EP1941056A2 - Procédé de commande de l'amplification enzymatique d'acides nucléiques par fragments par l'intermediaire de brins complémentaires incomplets - Google Patents

Procédé de commande de l'amplification enzymatique d'acides nucléiques par fragments par l'intermediaire de brins complémentaires incomplets

Info

Publication number
EP1941056A2
EP1941056A2 EP06805397A EP06805397A EP1941056A2 EP 1941056 A2 EP1941056 A2 EP 1941056A2 EP 06805397 A EP06805397 A EP 06805397A EP 06805397 A EP06805397 A EP 06805397A EP 1941056 A2 EP1941056 A2 EP 1941056A2
Authority
EP
European Patent Office
Prior art keywords
synthetic oligonucleotides
nucleic acid
groups
bonds
unnatural
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.)
Withdrawn
Application number
EP06805397A
Other languages
German (de)
English (en)
Inventor
Peter Bendzko
Stephen Heymann
Hans Joos
Beate Kraffert
Ralf Bergmann
Matthias Leiser
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.)
Invitek Gesellschaft fuer Biotechnik und Biodesign mbH
Original Assignee
Invitek Gesellschaft fuer Biotechnik und Biodesign mbH
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 Invitek Gesellschaft fuer Biotechnik und Biodesign mbH filed Critical Invitek Gesellschaft fuer Biotechnik und Biodesign mbH
Publication of EP1941056A2 publication Critical patent/EP1941056A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the invention relates to a novel method for controlling the segmental enzymatic nucleic acid amplification via incomplete
  • DNA analytics has become increasingly important for research and medical practice over the last decade and is increasingly pervading other areas of human activity.
  • Representative of the need and the growing repertoire of DNA analysis techniques are figures from human genetics: In 2002, tests were offered worldwide for approximately 750 polymorphic loci in the human genome (EPF 2002) 1 , so there are currently about 1870 such service Offers or proof kits available (OMIM) 1 . Due to the unambiguousness of the causal relationship and thus the informative value, allele diagnostics are the largest and most profitable group for monogenic hereditary diseases.
  • RNA analysis including the splicing and maturation processes of mRNA, RNA-based enzymes and bioactive tools of expression regulation up to the first therapeutics.
  • the reference sequence of the chromosomal DNA section S in question is known.
  • the sequence of S is sufficiently unique in the given isolate.
  • the mutated DNA section S ' may be strongly underrepresented, but need not be.
  • the nucleotide derivatives fulfill the function of substrates of the polymerases or ligases, of primers of the polymerase chain reaction and of probes in the assay systems.
  • the rough assignment gives the following picture:
  • Terminal modifications in probes are for selective detection purposes based on
  • Scorpions (Sigma-Aldrich) combine primer and probe function.
  • chip-based detection systems Asper
  • substrate analogue incorporation resulting in fragmentation, primer extension with synthesis stop, and fluorescence label insertion are combined into one technological process.
  • fluorescence label insertion is combined into one technological process.
  • cancer-relevant k-ras minor components either wild-type cleavage and differential probe thermostability for sensitive mutant detection via DNA Elisa (Invitek GmbH) or PCR plus SSCP (Nordiag SA) are carried out in succession.
  • the basic idea is to produce a PCR product fraction which receives the vector-complementary overhangs as it forms.
  • the split vector has two fours indentations. Accordingly, the amplification primers are 5'-sided equipped with the four complementary bases, z. B. the Hinprimer with the inner four bases AATT of EcoR I
  • the modification will be inserted only at a fraction (about 1/10 to 1/100) of the primer thereby on the one hand normal exponential amplification of the
  • the 'normal' amplicons evade cloning by end-to-end mismatching Vector.
  • the amplicon derivatives produced by occasional incorporation of stop variants of the primers have the desired cohesive ends.
  • Numerous genes provide more than one mature mRNA and the associated amino acid sequence by alternative splicing.
  • the accumulated distribution statistics of the number of transcripts and exons per gene as a function of gene length determined experimentally and bioinformatic (by EST alignment) are constantly updated for human genes (Ensembl V.32 ff) 22 .
  • N is any of the natural deoxyribonucleotides dA, dC, dG, dT, X for any of the natural ribonucleotides A, C, G, T; k and I are the monomer numbers of the deoxyribonucleotide segments in the synthetic chimeric oligonucleotide.
  • Nucleotide building blocks are known to have other reactive groups that can be altered in chemical and biochemical material transformation processes.
  • the effects according to the invention described in (2) also occur in different intensities if a) the natural sugars ribose or deoxyribose are replaced by arabinose, b) the natural phosphodiester bonds are modified by modifications which render them stable to hydrolysis, c) the sugar Phosphate backbone of the synthetic oligonucleotides is replaced by intermediate base bridges of other chemical nature with a stopping effect on the complementary strand synthesis, d) on the nitrogen bases chemical changes are made, which do not affect the formation of the complementary base pairs AT and GC, but not as a template component of the polymerase be accepted / tolerated, e) between the building blocks of the synthetic oligonucleotide additional chemical bonds exist that cause structural distortions and f) in the synthetic oligonucleotide mixed changes of types (2), (3) and (4a-e) available.
  • Base case 1 is realized according to the invention by the following approach: In parallel PCR batches of 25 ⁇ l reaction volume, 50 ng of isolated genomic DNA from SW620 cells are subjected to amplification in an Eppendorf Mastercycler gradient according to the following program: 94 ° C./5 'for initial denaturation of genomic templates; 94 ° C / 30 "for the Denaturations Colour in each cycle; 61 + 10 0 C / 30" of annealing of 12 identical reaction aliquots at variable temperature in the range of 51 to 71 0 C at a constant temperature raising or cooling rate of 3 ° C per second; 72 ° C / 1 "for primer elongation, 35 cycles, 72 ° C / 5 'to the final synthesis completion;.
  • G * represents the 2'-O-tert-butyldimethyl-silyl derivative of the guanosine ribonucleotide
  • G * represents the 2'-O-tert-butyldimethyl-silyl derivative of the guanosine ribonucleotide
  • G * means the 2'-O-tert-butyldimethyl-silyl derivative of the guanosine ribonucleotide and U * the 2'-O-tert-butyl Dimethyl-silyl derivative of the uridine ribonucleotide;
  • G * represents the 2'-O-tert-butyldimethyl-silyl derivative of the guanosine ribonucleotide
  • U * the 2'-O tert-butyl-dimethyl-silyl derivative of the uridine ribonucleotide
  • C * the cytidine arabinoside
  • a sixth identical aliquot of the master mix which is also sufficient for 12 aliquots, was spiked with a round primer (again 15 pmol relative to each aliquot of sample), at the same genomic position in the k-ras gene as primers 1.1 to 1.5, but in difference specific to these is the chromosome in SW620, which has a glycine-to-valine mutation (G12V) in codon 12 of the k-ras gene.
  • G12V glycine-to-valine mutation
  • Quantitative inhibition is observed in even series of stop functions (2 and 4), while odd numbered series (1 and 3) cause partial inhibition.
  • the intensity of the wild type and mutant amplificates from parallel runs with primers without any stop function (top left and bottom right) is used for the comparison of quantities.
  • Embodiment 1 is realized according to the invention with the following changes: Instead of the primer 1.2-1.5 becomes
  • Primer 2.1 suppresses the exponential amplification of the sequence of the k-ras wild-type allele, whereas it causes exponential amplification of the mutant allele when using a so-called proof reading polymerase, here Pfu polymerase.
  • the Pfu polymerase removes the 3'-terminal G * of primer 2.1 after binding to the mutant template to which it is not complementary. After 42 cycles of amplification, the selectively amplified mutant signal appears in the electrophoretic detection.
  • the embodiments 1 and 2 are extended according to the invention to the effect that in the primers 1.2-1.5 and 2.1 as a stop function a bulky additional group with conjugated electron pairs is used.
  • the advantage of this embodiment variant is the combinability of inventive control of the sectionwise enzymatic nucleic acid amplification with fluorescent detection principles.
  • Partial inhibition according to the invention.
  • the amount of amplicon when using a synthetic oligonucleotide with stop function (lower row) is reduced compared to the control (upper row).
  • the attached helper cloning site consists of the corresponding recognition sequence of the selected restrictase plus 3-4 arbitrary nucleotides used to ensure the full endonucleolytic Effect of the restriction enzyme can be used.
  • This method has the serious disadvantage that the auxiliary cloning sites in the PCR product must be unique, otherwise the amplificate becomes fragmented. The unicality is a problem especially with long amplicons of unknown sequence.
  • Nucleotidyltransferases add an additional 1 -3 nucleotides to the 3 'end of a newly synthesized complementary strand. As a rule of thumb, over 90% of the 3 'ends carry a supernatant nucleotide that is 85% dA; a second nucleotide (also usually dA) is found in about 1% of the synthesis products, a third in 0.01%.
  • Thermostable polymerases which, depending on the ion (Mn 2+ / Mg 2+ ) show a higher template tolerance to RNA, are more prone to prolong the product with additional nucleotides. This explains why it is absolutely unsatisfactory to introduce crude PCR products into a blunt-ended vector. Partially create here 3'-T-tailed vectors remedy.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procédés d'amplification enzymatique d'acides nucléiques dans lesquels sont utilisés des oligonucléotides synthétiques qui contiennent une ou plusieurs unités structurales à structure chimique non naturelle. L'utilisation de ces unités structurales empêche la synthèse enzymatique des brins d'acide nucléique qui sont complémentaires des fragments contenant ces unités structurales. Les procédés selon la présente invention permettent entre autres la détection de formes d'épissure alternatives de gènes, la détection de variants d'acide nucléique, la production de molécules d'acide nucléique marquées, la production immédiate de produits à double brin d'acide nucléique pourvus d'extrémités à simple brin pour les réactions de ligature ultérieures. Les champs d'application de la présente invention sont les domaines les plus divers de la recherche, la pratique biomédicale, l'analyse basée sur les acides nucléiques, en particulier de produits biotechnologiques, agricoles et alimentaires, ainsi que la criminalistique.
EP06805397A 2005-10-07 2006-10-09 Procédé de commande de l'amplification enzymatique d'acides nucléiques par fragments par l'intermediaire de brins complémentaires incomplets Withdrawn EP1941056A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005048503A DE102005048503B4 (de) 2005-10-07 2005-10-07 Verfahren zur Steuerung der abschnittsweisen enzymatischen Nukleinsäurevervielfältigung über inkomplette Komplementärstränge
PCT/DE2006/001779 WO2007042003A2 (fr) 2005-10-07 2006-10-09 Procede de commande de l'amplification enzymatique d'acides nucleiques par fragments par l'intermediaire de brins complementaires incomplets

Publications (1)

Publication Number Publication Date
EP1941056A2 true EP1941056A2 (fr) 2008-07-09

Family

ID=37865070

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06805397A Withdrawn EP1941056A2 (fr) 2005-10-07 2006-10-09 Procédé de commande de l'amplification enzymatique d'acides nucléiques par fragments par l'intermediaire de brins complémentaires incomplets

Country Status (4)

Country Link
US (1) US20070082343A1 (fr)
EP (1) EP1941056A2 (fr)
DE (1) DE102005048503B4 (fr)
WO (1) WO2007042003A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2753712B1 (fr) 2011-09-06 2017-03-22 Gen-Probe Incorporated Structures fermées d'acide nucléique

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9211979D0 (en) * 1992-06-05 1992-07-15 Buchard Ole Uses of nucleic acid analogues
US5550047A (en) * 1994-02-18 1996-08-27 University Of Massachusetts Oligonucleotides with anti-Epstein-Barr virus activity
WO2003062452A2 (fr) * 2002-01-23 2003-07-31 Proligo, Llc Methodes pour la synthese et la purification integrees d' oligonucleotides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007042003A3 *

Also Published As

Publication number Publication date
DE102005048503B4 (de) 2012-10-18
US20070082343A1 (en) 2007-04-12
DE102005048503A1 (de) 2007-04-19
WO2007042003A2 (fr) 2007-04-19
WO2007042003A3 (fr) 2007-08-23

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