
16. The unused wells on MultiScreen~-PCR plate were covered with tape sheet to prevent contamination. See note at step 29.
17. Using an 8-channel pipettor, 150 ~,1 nuclease-free HZ~ was transferred for each reaction to be purified to a MultiScreen~-PCR plate.
18. Using an 8-channel pipettor, the entire cDNA reaction was transferred to a MultiScreen~-PCR plate.
19. The MultiScreenOO -PCR plate was placed onto MultiScreen~ Resist Vacuum Manifold.
20. The manifold was connected to house vacuum and the samples were aspirated at 1 S" Hg for 20 minutes for the wells to completely dry.
2 0 21. The vacuum was released completely and the plate was removed from the manifold.
22. The bottom of the plate was blotted on I~imwipe~.
23. 25 pal lOmM TRIS buffer was aliquoted to each well with an 8-channel pipettor.
2 5 24. The plate was placed on a vortexer with a plate adapter and was voi-texed to resuspend the sample.
In some instances, the cl~NA samples were also transferred to a multiple-compartment container to serve as the templates for the in vitro transcription 2 0 for amplification reaction.
Results:
Using the method described herein for the isolation of substantially pure cl~NA , 2 5 resulting cRNA yields were consistently in the range of 20 ~,g per reaction. In contrast, the SPRI-based cDNA purification executed in a multiple-compartment plate gave yields of approximately 13 to 22 ~,g with a wide variation in yield (Table 1).
SPRI
refers to a cDNA purification method based on the binding of I~NA or RNA to carboxylate-modified paramagnetic micro-particles. Furthermore, 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).
Table 1: Purifaeation I~Ieth~ds F~11~w~ing eDIVr~ Synthesis Influence the field and variability of field as Reflected in cltl~TA
Yield,Standard Standard Sample Pr~eess Replicatesug DeviationA260/A280Deviation SW120-1 Multiple-compartment8 12.94 2.34 1.88 0.039 plate/SPRI
SW120-1 Multiple-compartment4. 21.9 3.01 1.99 0.014 filter unit SW120-1 Test tubes/SPRI2 17.65 0.19 1.8 0.043 SW120-2 Multiple-compartment 4 21.99 11.45 1.77 0.39 plate/SPRI
SW120-2 Multiple-compartment 4 19.82 1.08 1.97 0.023 filter unit Table 2: Expression Results f~r vari~us eDhTA Pnrifieation I~lethods Pr~eess Replicate 1'Iumber Present Average Frequency falls 2 5 Multiple-compartment1 4.7 4413 plate/SPRI
2 3.8 4438 Multiple-compartment 1 12.8 5518 filter unit 2 12.0 5099 Test tubes/SPRI 1 6.1 5013 2 9.9 4828 3 0 Example 2:
High throughput protocol for generating, Aff~rnetrix~ GeneChip~ Targets ~biotinylated cRNA) in 96-well format.
The following equipment and plasticware was used: Millipore MultiScreen~-PCR
Filter Plate (#MANLJ03010); Millipore MultiScreen~ Resist Vacuum Manifold (#MAVM0960R); 8-channel pipettors, 5-50 ~,1 and 50 - 300 ~l capacities;
Beckman Modular Reservoir-quarter module (either #372788 or #372790); Voutex mixer with plate adapter; MicrosealT~ 'A' film (MJ Research #MSA-5001); Polypropylene microtiter plate, 96-well format or 48-well format; Nuclease-free HZ~ (Ambion #9938); V-bottom assay plate (Coming #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) Step 1: Annealing of Primer:
1. The RNA samples were thawed at 65°C for 5 minutes.
2. 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 ~,1; BAC Pool 1X, 2.0 ~.1, DEPC HZO, sufficient to make the total 2 0 volmne to 11.0 pal. The plate was sealed with MicrosealT~ 'A' film.
3. The samples were incubated in thermal cycler at 70°C for 10 minutes.
4.. The temperature was dropped to 50°C.
Sten 2: First strand synthesis:
5. A first strand cocktail was prepared by mixing the following amounts for each reaction: SX 1St strand buffer such as Gibco #18057-018, 4.0 ~,1; 100mM DTT, 2.0 ~1; lOmM dNTPs, 1.0 ~,1; Rnase Inlubitor such as Rnase-outTM, 1.0 ~1;
reverse transcriptase such as Superscript II RT, 1.0 ~,1; (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 Beclanan 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 ~.1 cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 5-50 ~1, and mix.
8. The 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:
9. A second strand cocktail was prepared by mixing the following amounts of materials for each reaction: DEPC Hz~, 91.0 ~1; SX 2nd strand buffer Gibco #
10812-014, 30.0 ~l; lOmM dNTPs, 3.0 ~.1; E. coli DNA Ligase, 1.0 ~1; E. coli DNA Polymerase, 4.0 ~.1; Rnase H, 1.0 ~,1. (Total volmne 130 ~.1). Enough cocktail was prepared for one more than the total number of reactions.
10. The cocktail was dispensed into a Beclcman quarter-module reagent reservoir.
2 0 11. With the microtiter plate in thermal cycler, the plate was unsealed, 130 ~1 second strand cocktail was carefully aliquoted to each sample with an 8-channel pipettor, 50-300 ~1, and mixed.
12. The plate was unsealed and was incubated in a thermal cycler at 16°C for 2 hours.
2 5 13. With the microtiter plate in the thermal cycler, the temperature was dropped to 4°C, the plate was unsealed, 2 pal T4 DNA polymerise was aliquoted to each sample with an 8-channel pipettor, 5-50 ~1, and mixed.
14. The plate was resealed and was incubated in a thermal cycler at 16°C for 5 minutes.
15. The microtiter plate was removed from the thermal cycler and was placed immediately on ice.
Step 4: cDNA Purification:
16. The unused wells on MultiScreen~-PCR plate were covered with tape sheet to prevent contamination. See note at step 29.
17. Using an 8-channel pipettor, 150 ~,1 nuclease-free HZ~ was transferred for each reaction to be purified to a MultiScreen~-PCR plate.
18. Using an 8-channel pipettor, the entire cDNA reaction was transferred to a MultiScreen~-PCR plate.
19. The MultiScreenOO -PCR plate was placed onto MultiScreen~ Resist Vacuum Manifold.
20. The manifold was connected to house vacuum and the samples were aspirated at 1 S" Hg for 20 minutes for the wells to completely dry.
2 0 21. The vacuum was released completely and the plate was removed from the manifold.
22. The bottom of the plate was blotted on I~imwipe~.
23. 25 pal lOmM TRIS buffer was aliquoted to each well with an 8-channel pipettor.
2 5 24. The plate was placed on a vortexer with a plate adapter and was voi-texed to resuspend the sample.
25. 20 ~,l of eluate was collected with 8-channel pipettor, 5-50 ~,1.
26. The eluate was transferred to a polypropylene microtiter plate, in which the IVT reaction was subsequently carned out. The plate was stored on ice until ready to proceed.
27. The rows on the MultiScreen~-PCR plate that have been used were clearly marked so that the unused rows may be used in future.
Step 5' lia vitro transcription for amplification:
Step 5' lia vitro transcription for amplification:
28. ' An IVT cocktail was prepared by mixing the following volumes for each reaction: I~EPC HZ~, 16.2 ~1; lOX IVT buffer such as Ambion #81506, 6 ~,1;
rNTP mix #5, 6 ~.1; biotinylated UTP such as Bio-11 UTP, 2.4 ~,1; biotinylated CTP such as Bio-11 CTP, 2.4 ~,1; Rnase W hibitor, 2 ~1; 100mM DTT, 3 ~,1; T7 RNA Polymerase, 1 ~,1. (Total volume 40 p,l.) Enough cocktail was prepared for one more than total number of reactions.
rNTP mix #5, 6 ~.1; biotinylated UTP such as Bio-11 UTP, 2.4 ~,1; biotinylated CTP such as Bio-11 CTP, 2.4 ~,1; Rnase W hibitor, 2 ~1; 100mM DTT, 3 ~,1; T7 RNA Polymerase, 1 ~,1. (Total volume 40 p,l.) Enough cocktail was prepared for one more than total number of reactions.
29. The IVT cocktail was dispensed into a Beclcman quarter-module reagent reservoir.
30. 40 ~.1 IVT cocktail was carefully aliquoted to each well of a polypropylene microtiter plate containing 20 ~,1 cleaned cI~NA product with an 8-channel pipettor, 5-50 ~1, and mixed.
2 0 31. The microtiter plate was sealed with I~IicrosealTl~ 'A' film.
32. The plate was incubated in a thermal cycler at 37°C for 16 hours.
Step 6: IVT Purification:
2 5 33. The microtiter plate containing IVT reaction product was removed from the thermal cycler. The plate was placed on ice if not purifying immediately.
34. 120 ~,1 Nuclease-free H20 was added to each sample with an 8-channel pipettor and mixed.
35. The samples were transferred to a MultiScreen~-PCR plate.
36. Unused wells were covered with tape sheet to prevent contamination.
37. The MultiScreen~-PCR plate was placed on a MultiScreen~ Resist Vacuum Maiufold.
38. The vacuum was set to 15" Hg and the plate was aspirated for 20 minutes.
39. After all wells were dry, 100 ~1 Nuclease-free H20 was added to each well with an 8-chasmel pipettor.
40. 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.
41. The vacuum was released completely before removing the plate.
42. The bottom of the plate was blotted dry on I~imwipe~.
43. 50 ~,l IOmM TRIS was aliquoted to each well with an 8-channel pipettor.
44. The plate was placed on a vortexer with a plate adapter, and the plate was vortexed to resuspend the samples.
45. 50 ~1 eluate was carefully collected and was transferred to a Costar v-bottom assay plate with an 8-channel pipettor, 50-300 ~,1.
46. The plate was covered and was placed on ice.
47. The samples were diluted 1 to 20 and quantified. The samples were then 2 0 transferred from plate to properly labeled 1.5 ~1 snap-cap tubes for storage at -80°C.
Results:
2 5 Using the method described herein for the isolation of substantially pure cRNA, product yields were consistently in the range of 55 ~,g per reaction. In contrast, a SPRI
and a traditional column-based purification method with an RNeasy spin column resulted in yields of 45 and 25 to 38 ~.g, respectively, with a wide variation on results as measured by the standard variation of the yield of replicate cRNA reactions (Table 3). Furthermore, the samples prepared by the method of the present invention yielded expression results very similar to those obtained by a column purification protocol, which is widely used in gene expression analysis (Table 4).
Table 3: Purification Methods Following cRl~lA
Synthesis Influence the Yield and Variability of Yield as Reflected in cRhlA
Yield,Standard Standard Sample Process Replicatesug DeviationA260/A280Deviation Column 7 38.4016.00 1.96 0.026 Multiple-compartment 7 56.5 2.1 1.95 0.04 filter unit 2 44.9 7.2 2.07 0.005 Hela Column 8 24.759.27 2.14 0.042 Multiple-compartment 8 51.3 4.61 2.02 0.007 filter unit Liver Column 8 26.824.86 2.08 0.083 Multiple-compartment 8 54.823.22 2.01 0.02 filter unit ~0 Table 4: E~prcssion i~csults for Various cRFJA Purification Mctlaods Process Replicate hlumbcr Average Frequency Prcscnt Calls Multiple-compartment filter unit I I 8.42 64~ 15 2 28.19 6012 3 20.93 6076 4 36.29 6351
2 0 31. The microtiter plate was sealed with I~IicrosealTl~ 'A' film.
32. The plate was incubated in a thermal cycler at 37°C for 16 hours.
Step 6: IVT Purification:
2 5 33. The microtiter plate containing IVT reaction product was removed from the thermal cycler. The plate was placed on ice if not purifying immediately.
34. 120 ~,1 Nuclease-free H20 was added to each sample with an 8-channel pipettor and mixed.
35. The samples were transferred to a MultiScreen~-PCR plate.
36. Unused wells were covered with tape sheet to prevent contamination.
37. The MultiScreen~-PCR plate was placed on a MultiScreen~ Resist Vacuum Maiufold.
38. The vacuum was set to 15" Hg and the plate was aspirated for 20 minutes.
39. After all wells were dry, 100 ~1 Nuclease-free H20 was added to each well with an 8-chasmel pipettor.
40. 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.
41. The vacuum was released completely before removing the plate.
42. The bottom of the plate was blotted dry on I~imwipe~.
43. 50 ~,l IOmM TRIS was aliquoted to each well with an 8-channel pipettor.
44. The plate was placed on a vortexer with a plate adapter, and the plate was vortexed to resuspend the samples.
45. 50 ~1 eluate was carefully collected and was transferred to a Costar v-bottom assay plate with an 8-channel pipettor, 50-300 ~,1.
46. The plate was covered and was placed on ice.
47. The samples were diluted 1 to 20 and quantified. The samples were then 2 0 transferred from plate to properly labeled 1.5 ~1 snap-cap tubes for storage at -80°C.
Results:
2 5 Using the method described herein for the isolation of substantially pure cRNA, product yields were consistently in the range of 55 ~,g per reaction. In contrast, a SPRI
and a traditional column-based purification method with an RNeasy spin column resulted in yields of 45 and 25 to 38 ~.g, respectively, with a wide variation on results as measured by the standard variation of the yield of replicate cRNA reactions (Table 3). Furthermore, the samples prepared by the method of the present invention yielded expression results very similar to those obtained by a column purification protocol, which is widely used in gene expression analysis (Table 4).
Table 3: Purification Methods Following cRl~lA
Synthesis Influence the Yield and Variability of Yield as Reflected in cRhlA
Yield,Standard Standard Sample Process Replicatesug DeviationA260/A280Deviation Column 7 38.4016.00 1.96 0.026 Multiple-compartment 7 56.5 2.1 1.95 0.04 filter unit 2 44.9 7.2 2.07 0.005 Hela Column 8 24.759.27 2.14 0.042 Multiple-compartment 8 51.3 4.61 2.02 0.007 filter unit Liver Column 8 26.824.86 2.08 0.083 Multiple-compartment 8 54.823.22 2.01 0.02 filter unit ~0 Table 4: E~prcssion i~csults for Various cRFJA Purification Mctlaods Process Replicate hlumbcr Average Frequency Prcscnt Calls Multiple-compartment filter unit I I 8.42 64~ 15 2 28.19 6012 3 20.93 6076 4 36.29 6351
Claims (20)
1. A method of generating substantially pure cDNA in a multiple-compartment container, comprising 1) dispensing at least one total or poly A+ RNA sample into at least one compartment of said multiple-compartment container; 2) synthesizing cDNA using said RNA as a template; 3) transferring the synthesized cDNA to a multiple-compartment filter unit; and 4) collecting substantially pure cDNA.
2. The method of claim 1, wherein each of a plurality of RNA samples are dispensed into individual compartments of said multiple-compartment container.
3. The method of claim 2, wherein the synthesized cDNA is labeled for detection.
4. The method of claim 3, wherein the synthesized cDNA is biotinylated, labeled with a fluorescent molecule, or labeled through the incorporation of amino allyl deoxynucleoside triphosphates.
5. The method of claim 4, wherein the fluorescent molecule is fluorescein, Cyanine 3, or Cyanine 5.
6. The method of claim 2, wherein the multiple-compartment container is in a or 384-well microtiter plate format.
7. The method of claim 6, wherein the synthesized cDNA is labeled for detection.
8. The method of claim 7, where the detection is performed with microarrays.
9. The method of claim 7, wherein the synthesized cDNA is biotinylated, labeled with a fluorescent molecule, or labeled through the incorporation of amino allyl deoxynucleoside triphosphates.
10. A method of generating substantially pure cDNA in a multiple-compartment container, comprising 1) dispensing at least one total or poly A+ RNA sample into at least one compartment of said multiple-compartment container; 2) synthesizing cDNA using said RNA as a template; 3) transferring the synthesized cDNA to a multiple-compartment filter unit; 4) collecting substantially pure cDNA; 5) synthesizing amplified cRNA using the substantially pure cDNA as a template; 6) transferring the synthesized amplified cRNA to a multiple-compartment filter unit; and 7) collecting substantially pure cRNA.
11. The method of claim 10, wherein each of a plurality of RNA samples are dispensed into individual compartments of said multiple-compartment container.
12. The method of claim 11, wherein the synthesized cRNA is labeled for detection.
13. The method of claim 12, wherein the synthesized cRNA is biotinylated, labeled with a fluorescent molecule, or labeled through the incorporation of amino allyl deoxynucleoside triphosphates.
14. The method of claim 13, wherein the fluorescent molecule is fluorescein, or Cyanine 3, or Cyanine 5.
15. The method of claim 11, wherein the multiple-compartment container is in a 96-or 384-well microtiter plate format.
16. The method of claim 15, wherein the synthesized cRNA is labeled for detection.
17. The method of claim 16, where the detection is performed with microarrays.
18. The method of claim 16, wherein the synthesized cRNA is biotinylated, labeled with a fluorescent molecule, or labeled through the incorporation of amino allyl deoxynucleoside triphosphates.
19. A method of generating fragmented and labeled genomic DNA suitable for hybridization to an oligonucleotide array designed for genetic analysis, comprising the following steps in a multiple-compartment container:
a) dispensing at least one genomic DNA sample into at least one compartment of said multiple-compartment container and digesting the genomic DNA with a restriction enzyme that generates fragments in the 0.2 - 1 kb size range, b) ligating adapters to the digested DNA to form adapter-modified DNA
fragments, c) transferring the adapter-modified DNA fragments to a multiple-compartment filter unit and collecting substantially pure adapter-modified DNA fragments, d) PCR amplifying the substantially pure adapter-modified DNA
fragments, e) transferring the pure amplified adapter-modified DNA to a multiple-compartment filter unit and collecting the substantially pure amplified adapter-modified DNA, f) digesting partially the substantially pure amplified adapter-modified DNA with a DNase to generate single stranded fragments, g) end-labeling the digested single stranded fragments with a label using terminal transferase; and h) transferring the labeled single stranded DNA fragments to a multiple-compartment filter unit and collecting the substantially pure fragmented and labeled DNA.
a) dispensing at least one genomic DNA sample into at least one compartment of said multiple-compartment container and digesting the genomic DNA with a restriction enzyme that generates fragments in the 0.2 - 1 kb size range, b) ligating adapters to the digested DNA to form adapter-modified DNA
fragments, c) transferring the adapter-modified DNA fragments to a multiple-compartment filter unit and collecting substantially pure adapter-modified DNA fragments, d) PCR amplifying the substantially pure adapter-modified DNA
fragments, e) transferring the pure amplified adapter-modified DNA to a multiple-compartment filter unit and collecting the substantially pure amplified adapter-modified DNA, f) digesting partially the substantially pure amplified adapter-modified DNA with a DNase to generate single stranded fragments, g) end-labeling the digested single stranded fragments with a label using terminal transferase; and h) transferring the labeled single stranded DNA fragments to a multiple-compartment filter unit and collecting the substantially pure fragmented and labeled DNA.
20. The method of claim 19, wherein the label is selected from the group consisting of dideoxy triphosphate, biotin, fluorescein, Cyanine 3 and Cyanine 5.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US34240103A | 2003-01-15 | 2003-01-15 | |
| US10/342,401 | 2003-01-15 | ||
| PCT/US2004/000538 WO2004065573A2 (en) | 2003-01-15 | 2004-01-12 | Novel high throughput method of generating and purifying labeled crna targets for gene expression analysis |
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| CA2509512A1 true CA2509512A1 (en) | 2004-08-05 |
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| AU (1) | AU2004206206A1 (en) |
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| WO (1) | WO2004065573A2 (en) |
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| WO2007020847A1 (en) * | 2005-08-19 | 2007-02-22 | Sumitomo Bakelite Co., Ltd. | Method for producing cdna and rna chains and nucleotide-immobilized support |
| ES2358473T3 (en) | 2007-05-11 | 2011-05-11 | Decathlon | ARTICLE OF CONFECTION WITH EFFECT OF HETEROGENIC CONTAINMENT FOR THE PRACTICE OF A SPORT. |
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