[go: up one dir, main page]

WO2012013932A1 - Procédé race perfectionné - Google Patents

Procédé race perfectionné Download PDF

Info

Publication number
WO2012013932A1
WO2012013932A1 PCT/GB2011/001131 GB2011001131W WO2012013932A1 WO 2012013932 A1 WO2012013932 A1 WO 2012013932A1 GB 2011001131 W GB2011001131 W GB 2011001131W WO 2012013932 A1 WO2012013932 A1 WO 2012013932A1
Authority
WO
WIPO (PCT)
Prior art keywords
cdna
adapter
molecule
sequence
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2011/001131
Other languages
English (en)
Inventor
Ian Alistair Johnston
Neil Bower
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of St Andrews
Original Assignee
University of St Andrews
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of St Andrews filed Critical University of St Andrews
Publication of WO2012013932A1 publication Critical patent/WO2012013932A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the present invention relates to a method of generating a cDNA molecule or pool of cDNA molecules which comprise adapter sequences 5' and 3' of the sequence corresponding to the 5' or 3' ends of the original mRNA molecule. There is further provided a method of amplifying a desired 5' or 3' end of said modified cDNA or pool of cDNA molecules whilst minimising the generation of false positives and/or non-specific amplification. There is also provided a kit for carrying out the method of the present invention.
  • Cloning of novel cDNAs typically results in sequences that only partially represent the mRNA species, with the 3' ends usually well represented and the 5' end of the sequence often incomplete. This tendency can be due to RNA degradation, or is a result of the cDNA synthesis reaction not reaching completion. When working with novel organisms, where the genome has not been sequenced, obtaining the 5' end of the cDNA, although in theory quite simple, can often prove to be technically challenging.
  • the unknown 5' sequence from the ends of cDNA molecules can be cloned using a PCR technique know as rapid amplification of cDNA ends (RACE) (2).
  • RACE rapid amplification of cDNA ends
  • homopolymer tailing of the cDNA is used to append a linker sequence to the cDNA terminus.
  • Extension of unknown regions of the cDNA is then achieved through PCR using a gene specific primer and a primer that can bind and prime DNA synthesis from the linker sequence.
  • New RACE an anchor primer is ligated to the 5' end of the mRNA molecule prior to reverse transcription, which subsequently synthesised cDNA molecules incorporate into their sequence (3). Therefore only full length cDNA molecules are amplified by PCR using the anchor primer and gene specific primer.
  • Cap-switch RACE Cap finder
  • MMLV Murine moloney leukaemia virus
  • dUTP is incorporated into all amplicons by substituting dUTP for dTTP in PCR reactions.
  • Treatment of PCR products with UDG prior to thermal cycling cleaves DNA at uracil bases thereby inhibiting amplification of carryover contamination from previous PCR reactions (10). This function can be exploited to remove unwanted DNA sequences from reaction mixtures.
  • the use of dUTP and UDG to increase the specificity of 3' RACE reactions has been reported (11 , see also US5334515), however, no methods using dUTP/UDG for 5' RACE are currently available.
  • the present invention is based on the development of a modified version of the RACE protocol which enables the amplification reaction to be simplified by degrading non- target modified cDNA sequences using uracil DNA glycosylase (UDG).
  • UDG uracil DNA glycosylase
  • a double stranded cDNA is synthesised to incorporate dUTP, and this then serves as template for subsequent amplification of cDNA ends of any target gene.
  • PCR primer sites (where dUTP replaces dTTP) are incorporated at the 3' and 5' ends of each cDNA molecule. These primer sites are incorporated at the 3' and 5' ends by way of adapter sequences.
  • the modified double stranded cDNA, incorporating dUTP now serves as a universal pool for PCR amplification of cDNA ends for any gene of interest.
  • a method for generating a modified cDNA molecule comprising sequence corresponding to the 5' and 3' ends of an mRNA molecule comprising the steps of: a) annealing a first adapter oligonucleotide to an mRNA molecule, said first adapter comprising a 3' portion which is substantially complimentary to the 3' poly A tail of the mRNA molecule and a 5' portion which comprises one or more uracil residues; b) extending from said first adapter, utilizing dNTPs which include dUTP, so as to generate a first modified cDNA strand, which comprises one or more, typically a plurality of uracil residues in its sequence; c) incorporating a second adapter, which is different to the first adapter, at the 3' end of the first cDNA strand, wherein the second adapter comprises at least a 3' portion which is not specific to the mRNA molecule and which comprises one or more uracil
  • the method may be conducted on a pool of mRNA molecules, thereby generating a single pool of cDNA molecules, each cDNA molecule from the pool comprising the same 5' and 3' adapter sequences respectively
  • modified cDNA molecule relates to a cDNA molecule which comprises one or more, typically many uracil molecules throughout its sequence.
  • the modified cDNA molecules of the present invention comprise uracils in place of many thymidine residues.
  • typically at least 1 in 1 , to 1 in 20, such as 1 in 1 to 1 , in 4, especially 1 in 1.05 to 1 in 2 thymidine residues are replaced by a uracil residue.
  • the first adapter oligonucleotide typically comprises a 3' portion which comprises a stretch of T residues, so as to be able to complimentary base pair with the poly A tail of the mRNA molecule.
  • the stretch will be 10-40 Ts in length, such as 15-30 Ts in length.
  • the 3' end of the first adapter may be a T or may incorporate other residues to ensure proper annealing and extension from the first adapter. Indeed a pool of first adapters may be provided which include possible variations at the 3' end so as to ensure proper annealing and extension from the adapter.
  • the 5' end of the first adapter sequence is designed to be not complimentary to eukaryotic sequences based on blast homology searches and generally not to be specific to the mRNA sequence.
  • any nucleotide sequence as described herein will comprise a complimentary portion which is at least 95%, such as 97-99% identical with the sequence to which it is to bind.
  • the extension from the first adapter is carried out using dNTPs which comprise dUTP, but as mentioned above, conveniently dTTP may also be present, albeit in much lower (e.g. ⁇ 10%) amounts than dUTP, in the dNTP mix.
  • the extension reaction may be catalysed using a variety of reverse transcriptases, such as avian myeloblastosis virus (A V) or Moloney murine leukaemia virus (M-MLV).
  • the reverse transcriptase is M-MLV reverse transciptase.
  • M- MLV reverse transcriptase adds 2-4 cytosine residues to the 3' ends of cDNA molecules and by using the cap switching techniques previously described (5, 6), it is possible to incorporate a second adapter (containing uracil) at the 3' end of the first cDNA strand.
  • RNA ligase method previously described (3), however to make this compatible with the technique described herein, dUTP should replace dTTP in the dNTP mix used for first strand cDNA synthesis.
  • RNA of the RNA/cDNA hybrid may be removed by way of RNAse H treatment to degrade RNA of the RNA/cDNA hybrid and the cDNA purified from degraded RNA and unincorporated adapter molecules by way of affinity purification (e.g. silica-based purification) using a suitable column purification method, such as by using QIA quick (Qiagen Inc., Chatsworth, CA, USA).
  • affinity purification e.g. silica-based purification
  • suitable column purification method such as by using QIA quick (Qiagen Inc., Chatsworth, CA, USA).
  • the second cDNA strand may be easily generated by copying the first cDNA strand using a first primer (where uracil replaces thymidine) which is able to specifically bind to the second adapter sequence at 3' end of the first cDNA strand, using a suitable DNA polymerase, such as Taq polymerase and a dNTP mix comprising dUTP.
  • a first primer where uracil replaces thymidine
  • a suitable DNA polymerase such as Taq polymerase and a dNTP mix comprising dUTP.
  • the resulting double stranded cDNA can be purified by way of affinity purification (e.g. silica-based purification) using a suitable column purification method, such as by using QIA quick (Qiagen Inc., Chatsworth, CA, USA) and then used as a template for the specific amplification of the ends of target genes.
  • affinity purification e.g. silica-based purification
  • suitable column purification method such as by using QIA quick (Qiagen Inc., Chatsworth, CA, USA) and then used as a template for the specific amplification of the ends of target genes.
  • the modified resulting double stranded cDNA molecule(s) can be amplified further by using said first primer and a second primer which is able to specifically bind to the sequence at the 3' end of the second strand cDNA strand and which second primer comprises one or more uracil residues, and the dNTP mix contains dUTP, so as to generate an amplified cDNA molecule(s), which comprises a plurality of uracil residues within the first and second strands of the amplified cDNA molecule.
  • Further rounds of amplification may be carried out, such as 20-25 cycles of PCR, so as to increase the cDNA copies.
  • an asymmetric amplification reaction using a single gene specific primer and standard dNTPs (without dUTP) is performed.
  • the reaction mixture is then treated with UDG to simplify the PCR reaction mixture by degrading non-target cDNAs.
  • the present invention provides a method of amplifying a 5' or 3' end of an mRNA molecule for enabling identification of sequence at a respective 5' or 3' end of the mRNA molecule, the method comprising: a) providing the double stranded cDNA molecule or pool of cDNA molecules according to the first aspect, or amplified copies thereof; b) asymmetrically amplifying the 5' or 3' end of said cDNA molecule or one of said cDNA molecules in the pool using a gene specific primer which is substantially complementary to an internal portion of the cDNA sequence, using dNTPs which do not include dUTP, so as to generate an amplified gene specific single stranded DNA molecule; c) adding UDG so as to degrade sequences which comprise uracil thereby leaving only an amplified gene
  • Step d) is preferable in order to increase specificity and yield of the amplified 5' or 3' end to be detected
  • the gene specific primers are generally comprised only of DNA bases (i.e. A, C, G and T) and may be designed to hybridise specifically to a desired mRNA sequence.
  • the gene specific primers may be designed and synthesised based on a known mRNA sequence, or may be based on a consensus sequence ascertained from previously sequenced gene sequences from other organisms.
  • the skilled addressee is very familiar with asymmetric amplification or PCR and a number of cycles (e.g. 10-40) may be performed to linearly amplify the sequence of interest and hence the 5' or 3' end of the cDNA. It is to be appreciated that the gene specific primer can be chosen to anneal to either particular strand of the modified cDNA molecule and hence generate amplified copies of the 5' or 3' end of a particular mRNA, as desired.
  • UDG is added so as to degrade the modified cDNA molecule(s) present once the asymmetric amplification step has been completed.
  • the amplified single stranded DNA molecule which includes a complimentary DNA copy representative of the desired 5' or 3' end of the mRNA molecule remains as a viable target for a second exponential PCR amplification.
  • Exponential PCR amplification of the desired amplified single stranded DNA molecule is achieved using a second nested gene specific primer and a primer which is able to specifically bind to the sequence at the 5' or 3' end of the amplified single stranded DNA molecule. Further rounds of amplification may be carried out as necessary.
  • the 5' end of the mRNA molecule is amplified.
  • the appropriately amplified 5' or 3' end sequence can easily be ascertained by conventional cloning and gene sequencing techniques known to the skilled addressee, utilizing a suitable primer.
  • kits for use in generating the modified cDNA molecule(s) and/or the amplified DNA molecule which includes the amplified 5' or 3' end may comprise first and second adapter molecules, a dNTP mix which includes dUTP and first and second primer molecules for amplifying the modified cDNA molecule.
  • a kit for generating an amplified DNA molecule which comprises a sequence corresponding to a 5' or 3' end of a mRNA molecule may comprise UDG and a primer for use in amplifying the 5' or 3' end.
  • kits may also comprise other suitable reagents such as buffers, salts solutions and the like, as well as instructions for carrying out the methods as described hereinabove.
  • FIG. 1 shows a diagrammatic representation of a double stranded template (T-RACE cDNA) synthesis protocol according to the present invention.
  • First strand cDNA synthesis using a dNTP mix which includes dUTP is primed using an oligo-dT adapter- A primer.
  • oligo(dC) is added to the end of the cDNA by the M-MLV reverse transcriptase.
  • Adapter A and B both contain dUTP instead of dTTP.
  • Adapter A and B serve as priming sites for PCR amplification using primers A and B, which also have dTTP replaced by dUTP.
  • T-RACE ready cDNA contains dUTP residues throughout and dUTP containing adapters at the 3' and 5' ends.
  • Figure 2 shows a diagrammatic representation of the 5' specific amplification of cDNA ends using T-RACE.
  • T-RACE ready cDNA serves as template for an asymmetric PCR reaction using a single gene specific primer (GSP1 ) and standard dNTPs (without dUTP). This reaction produces single stranded target cDNAs which include the adapter sequence comprised of standard dNTPs.
  • FIG. 1 shows (A) Specific amplification of 5' cDNA ends from Atlantic salmon (Ss) and Zebrafish (Dr) fast muscle. Lane 1 STAC3 (Ss), Lane 2 IGF-I (Ss) Lane 3 CMYA5 (Dr), Lane 4 MAFbx (Ss), Lane 5 STAC3 (Ss). DNA size marker of 5000, 2000, 850, 400 and 200 bp is shown in Lane M.
  • Figure 4 shows for background purposes non-specific amplification of products without UDG treatment.
  • the T-RACE ready protocol is depicted in figure 1 and the primers used listed in table 1.
  • RNAse inhibitor RNase OUT, Invitrogen, Carlsbad, CA, USA), 200 U Superscript II (Invitrogen, Carlsbad, CA, USA) and
  • RNA/cDNA hybrid was achieved by addition of 1 pi RNAse H (Invitrogen, Carlsbad, CA, USA) and incubation at 37 °C for 15 minutes.
  • the reaction was purified through a QIAquick PCR purification column and eluted in 30 ⁇ elution buffer as per manufacturer's guidelines (Qiagen Inc., Chatsworth, CA, USA).
  • Second strand synthesis was achieved by PCR using the priming sites incorporated at the 3' (Adapter B) and 5' (Adapter A) ends of the first strand cDNA.
  • 2 ⁇ of the purified first strand cDNA was used as template, and amplified in the following reaction (50 ⁇ ): 2.5 mM MgCI2, 1.0 mM dUTP, 0.5 m dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.1 mM dTTP, 0.5 ⁇ primer A, 0.5 ⁇ primer B (table 1 ).
  • the reaction mixture was mixed and heated to 95 °C before addition of 5 units of Taq DNA polymerase (Bioline, London, UK).
  • the reaction mixture was then incubated at 95 °C for a further 2 minutes, followed by 20 cycles of 95 °C denaturing for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C 12 minutes.
  • the reaction was then chilled on ice and purified through a QIAquick PCR purification column (Qiagen Inc., Chatsworth, CA, USA) and eluted in 10Oul elution buffer as per manufacturer's guidelines.
  • the purified double stranded cDNA, incorporating dUTP and 5' and 3' adapter sequences serves as template for the following PCR reactions.
  • Asymmetric PCR was performed using a gene specific primer (GSP1 ) and standard dNTPs (dATP, dTTP, dGTP, dCTP) in the following hot start 20 ⁇ reaction: 0.5 ⁇ GSP1 , 2 ⁇ T-RACE ready cDNA, 16 mM (NH 4 ) 2 S0 4 , 67 mM Tris-HCI, 3 mM MgCI 2 , 0.25 mM dNTPs.
  • the reaction mixture was heated to 95 °C for 2.5 min before addition of 1 unit of Taq DNA polymerase (Bioline, London, UK), and then cycled 20 times with the following conditions: 95 °C 30 s, 60 °C 30 s, 72 °C 2 min.
  • the reaction was chilled on ice, purified through a MinElute PCR purification column (Qiagen Inc., Chatsworth, CA, USA) and eluted in 11 ⁇ water.
  • the length of time for the 72 °C extension step of the PCR should be varied according to expected amplicon length.
  • the entire asymmetric reaction is used in the following steps using a nested gene specific primer (GSP2), and the primers used for PCR do not contain dUTP.
  • GSP2 nested gene specific primer
  • cDNAs incorporating dUTP were removed by UDG treatment to leave only those cDNAs synthesised with standard dNTPs during the asymmetric PCR.
  • the following 17.8 ⁇ reaction was performed: 10 ⁇ asymmetric PCR product, 2 ⁇ (2 units) Uracil DNA glycosylase, 2 ⁇ 10 x PCR buffer (160 mM (NH 4 ) 2 S0 4l 670 mM Tris-HCI), 1.2 ⁇ 50 mM MgCI 2 , 1 ⁇ nested GSP2, 1 ⁇ of 10 ⁇ T-RACE primer (table 1), 0.6 ⁇ water, and incubated at 37 °C for 30 minutes. Depending on whether 3' or 5' cDNA ends are targeted, the 3' or 5' T-RACE primer is used for amplification.
  • T-RACE PCR Targeted amplification of cDNA ends
  • the UDG treated cDNA mixture was heated to 95 °C before addition of 2 ⁇ 2.5 mM standard dNTPs and 0.2 ⁇ (1unit) of Taq DNA polymerase (Bioline, London, UK).
  • the reaction was denatured at 95 °C for 5 min followed by 35 cycles of 95 °C 30 s, 60 °C 30s, 72 °C 2 min, followed by a final extension of 7 min at 72 °C.
  • the length of time for the 72 °C extension step of the PCR should be varied according to expected amplicon length.
  • PCR products were separated on a 1.2% (m/v) agarose gel, and the bands excised and purified using a QIAquick gel extraction kit (Qiagen Inc., Chatsworth, CA, USA).
  • the PCR products were cloned into a T/A pCR4-TOPO vector (Invitrogen, Carlsbad, CA, USA) and transformation of chemically competent TOP10 Escherichia coli cells (Invitrogen, Carlsbad, CA, USA). Individual colonies were grown and plasmids purified using a QIAprep Spin Miniprep Kit (Qiagen Inc., Chatsworth, CA, USA).
  • This asymmetric PCR using only a gene specific primer produces a cDNA fragment which includes the 5' adapter sequence, and is comprised of standard dNTPs (Fig. 2).
  • Degradation of dUTP containing cDNA molecules by Uracil DNA glycosylase (UDG) treatment leaves only the molecules comprised of standard dNTPs primed by the gene specific primer (Fig. 2).
  • UDG Uracil DNA glycosylase
  • CMYA5 zebrafish myospryn
  • IGF-I insulin-like growth factor-l
  • Standard RACE methods often result in non specific amplification giving false positive results (6, 7, 8).
  • cDNA is synthesised using oligo dT
  • the subsequent incorporation of the adapter sequence for 5' RACE through terminal transferase tailing or cap finder methods
  • mispriming events during PCR will often lead to exponential amplification, and smears, rather than distinct bands are often obtained.
  • the use of a nested PCR reaction should increase the specificity of the RACE reaction, however non-specific products are frequently obtained.
  • the T-RACE method described herein degrades all of the non-target cDNA molecules, except for those that were primed by the gene specific primer and synthesised using standard dNTPs, thereby significantly simplifying the templates present in the PCR reaction.
  • the gene specific primer is used in an asymmetric PCR, where higher annealing temperatures are permitted, rather than during cDNA synthesis, the specificity of the reaction is increased.
  • a nested primer in the second PCR reaction specific amplification of the desired molecule is achieved.
  • Taq polymerase is less efficient at incorporating dUTP than dTTP (10) which could impact on the double stranded cDNA synthesis. It was observed that a ratio of dUTP:dTTP of 8:1 , improved the efficiency of the T-RACE reaction, but still resulted in sufficient degradation of non- target cDNAs to give specific amplification. We also observed an improvement in the specificity of the PCR reactions by adding the MnCI 2 and adapter-A after cDNA synthesis has completed. Presumably this modification decreases the chances of adapter-A priming cDNA synthesis during the first strand cDNA reaction which could lead to spurious amplification products in subsequent reactions.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de production d'une molécule d'ADNc ou d'un pool de molécules d'ADNc qui comprennent des séquences d'adaptateur 5' et 3' de la séquence correspondant aux extrémités 5' et 3' de la molécule d'ARNm originelle. L'invention concerne en outre un procédé d'amplification d'une extrémité 5' ou 3' désirée dudit ADNc modifié ou du pool de molécules d'ADNc tout en réduisant la production de faux positifs et/ou l'amplification non sélective. L'invention concerne également un kit pour mettre en œuvre le procédé de la présente invention.
PCT/GB2011/001131 2010-07-29 2011-07-28 Procédé race perfectionné Ceased WO2012013932A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1012748.8A GB201012748D0 (en) 2010-07-29 2010-07-29 Improved RACE
GB1012748.8 2010-07-29

Publications (1)

Publication Number Publication Date
WO2012013932A1 true WO2012013932A1 (fr) 2012-02-02

Family

ID=42799293

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2011/001131 Ceased WO2012013932A1 (fr) 2010-07-29 2011-07-28 Procédé race perfectionné

Country Status (2)

Country Link
GB (1) GB201012748D0 (fr)
WO (1) WO2012013932A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013191775A2 (fr) 2012-06-18 2013-12-27 Nugen Technologies, Inc. Compositions et procédés pour la sélection négative de séquences d'acide nucléique indésirable
CN106636074A (zh) * 2017-02-23 2017-05-10 厦门大学 针对3’端带有a 重复序列的情况下获得完整3’末端序列的3’ race 方法
US9650628B2 (en) 2012-01-26 2017-05-16 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library regeneration
US9745614B2 (en) 2014-02-28 2017-08-29 Nugen Technologies, Inc. Reduced representation bisulfite sequencing with diversity adaptors
US9822408B2 (en) 2013-03-15 2017-11-21 Nugen Technologies, Inc. Sequential sequencing
WO2018006567A1 (fr) * 2016-07-08 2018-01-11 北京全式金生物技术有限公司 Procédé hautement efficace pour lier un lieur d'adn
US10102337B2 (en) 2014-08-06 2018-10-16 Nugen Technologies, Inc. Digital measurements from targeted sequencing
US10570448B2 (en) 2013-11-13 2020-02-25 Tecan Genomics Compositions and methods for identification of a duplicate sequencing read
US11028430B2 (en) 2012-07-09 2021-06-08 Nugen Technologies, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system
US12492430B2 (en) 2017-04-11 2025-12-09 Tecan Genomics, Inc. Library quantitation and qualification

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5035996A (en) 1989-06-01 1991-07-30 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
WO1992018521A1 (fr) * 1991-04-10 1992-10-29 Life Technologies, Inc. Procede d'amplification et de modification d'une sequence d'arn
US5683896A (en) 1989-06-01 1997-11-04 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5035996A (en) 1989-06-01 1991-07-30 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
US5683896A (en) 1989-06-01 1997-11-04 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
US5945313A (en) 1989-06-01 1999-08-31 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
WO1992018521A1 (fr) * 1991-04-10 1992-10-29 Life Technologies, Inc. Procede d'amplification et de modification d'une sequence d'arn
US5334515A (en) 1991-04-10 1994-08-02 Life Technologies, Inc. Method for altering a nucleotide sequence

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
BOWER NEIL I ET AL: "Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences.", NUCLEIC ACIDS RESEARCH, vol. 38, no. 21, E194, November 2010 (2010-11-01), pages 1 - 7, XP002661009, ISSN: 1362-4962, DOI: 10.1093/nar/gkq816 *
BUCHMAN G W ET AL: "Selective RNA amplification: a novel method using dUMP-containing primers and uracil DNA glycosylase.", PCR METHODS AND APPLICATIONS, vol. 3, no. 1, August 1993 (1993-08-01), pages 28 - 31, XP002661010, ISSN: 1054-9803 *
BUCHMAN, G.W., SCHUSTER, D.M., RASHTCHIAN, A.: "Selective Rna Amplification - a Novel Method Using Dump-Containing Primers and Uracil DNA Glycosylase", PCR METHODS APPL., vol. 3, 1993, pages 28 - 31, XP002661010
FROHMAN, M.A., DUSH, M.K., MARTIN, G.R.: "Rapid Production of Full-Length Cdnas from Rare Transcripts - Amplification Using a Single Gene-Specific Oligonucleotide Primer", PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 8998 - 9002, XP000604678, DOI: doi:10.1073/pnas.85.23.8998
FROMONT-RACINE, M., BERTRAND, E., PICTET, R., GRANGE, T.: "A Highly Sensitive Method for Mapping the 5' Termini of mRNAs", NUCLEIC ACIDS RES., vol. 21, 1993, pages 1683 - 1684
KWOK, S., HIGUCHI, R.: "Avoiding False Positives with Pcr", NATURE, vol. 339, 1989, pages 237 - 238, XP001172940, DOI: doi:10.1038/339237a0
LIPOVICH, L., LYNCH, E.D., LEE, M.K., KING, M.C.: "A novel sodium bicarbonate cotransporter-like gene in an ancient duplicated region: SLC4A9 at 5q31", GENOME BIOL., 2001
LONGO M C ET AL: "Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions", GENE, vol. 93, no. 1, 1 September 1990 (1990-09-01), ELSEVIER, AMSTERDAM, NL, pages 125 - 128, XP023545615, ISSN: 0378-1119, [retrieved on 19900901], DOI: 10.1016/0378-1119(90)90145-H *
LONGO, M.C., BERNINGER, M.S., HARTLEY, J.L.: "Use of Uracil DNA Glycosylase to Control Carry-over Contamination in Polymerase Chain-Reactions", GENE, vol. 93, 1990, pages 125 - 128, XP023545615, DOI: doi:10.1016/0378-1119(90)90145-H
SALEHI-ASHTIANI, K., LIN, C.W., HAO, T., SHEN, Y., SZETO, D., YANG, X.P., GHAMSARI, L., LEE, H., FAN, C.Y., MURRAY, R.R. ET AL.: "Large-scale RACE approach for proactive experimental definition of C. elegans ORFeome", GENOME RES., vol. 19, 2009, pages 2334 - 2342
SAMBROOK, J, FRITSCH, E.F., MANIATIS, T.: "Molecular cloning: a laboratory manual", 1989, COLD SPRING HARBOR LABORATORY, pages: 1.74 - 1.84,7.3
SCHAEFER, B.C.: "Revolutions in Rapid Amplification of Cdna Ends - New Strategies for Polymerase Chain-Reaction Cloning of Full-Length Cdna Ends", ANAL BIOCHEM., vol. 227, 1995, pages 255 - 273, XP009012046, DOI: doi:10.1006/abio.1995.1279
SCHMIDT W M ET AL: "CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.", NUCLEIC ACIDS RESEARCH, vol. 27, no. 21, E31, 1 November 1999 (1999-11-01), pages I - IV, XP002661269, ISSN: 1362-4962 *
SCHMIDT, W.M., MUELLER, M.W.: "CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs", NUCLEIC ACIDS RES., vol. 27, 1999, pages E31, XP002661269, DOI: doi:10.1093/nar/27.21.e31
SCHRAMM G ET AL: "A simple and reliable 5'-RACE approach.", NUCLEIC ACIDS RESEARCH, vol. 28, no. 22, E96, 15 November 2000 (2000-11-15), pages 1 - 4, XP002661255, ISSN: 1362-4962 *
SCHRAMM, G., BRUCHHAUS, I., ROEDER, T.: "A simple and reliable 5'-RACE approach", NUCLEIC ACIDS RES., vol. 28, 2000, pages E96

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9650628B2 (en) 2012-01-26 2017-05-16 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library regeneration
US10876108B2 (en) 2012-01-26 2020-12-29 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation
US10036012B2 (en) 2012-01-26 2018-07-31 Nugen Technologies, Inc. Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation
EP2861787A4 (fr) * 2012-06-18 2016-03-02 Nugen Technologies Inc Compositions et procédés pour la sélection négative de séquences d'acide nucléique indésirable
US9957549B2 (en) 2012-06-18 2018-05-01 Nugen Technologies, Inc. Compositions and methods for negative selection of non-desired nucleic acid sequences
WO2013191775A2 (fr) 2012-06-18 2013-12-27 Nugen Technologies, Inc. Compositions et procédés pour la sélection négative de séquences d'acide nucléique indésirable
US11697843B2 (en) 2012-07-09 2023-07-11 Tecan Genomics, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US11028430B2 (en) 2012-07-09 2021-06-08 Nugen Technologies, Inc. Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US10619206B2 (en) 2013-03-15 2020-04-14 Tecan Genomics Sequential sequencing
US9822408B2 (en) 2013-03-15 2017-11-21 Nugen Technologies, Inc. Sequential sequencing
US10760123B2 (en) 2013-03-15 2020-09-01 Nugen Technologies, Inc. Sequential sequencing
US10570448B2 (en) 2013-11-13 2020-02-25 Tecan Genomics Compositions and methods for identification of a duplicate sequencing read
US11098357B2 (en) 2013-11-13 2021-08-24 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
US11725241B2 (en) 2013-11-13 2023-08-15 Tecan Genomics, Inc. Compositions and methods for identification of a duplicate sequencing read
US9745614B2 (en) 2014-02-28 2017-08-29 Nugen Technologies, Inc. Reduced representation bisulfite sequencing with diversity adaptors
US10102337B2 (en) 2014-08-06 2018-10-16 Nugen Technologies, Inc. Digital measurements from targeted sequencing
WO2018006567A1 (fr) * 2016-07-08 2018-01-11 北京全式金生物技术有限公司 Procédé hautement efficace pour lier un lieur d'adn
CN106636074A (zh) * 2017-02-23 2017-05-10 厦门大学 针对3’端带有a 重复序列的情况下获得完整3’末端序列的3’ race 方法
US12492430B2 (en) 2017-04-11 2025-12-09 Tecan Genomics, Inc. Library quantitation and qualification
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
US12059674B2 (en) 2020-02-03 2024-08-13 Tecan Genomics, Inc. Reagent storage system

Also Published As

Publication number Publication date
GB201012748D0 (en) 2010-09-15

Similar Documents

Publication Publication Date Title
WO2012013932A1 (fr) Procédé race perfectionné
Chenchik et al. Full-length cDNA cloning and determination of mRNA 5′ and 3′ ends by amplification of adaptor-ligated cDNA
Stewart et al. Characterization of mature mitochondrial transcripts in Drosophila, and the implications for the tRNA punctuation model in arthropods
US5830712A (en) Selective template deletion method
Yeku et al. Rapid amplification of cDNA ends (RACE)
US20070117121A1 (en) cDNA library preparation
CN106062209B (zh) 合成性长阅读dna测序
CN112689673A (zh) 转座体使能的dna/rna测序（ted rna-seq）
US20140004569A1 (en) Use of template switching for dna synthesis
JP2017503521A (ja) 高温核酸合成で使用するための新規な逆転写酵素
US20060240451A1 (en) Compositions and methods employing 5' phosphate-dependent nucleic acid exonucleases
JP6583796B2 (ja) 核酸の増幅方法
US10619199B2 (en) Nucleic acid amplification and library preparation
JP2023002557A (ja) シングルプライマーからデュアルプライマーのアンプリコンへのスイッチング
JP2023541511A (ja) 活性および熱安定性が向上した逆転写酵素変異体
JP4284063B2 (ja) 核酸塩基配列決定方法
US20080145844A1 (en) Methods of cDNA preparation
KR20240051994A (ko) 레트로트랜스포존 및 이의 기능적 단편을 포함하는 시스템, 조성물, 및 방법
Bower et al. Targeted rapid amplification of cDNA ends (T-RACE)—an improved RACE reaction through degradation of non-target sequences
CN114364813B (zh) 多重等温扩增核酸序列的方法
US20240368586A1 (en) Guide rna sequencing confirmation
WO2002059353A2 (fr) Amplification par reaction en chaine de la polymerase (pcr) asymetrique
Fehr et al. Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase 6: an improved method to determine 5′-and 3′-cDNA ends
Chutia et al. Recent developments in RACE-PCR for the full-length cDNA identification
JP5532925B2 (ja) 一本鎖又は二本鎖ｄｎａの合成方法並びに合成用キット

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11743571

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11743571

Country of ref document: EP

Kind code of ref document: A1