WO1996038591A1 - Procede ameliore pour obtenir des sequences d'adn complementaire longues - Google Patents

Procede ameliore pour obtenir des sequences d'adn complementaire longues Download PDF

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WO1996038591A1
WO1996038591A1 PCT/US1996/008501 US9608501W WO9638591A1 WO 1996038591 A1 WO1996038591 A1 WO 1996038591A1 US 9608501 W US9608501 W US 9608501W WO 9638591 A1 WO9638591 A1 WO 9638591A1
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cdna
nucleic acid
pcr
sequence
primer
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PCT/US1996/008501
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English (en)
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Karl J. Guegler
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Incyte Pharmaceuticals, Inc.
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Priority claimed from US08/459,046 external-priority patent/US6008039A/en
Application filed by Incyte Pharmaceuticals, Inc. filed Critical Incyte Pharmaceuticals, Inc.
Priority to EP96917035A priority Critical patent/EP0832282A1/fr
Priority to AU59729/96A priority patent/AU5972996A/en
Priority to JP8536785A priority patent/JPH11506332A/ja
Publication of WO1996038591A1 publication Critical patent/WO1996038591A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is in the field of molecular biology and more particularly, in the field of recombinant DNA technology.
  • PCR has become a widely used nucleic acid amplification technique since it was first presented by Kary Mullis at the Cold Spring Harbor Symposium (Mullis K et al (1986) Cold Spring Harbor Symp Quant Biol 51: 263-273) .
  • PCR requires that a pair of primers be generated from known sequences.
  • sequence is available only from one end of a DNA segment.
  • Several methods have been developed to sequence an entire gene once a partial nucleotide sequence is available. As more partial cDNA sequences become available in the world' s genetic databanks, more efficient and economical methods will be sought for then obtaining the complete gene.
  • PCR has become a widely used technique to complete genes for which a partial sequence is already known.
  • Gene-specific primers and primers located in the vector into which the cDNAs have been cloned are used for this purpose.
  • this method is limited by the use of primers complementary to vector sequence which is common to all clones in the library. This results in an abundance of non-specific PCR-products which have to be cloned and sequenced. Multiple rounds of amplifications with nested primers might be required. These additional operations increase the incorporation of errors.
  • Gobinda, Turner and Bolander (1993) in PCR Methods and Applications 2:318-22 disclose "restriction-site PCR" as a direct method of retrieving unknown sequence which is adjacent to a known locus by using universal primers.
  • genomic DNA is amplified in the presence of restriction site oligonucleotides and a primer specific to the known region.
  • those products are subjected to a second round of PCR with the same restriction site oligonucleotides and another specific primer internal to the first one.
  • the products of the last round of PCR are transcribed with an appropriate RNA polymerase and sequenced with a reverse transcriptase and an end-labeled specific primer internal to the second specific PCR primer.
  • Gobinda et al. present data concerning Factor IX for which they identified a conserved stretch of 20 nucleotides in the 3' noncoding region of the gene.
  • Inverse PCR is the first method that reported successful acquisition of unknown sequences starting with primers based on a known region (Triglia T, Peterson MG, and Kemp DJ (1988) Nucleic Acids Res. 16:8186) .
  • Inverse PCR employs a strategy in which several restriction enzymes are used to generate a suitable fragment in the known region. The segment is then circularized by intramolecular ligation and used as a PCR template with divergent primers created from the known region.
  • the requirement of multiple restriction enzyme digestions followed by multiple ligations make the procedure slow and expensive (Gobinda et al. Supra) .
  • Capture PCR first disclosed by Lagerstrom M, Parik J, Malmgren H, Stewart J, Patterson U and Landegren ⁇ (1991) PCR Methods Applic. 1:111-19, is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and YAC DNA. As noted by Gobinda et al. supra, that method also requires multiple restriction enzyme digestions and ligation of an engineered double-stranded primer before PCR. Although the restriction and ligation reactions are carried out simultaneously in this method, the requirement of extension reaction, immobilization of the extended product, two rounds of PCR and purification of template prior to sequencing render it cumbersome and time consuming as well.
  • cDNA complementary DNA
  • This cDNAs are subsequently cloned into a vector (plasmid or Lambda) and amplified by transfection into E.coli cells resulting in a so-called cDNA library.
  • plasmid or Lambda plasmid or Lambda
  • the enzymes used in converting mRNA into cDNA are limited in their ability to produce complete copies of the existing mRNAs. This requires the researcher to isolate multiple cDNA clones of the gene of interest using specific probes and analyze each of these isolates for a complete cDNA of the gene of interest. This process is called screening of cDNA libraries.
  • the method may be used for extending known DNA sequences of genomic or cDNA origin.
  • the method utilizes the polymerase chain reaction (PCR) and includes the steps of: a) combining a first and second PCR primer with nucleic acid from a cDNA library, or pools of cDNA libraries, expected to contain said partial cDNA, or said partial cDNA that has been extended, or a genomic library, under conditions suitable for synthesis of nucleic acid PCR products from the first and second primers, wherein said first and second primers are capable of annealing to opposite strands of the partial cDNA or genomic DNA and initiating nucleic acid synthesis in an outward manner and wherein the first primer is capable of being extended by DNA polymerase in an antisense direction and the second primer is capable of being extended in a sense direction, b) purifying the PCR products, and c) identifying extended nucleotide sequences derived from said partial cDNA or said
  • the method of identifying the extended nucleotide sequences comprises nucleic acid sequencing. In another embodiment of the present invention, the method proceeds with repeating steps 6a through 6c on the nucleotide sequences identified in step 6c.
  • a method for extending the nucleotide sequence of a partial complementary DNA (cDNA) using polymerase chain reaction (PCR) comprising the steps of a) combining a first and second PCR primer with nucleic acid from a cDNA library, or pools of cDNA libraries, expected to contain said partial cDNA, or said partial cDNA that has been extended, or a genomic DNA library, under conditions suitable for synthesis of nucleic acid PCR products from the first and second primers, wherein said first and second primers are capable of annealing to- opposite strands of the partial cDNA and initiating nucleic acid synthesis in an outward manner and wherein the first primer is capable of being extended by DNA polymerase in an antisense direction and the second primer is capable of being extended in a sense direction, b) purifying the PCR products, c) ligating the purified PCR products under conditions suitable for the formation of circular, closed nucleic acid, d) transforming a host
  • the present invention also provides a method for extending known genomic DNA sequences which may be used for the detection and amplification of 5' untranslated nucleotide sequences and/or promoter sequences .
  • an isolated DNA molecule comprising SEQ ID NO:11, the DNA for a novel human purinergic P2U receptor.
  • Figure 1 is a flow chart of the steps in the inventive method.
  • Figure 2 shows a typical plasmid obtained from the excision process of a lambdaZAP cDNA library. Typically 250-300 base pairs of the sequence are obtained in the high-throughput sequence operation. The clone is partially sequenced from the 5' end with T3 as a sequencing primer.
  • Figure 3 is a representation of the next step, in which pBLUESCRIPT SK plasmids in a cDNA library are used as a template and the two specially designed primers (XLR and XLS) amplify plasmids containing the gene of interest. Only plasmids containing priming sites for both XL-PCR primers and the gene of interest will be amplified during the XL-PCR reaction.
  • Figure 4 is a representation of the amplified DNA segments which have been obtained through the XL-PCR reaction and consequently purified after separating the products on an agarose gel.
  • the cDNA library used as a template should be synthesized by random priming to assure the availability in this step of different amplified length of DNA (3' end) between the XLS priming site and the T7 priming site in the vector.
  • the length of the 5' end (between the XLR priming site and the T3 priming site) in the vector will vary in size depending on how much of the mRNA of the gene of interest had been converted into cDNA during the cDNA library synthesis.
  • Figure 5 shows how the purified DNA segments containing the plasmid and the gene of interest are religated to form a circular plasmid and transformed into bacteria for amplification.
  • chemically competent E. coli cells were transformed and grown on petri dishes containing LB agar and 25 mg/L carbenicillin (2XCarb) for antibiotic selection.
  • Figure 6 shows schematically how pure samples of clones were obtained from the different E. coli colonies grown in the procedure shown in Figure 5 (also Step 1 purification, Step 2 religation and Step 3 transformation in Figure 6) . These clones are screened in Step 4 for additional sequence of the gene of interest at the 5' end. For this purpose the clones were analyzed by a PCR reaction employing the XLR primer and the T3 vector primer. The size of the resulting product will indicate how much additional sequence upstream of the XLR priming site each clone contains.
  • Figures 7A through 7H show the results of the inventive method, in which a partial sequence from Incyte clone 14770, which was similar to heat shock protein 90, was successively sequenced to obtain a full-length cDNA.
  • Figures 8A through 8F show the results of the inventive method, in which a partial sequence from Incyte clone 87058 which was similar to cathepsin was successively sequenced to obtain extensions of the cDNA.
  • the present method provides a way to utilize a genomic DNA library or a plasmid cDNA library (either obtained by cloning cDNAs directly into a plasmid vector or by converting a Lambda library into a plasmid library by known methods e.g. Lambda ZAP excision or Lambda ZIPLOCK conversion) which has been used for sequencing cDNAs, as a source to obtain much longer DNAs and in certain cases complete genes of partially known DNA sequences.
  • a genomic DNA library or a plasmid cDNA library either obtained by cloning cDNAs directly into a plasmid vector or by converting a Lambda library into a plasmid library by known methods e.g. Lambda ZAP excision or Lambda ZIPLOCK conversion
  • This new method utilizes PCR kits which enable the researcher to amplify long pieces of DNA.
  • the XL-PCR amplification kit (Perkin-Elmer) was employed. However, equivalent products may be available from other major suppliers.
  • This novel method allows one person to process multiple genes (up to 96 genes) at a time and obtain extended or complete sequence (possibly full-length) of the cDNAs of interest within 6-10 days. This compares very favorably with current competitive methods like screening with labelled probes which allow one worker to process only about 3-5 genes and obtain initial results in 14-40 days. This represents an increase in throughput of at least 1000%.
  • primer design and synthesis (based on a known partial sequence) can be performed in about two days.
  • the PCR amplification can be performed in 6-8 hours.
  • Multiple libraries can be pooled and therefore screened at the same time.
  • the next steps of purification and ligation take about one day.
  • transformation and growing up the bacteria take one day.
  • screening for clones with additional sequence of the genes of interest by PCR takes approximately five hours.
  • the next steps of DNA preparation and sequencing of the selected clones can be performed in about one day. This totals 6-7 days.
  • This method presumes that one already has partial cDNA sequences, either from a publicly available database or the scientist' s own earlier research, including but not limited to earlier preparation of a cDNA library whose cDNAs have been partially sequenced.
  • the cDNA library may have been prepared with oligo dT or random primers.
  • the difference between oligo dT and randomly primed libraries is that a randomly primed library will have more sequences which contain 5' ends of cDNAs.
  • a randomly primed library may be particularly useful for further work when the oligo dT library does not yield a complete gene. Random priming of the library also helps yield more cDNA sequences of different lengths. Library preparation techniques which promote longer insert sizes will in turn permit the sequencing of more complete cDNAs. Obviously, the larger the protein, the less likely it is that the complete cDNA will be found in a single plasmid.
  • Figure 2 shows a typical plasmid containing a cDNA which had been partially sequenced from the 5' end with T3 as a primer.
  • the top darkened portion represents the insert containing the gene of interest.
  • Step 1 PCR-amplification of cDNA-clones containing the ⁇ ene of interest
  • the first step of this method requires the design of two primers based on the known sequence.
  • the known sequence can be obtained by those skilled in the art either by a wet lab method or from the many publicly available DNA databases.
  • One primer is synthesized to be extended in an antisense direction (XLR) and the other in the sense direction (XLS or XLF) .
  • XLR antisense direction
  • XLS sense direction
  • XLF sense direction
  • the primers are designed to anneal to either end of the known sequence and to be extended "outward" from there to generate amplicons containing new, unknown sequences of the genes of interest. This is different from typical PCR, in which the primers are designed to amplify a known sequence in a direction "inward" toward each other.
  • primers need to be designed in a way displaying optimal criteria for extra long PCR.
  • a program like Oligo 4.0s can be employed for this purpose.
  • primers should be 22-30 nucleotides in length, consist of a GC content of 50% or more and anneal at 68°C-72°C to the target. Hairpin structures and primer-primer dimerizations must be avoided. Primers varying from the conditions described above may result in amplification of the desired targets providing extension conditions have been adjusted.
  • Figure 3 shows the next step, in which a cDNA library is used as a template and the two primers (XLR and XLS) amplify plasmids containing the gene of interest.
  • a cDNA library is used as a template and the two primers (XLR and XLS) amplify plasmids containing the gene of interest.
  • XLR and XLS primers
  • PCR enzymes which provide high fidelity and copy long sequences, such as that provided in the XL-PCR kit (Part No.
  • kit instructions should be followed, including suggestions to optimize concentrations of various reagents. In the examples disclosed infra, 25pMol of each primer worked well. Template (plasmid library) concentrations can be varied (see Examples infra for details) . It is essential to thoroughly resuspend the enzyme in solution prior to use, especially if the solution has been stored at -20°C. If the enzyme is not adequately resuspended, its effectiveness is impaired.
  • the preferred system is setup initially in two layers, employing Ampliwax" PCR Gems. However, efficiency can be increased by avoiding the use of these Gems and initiating amplification by using the "hot-start” technique by adding Magnesium, which is essential for amplification, at 82° C.
  • thermocycler MJ Research, Watertown, MA
  • Times and temperatures may be varied to optimize conditions in different thermocyclers.
  • Step 1 94° for 60 sec (initial denaturation)
  • Step 2 94° for 15 sec
  • Step 5 Repeat step 2-4 for 15 additional times
  • Step 6 94° for 15 sec
  • Step 7 65° for 1 min
  • Step 8 68° for 7 min + 15 sec/cycle
  • Step 9 Repeat step 6-8 for 11 additional times
  • Step 11 4° for 0.00 sec (to hold at 4°) At the end of these 28 cycles, 50 ⁇ l of the reaction mix is removed; on the remaining reaction mix, an additional 10 additional cycles are run, as outlined below:
  • Step 1 94° for 15 sec
  • Step 2 65° for 1 min
  • Step 3 68° for (10 min + 15 sec) /cycle
  • Step 4 Repeat step 1-3 for 9 additional times
  • Step 5 72° for 10 min
  • a 5-10 ⁇ l aliquot of the reaction mixture can be analyzed on a mini-gel to determine which reactions were successful.
  • Step 2 Purification of amplicons containing the ⁇ ene of interest
  • Figure 4 is a graphical representation of the amplified cDNA segments which have been separated on an agarose gel. Note that there are a variety of lengths of cDNA. Although the rest of the method could be performed using all extended cDNA species, the method can proceed optionally after selecting the largest products (likeliest to provide the remainder of the full-length gene) . Some of the larger species may in fact be hybrid clones which contain two cDNA inserts as a result of malfunction during the cDNA library construction which may represent an incomplete digestion with the restriction enzyme at the end of the cDNA synthesis. Such amplified hybrid clones, also called chimera, could result in overlooking the correct targeted extensions.
  • Successful reaction products should be purified on an agarose gel (preferentally low agarose concentrations 0.6-0.8% should be used) or other appropriate method.
  • An appropriate volume of reaction mixture should be loaded to obtain good separation of the products and to separate them from the plasmid library (template) still in the reaction mixture. Contamination with the template cDNA library will result in transformants which don't contain the desired gene and will require an extensive screening of many colonies.
  • the bands representing the genes of interest are then cut out of the gel and purified using a method like the QIAQuick gel extraction kit (Qiagen, Inc., Chatsworth, CA) .
  • Step 3 Cloning of amplicons containing the gene of interest Eventual overhangs are converted into blunt ends to facilitate religation and cloning of the products.
  • Klenow enzyme (3 units/reaction mixture) and dNTP' s (0.2 mM final concentration) are added and the reaction is incubated at room temperature for 30 min. The Klenow enzyme is then inactivated by incubating the reaction at 75° for 15 min.
  • Step 4 Screening of cloned products
  • each well contains 150ml of LB/2XCarb medium.
  • each row of the microtiter plate contains twelve clones from the same extension reaction.
  • the cells are grown over night at 37°C.
  • 5 ⁇ l of these overnight cultures are tranferred into a non-sterile 96-well plate (Falcon 3911 Microtest IIITM, Flexible Assay Plate, Becton Dickinson, Oxnard, CA) and diluted 1:10 with water. 5 ⁇ l of each dilution are then transferred into a PCR array (e.g., Cycleplate, Robbins Scientific Corp., Sunnyvale, CA) .
  • a PCR array e.g., Cycleplate, Robbins Scientific Corp., Sunnyvale, CA
  • Amplification generally was performed using the following conditions:
  • Step 1 9 °C for 60sec
  • Step 2 94°C for 20sec
  • Step 5 repeat steps 2-4 for an additional 29 times
  • Step 6 72°C for 180sec Step 7 4°C for ever
  • PCR products will allow direct determination of how much additional sequence the selected clones contain compared to the original partial cDNA. The efficiency of the method has been further improved by using the resulting PCR-products directly for sequencing thus avoiding the necessity of preparing plasmids.
  • the appropriate clones are selected and grown for plasmid preparation and sequencing. Plasmid preparations are made with standard kits familiar to those skilled in the art. Examples include the PROMEGA Magic
  • Sequencing is performed employing standard automated ABI sequencing equipment and protocols using either dye-primer or dye-terminator kits.
  • Sequence processing and assemblage of the sequencing data are performed using standard ABI software, including INHERITTM analysis and the Power assembler.
  • INHERITTM analysis and the Power assembler.
  • a THP-1 cDNA library constructed into the LambdaZAP vector (Stratagene) was converted into a plasmid library following the mass excision protocol. Plasmids of the excised libraries were prepared using the Quiagen Midi plasmid purification kit. 1.3 XL-PCR reaction set-up
  • the extension reactions were prepared following the instructions provided with the GeneAmp XL PCR Kit (Part No. N808-0182) from Perkin Elmer.
  • a two layer system was set up as follows: The lower reagent mix was prepared by pipetting the following components into a 0.2ml MicroAmp reaction tube.
  • Lower reagent mix preparation ⁇ Water 13.6 ⁇ l 3.3X buffer 12.0 ⁇ l dATP (lOmM) 2.0 ⁇ l dCTP (lOmM) 2.0 ⁇ l dGTP (lOmM) 2.0 ⁇ l dTTP (lOmM) 2.0 ⁇ l
  • the template DNA (excised library) was diluted to an appropriate concentration in water and then added to the upper mix. Mixing of the components is not necessary.
  • Step 1 94° for 60 sec (initial denaturation)
  • Step 2 94° for 15 sec
  • Step 4 68° for 7 min
  • Step 5 Repeat step 2-4 for 15 additional times
  • Step 8 68° for 7 min + 15 sec/cycle
  • Step 9 Repeat step 6-8 for 11 additional times Step 10 72° for 8 min
  • Step 11 4° for 0.00 sec (to hold at 4°)
  • Klenow enzyme (3 units/reaction) and dNTP's (0.2mM final concentration) were added and the reactions were incubated at room temperature for 30 min followed by incubation at 75° C for 15 min. The products were then ethanol precipitated and redissolved in 13 ⁇ l of ligation buffer containing ImM ATP.
  • T4-DNA ligase (15 units) and T4 Polynucleotide kinase (5 units) were added, and the reaction was incubated at room temperature for 3 hours. 3 ⁇ l of the ligation mixture were transformed into 40 ml of competent E.coli cells. After heatshocking the cells at 42° C for 45 seconds, 80 ⁇ l of SOC medium were added, and the cells were allowed to recover at 37° C for 1 hour. The whole transformation mixture then was plated on LB-agar/2XCarb-containing petri dish plates.
  • the 1.33 x concentrated PCR mix contained the following components: 10X PCR-buffer 2.0 ⁇ l
  • Step 1 94° C for 60sec
  • Step 2 94° C for 20sec
  • Step 3 55° C for 30sec
  • Step 4 72° C for 90sec
  • Step 5 repeat steps 2-4 for an additional 29 times Step 6 72° C for 180 sec Step 7 4° C for ever
  • the DNA of the selected clones was prepared using the WizardTM Minipreps DNA Purification System (Promega Corporation, Madison, WI) following the instructions of the manufacturer. Sequencing reactions were performed using the PRISMTM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Part No 401628, Perkin Elmer, Applied Biosystems, Foster City, CA) . 1.9 Analysis of sequenced products
  • FIG. 14A-7H shows an alignment of the obtained sequences with the published human Hsp 90 nucleotide sequence.
  • Clones 14201.3 and 14201.5 contain part of the 5' untranslated region and therefore the full coding region of the gene has been obtained.
  • Example 2 For further method evaluation, a second known gene was selected. A partial sequence from a liver library was found to be related to that of the human cathepsin B gene (accession L16510, HUMCATHB, SEQ ID NO: 6) . This partial sequence (Incyte clone 87058, SEQ ID NO:7) initiated at base 1066 of the sequence with accession number L16510.
  • the extension reactions were prepared following the instructions provided with the GeneAmp XL PCR Kit (Part No. N808-0182) from Perkin Elmer. A two layer system was set up as described below. The lower reagent mix was prepared by pipetting the following components into a 0.2ml MicroAmp reaction tube. Lower reagent mix preparation:
  • the template DNA (excised library) was diluted to an appropriate concentration in water and then added to the upper mix. Mixing of the components is not necessary. Template (6.25ng/ ⁇ l) 40.0 ⁇ l
  • Step 1 94° for 60 sec (initial denaturation)
  • Step 2 94° for 15 sec
  • Step 5 Repeat step 2-4 for 15 additional times
  • Step 6 94° for 15 sec
  • Step 7 65° for 1 min
  • Step 8 68° for 7 min + 15 sec/cycle
  • Step 9 Repeat step 6-8 for 11 additional times
  • Step 11 4° for 0.00 sec (to hold at 4° ) 2.5 Purification of amplified products
  • T4-DNA ligase (15 units) and T4 Polynucleotide kinase (5 units) were added, and the reaction was incubated at room temperature for 3 hours.
  • the 1.33 x concentrated PCR mix contained the following components:
  • Step 1 94°C for 60sec Step 2 94°C for 20sec Step 3 55°C for 30sec Step 4 72°C for 90sec Step 5 repeat steps 2-4 for an additional 29 times Step 6 72°C for 180sec Step 7 4°C for ever
  • the DNA of the selected clones was prepared using the WizardTM Minipreps DNA Purification System (Promega Corporation, Madison, WI) following the instructions of the manufacturer. Sequencing reactions were performed using the PRISMTM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Part No 401628, Perkin Elmer, Applied Biosystems, Foster City, CA) .
  • Figures 8A through 8F show an alignment of the obtained sequences with the published human Hsp 90 nucleotide sequence.
  • Clone 87058.16 contains part of the 5'UT and therefore the full coding region of the gene.
  • Example 3 In Example 3, a full length cDNA (Seq ID NO 11) of a novel P2U purinergic receptor homolog was obtained by the inventive method and is the subject of U.S. Patent Application 08/459,046 filed June 2, 1995, which is hereby incorporated by reference.
  • Incyte Clone 179696 was extended to full length using a modified XL-PCR (Perkin Elmer) procedure. Primers were designed based on known sequence; one primer was synthesized to initiate extension in the antisense direction (XLR) and the other to extend sequence in the sense direction (XLF) .
  • XLR antisense direction
  • XLF sense direction
  • the primers allowed the sequence to be extended "outward" from the known sequence, thus generating amplicons containing new, unknown nucleotide sequence comprising the gene of interest.
  • the primers were designed using Oligo 4.0 (National Biosciences Ine, Plymouth MN) to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • cDNA library was used as a template, and XLR (bases 278-298) and XLF (bases 587-610) primers were used to extend and amplify the 179696 sequence.
  • XLR bases 278-298
  • XLF bases 587-610
  • Step 3 65° C for 1 min
  • Step 4 68° C for 7 min
  • Step 5 Repeat step 2-4 for 15 additional cycles
  • Step 7 65° C for 1 min
  • Step 8 68° C for 7 min + 15 sec/cycle
  • Step 9 Repeat step 6-8 for 11 additional cycles
  • Step 1 94° C for 15 sec
  • Step 3 68° C for (10 min + 15 sec) /cycle
  • Step 4 Repeat step 1-3 for 9 additional cycles
  • Competent E. coli cells (in 40 ⁇ l of appropriate media) were transformed with 3 ⁇ l of ligation mixture and cultured in 80 ⁇ l of
  • Step 1 94° C for 60 sec
  • Step 2 94° C for 20 sec
  • Step 3 55° C for 30 sec
  • Step 4 72° C for 90 sec
  • Step 5 Repeat steps 2-4 for an additional 29 cycles
  • Step 6 72° C for 180 sec
  • Step 7 4° C (and holding)
  • the inventive method was used to obtain a novel full length cDNA from the partial sequence found in Incyte clone 08118 which was found to be somewhat homologous to the GenBank sequence of C5a anaphylatoxin receptor, a G-protein coupled surface receptor from dog (Perret J et al (1995) Biochem J 288:911-17) .
  • Example 3 Essentially the same method outlined in Example 3 above was used to extend the partial sequence of 8118 to obtain the full length sequence (Seq ID NO:12) of a novel C5a-like receptor homolog which is the subject of a U.S. Patent Application 08/462,355 filed June 5, 1995, and whose disclosure is incorporated by reference.
  • CTATTTATTC CTCGTCGGGC TCCCTTTGAC CTTTTTGAGA ACAAGAAGAA AAAGAACAAC 1140
  • GAGATTTTCC TTCGGGAGTT GATCTCTAAT GCTTCTGATG CCTTGGACAA GATTCGCTAT 240
  • CATGCTGTGG CAGATGTGTGTG GGGACGGCTG TAATGGTGGC TATCCTGCTG AAGCTTGGAC 300
  • ATCATTGTCC TCAACATGTT TGGCAGTGTC TTCCTGCTTA CTGCCATTAG CCTGGATCGC 360
  • GGCCAATTCA CAGATGACGA TCAAGTGCCA ACACCCCTCG TGGCAATAAC GATCACTAGG 1020
  • CTAGTGGTGG GTTTCCTGCT GCCCTCTGTT ATCATGATAG CCTGTTACAG CTTCATTGTC 1080

Abstract

L'invention décrit un procédé permettant d'obtenir des séquences d'ADNc plus longues. Ledit procédé fait intervenir une séquence d'ADN génomique connue ou une séquence d'ADNc partiel, telle que celles que l'on peut obtenir à partir d'ADNc partiels de GenBank. Deux amorces d'ACP sont mises au point pour correspondre aux extrémités de la séquence partielle connue et pour s'hybrider à l'ADN dans une bibliothèque d'ADNc afin d'amorcer l'extension loin de l'ADNc connu et de l'autre amorce. Les amorces sont ajoutées à une bibliothèque d'ADNc à l'aide d'enzymes appropriées et s'étendent à travers une séquence d'ADN supplémentaire pour produire des produits d'ACP qui sont ensuite purifiés et séquencés pour donner de nouvelles séquences. Ces nouvelles séquences sont ensuite comparées avec la séquence d'ADNc partiel connue pour trouver des zones de chevauchement, et la séquence est étendue au-delà des zones de chevauchement pour donner une séquence d'ADN plus longue.
PCT/US1996/008501 1995-06-02 1996-06-03 Procede ameliore pour obtenir des sequences d'adn complementaire longues WO1996038591A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP96917035A EP0832282A1 (fr) 1995-06-02 1996-06-03 Procede ameliore pour obtenir des sequences d'adn complementaire longues
AU59729/96A AU5972996A (en) 1995-06-02 1996-06-03 Improved method for obtaining full-length cdna sequences
JP8536785A JPH11506332A (ja) 1995-06-02 1996-06-03 全長ｃＤＮＡ配列を獲得する改良された方法

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US08/459,046 US6008039A (en) 1995-06-02 1995-06-02 Polynucleotide encoding a novel purinergic P2U receptor
US08/459,046 1995-06-02
US46235595A 1995-06-05 1995-06-05
US08/462,355 1995-06-05
US48711295A 1995-06-07 1995-06-07
US08/487,112 1995-06-07
US680995P 1995-11-15 1995-11-15
US60/006,809 1995-11-15
US56633495A 1995-12-01 1995-12-01
US08/566,334 1995-12-01

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WO1999027080A2 (fr) * 1997-11-25 1999-06-03 Incyte Pharmaceuticals, Inc. Proteine kinase humaine et inhibiteurs de kinase
WO2000052200A1 (fr) * 1999-02-26 2000-09-08 Shanghai Biorigin Gene Development Co., Ltd. Procede de clonage et de sequençage d'adnc a grande echelle grace a la reduction cyclique
WO2000060076A2 (fr) 1999-04-02 2000-10-12 Corixa Corporation Compositions pour le traitement et le diagnostic du cancer du sein et leurs procedes d'utilisation
WO2001032932A2 (fr) * 1999-11-02 2001-05-10 Curagen Corporation Procede d"identification d"une sequence d"acide nucleique
WO2001059101A1 (fr) * 2000-02-10 2001-08-16 The Penn State Research Foundation Procede d'amplification de sequences polynucleotidiques monocatenaires completes
WO2001098460A2 (fr) 2000-06-20 2001-12-27 Corixa Corporation Proteines de fusion du mycobacterium tuberculosis
WO2003053220A2 (fr) 2001-12-17 2003-07-03 Corixa Corporation Compositions et procedes applicables a la therapie et au diagnostic pour la maladie intestinale inflammatoire
US6703228B1 (en) 1998-09-25 2004-03-09 Massachusetts Institute Of Technology Methods and products related to genotyping and DNA analysis
WO2004062599A2 (fr) 2003-01-06 2004-07-29 Corixa Corporation Composes d'aminoalkyle glucosaminide phosphate et leur utilisation
WO2006046994A2 (fr) 2004-07-30 2006-05-04 Mount Sinai School Of Medicine Of New York University Formes alternatives d'epissure de klf6 et polymorphisme d'adn de klf6 de cellules germinales associes a un risque accru de cancer
WO2006117240A2 (fr) 2005-04-29 2006-11-09 Glaxosmithkline Biologicals S.A. Nouvelle methode de prevention ou de traitement d'une infection par m tuberculosis
EP1961819A2 (fr) 2000-06-28 2008-08-27 Corixa Corporation Composition et procédés pour la thérapie et le diagnostic du cancer des poumons
EP1975231A1 (fr) 2002-01-22 2008-10-01 Corixa Corporation Compositions et procédés de détection, diagnostic et de thérapie de malignancies hématologiques
EP1988097A1 (fr) 2001-05-09 2008-11-05 Corixa Corporation Compositions et procédés pour le traitement et le diagnostic du cancer de la prostate
EP2003201A2 (fr) 1998-03-18 2008-12-17 Corixa Corporation Composés et méthodes thérapeutiques et diagnostiques du cancer du poumon
EP2022800A2 (fr) 2000-07-17 2009-02-11 Corixa Corporation Compositions utilisés dans la thérapie et le diagnostic du cancer des ovaires
EP2028190A1 (fr) 1999-04-02 2009-02-25 Corixa Corporation Composés et procédés de thérapie et de diagnostic du cancer du poumon
EP2105502A1 (fr) 2000-12-12 2009-09-30 Corixa Corporation Composés et procédés de thérapie et de diagnostic du cancer du poumon
EP2192128A2 (fr) 2000-04-21 2010-06-02 Corixa Corporation Composés et méthodes pour le traitement et le diagnostique des infections de Chlamydia
EP2218733A1 (fr) 1998-12-08 2010-08-18 Corixa Corporation Composés et procédés pour le traitement et le diagnostic des infections à chlamydia
WO2011092253A1 (fr) 2010-01-27 2011-08-04 Glaxosmithkline Biologicals S.A. Antigènes modifiés de la tuberculose
EP2386314A1 (fr) 2005-03-31 2011-11-16 GlaxoSmithKline Biologicals SA Vaccins contre l'infection à chlamydia
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WO2015197737A1 (fr) 2014-06-25 2015-12-30 Glaxosmithkline Biologicals S.A. Composition immunogène de clostridium difficile

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