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WO2007016929A1 - Procédé de préparation d’une échelle d'adn en utilisant une acp et son optimisation par sa modelisation numérique - Google Patents

Procédé de préparation d’une échelle d'adn en utilisant une acp et son optimisation par sa modelisation numérique Download PDF

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Publication number
WO2007016929A1
WO2007016929A1 PCT/EG2006/000015 EG2006000015W WO2007016929A1 WO 2007016929 A1 WO2007016929 A1 WO 2007016929A1 EG 2006000015 W EG2006000015 W EG 2006000015W WO 2007016929 A1 WO2007016929 A1 WO 2007016929A1
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WO
WIPO (PCT)
Prior art keywords
dna
pcr
length
primers
fragments
Prior art date
Application number
PCT/EG2006/000015
Other languages
English (en)
Inventor
Yasser Refaat Abdel-Fattah
Ahmed Abo El-Einin Gaballa
Mahmoud Mohamed El-Sayed Berekaa
Original Assignee
Mubarak City For Scientific Research & Technology Applications
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 Mubarak City For Scientific Research & Technology Applications filed Critical Mubarak City For Scientific Research & Technology Applications
Publication of WO2007016929A1 publication Critical patent/WO2007016929A1/fr

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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 patent pending is in the field of molecular biology.
  • the request of invention relates to a method for preparing a DNA ladder in the range (100 - 2500) base with ten marker fragments by applying the polymerase chain reaction.
  • optimization of the PCR yield by applying numerical modeling.
  • DNA ladders are common reagents in molecular biology, useful for determining the size of DNA fragments.
  • a DNA ladder comprises two or more DNA fragments of known size.
  • a DNA sample and a DNA ladder are loaded in adjacent wells of an agarose gel.
  • the DNA is separated by electrophoresis through the gel.
  • the gel is stained with a flourescent dye, such as ethidium bromide, and exposed to ultraviolet light.
  • the size of the sample DNA fragments are determined by comparing their migration with the bands of known size in the DNA ladder.
  • DNA ladders are commercially available from numerous vendors, including Sigma, Pharmacia, Life Technologies, Promega, Boerhinger-Mannheim, Amersham, New England Biolabs, Stratagene, and Invitrogen.
  • the special plasmid contains an insert of tandem repeats of a DNA fragment.
  • the same unique restriction site lies at each junction of the repeat units. Partial restriction digestion of this plasmid produces a ladder containing multimers of the repeated DNA fragment.
  • DNA ladder preparation is the restriction digestion of ⁇ phage viruse with an appropriate restriction enzyme to create DNA fragments with specified lengthes depending on the restriction enzyme used.
  • PCR polymerase chain reaction
  • oligonucleotides representing one forward and ten reverse primers, was designed based on the sequence of the template DNA used (plasmid DNA).
  • sequence of the primers is represented as follows:
  • AGAGGCCCCAAGGfiGTTAT R1 : GGCCGCTCGAGCAGATC
  • R2 ACAGCAAATGGGTCGGGAT
  • R3 CGAAATTAATACGACTCACTAT
  • R4 GGCAACCCCGCCAGCCTA
  • R5 ACCGAAGACCATTCATGTTGT
  • R6 GAGAGAGGATGCTCACGAT
  • R7 TGTCAGAGGTTTTCACCGTC
  • R8 TGTCGGGTTTCGCCACCT
  • CTGCGCGTAATCTGCTGCT R10: TGTAACTCGCCTTGATCGTT
  • Fig. 1 illustrates the DNA sequence of the plasmid used as target DNA along with the location of primers annealing.
  • Preliminary amplification reactions (50 ⁇ l) were done as follows : 25 ⁇ l of 2X PCR master mix; 25 pmoles of each primer; 50 ng of template DNA. This method is characterized by its ease of application beside low price of chemicals (which are no longer expensive biochemicals). Besides, it gives the possibility and flexibility of producer to creat landmark fragments within the ladder by increasing the concentration of specified fragments upon mixing. Moreover, it facilitates the preparation of customised ladder rather than defaulted one.
  • Fig. 3-A represents 2% agarose gel electrophoresis of the PCR amplified fragments that constitutes the DNA synthesized marker.
  • the second part of this work describes a method for optimization of PCR product by applying numerical modeling and statistically designed experiments.
  • This method could be an economical method for increasingt he yield of PCR product specially when PCR yield is a target response as in the present case (DNA ladder).
  • a response surface methodology based on numerical modeling was applied to optimize the production of 2 Kb fragment (which has been showed lower yield of production in comparison with other fragments).
  • a Box-Behnken design (1960) based on response surface methodology was applied.
  • Six variables were tested in this experiment, namely: primers concentration, number of cycles, Taq concentration, magnesium concentration, annealing temperature, and extension time.
  • Table 1 represents the design matrix of a 46 trials experiment with the real values of the tested variables, where factors were prescribed into three settings, middle and high concentrations (or values).
  • Annealing primer annealing temperature
  • Fig. 2 illustrates the three dimensional surface response showing the correlation between studied variables and the PCR yield (response).
  • Fig. 3-B illustrates the prepared DNA marker after optimization on running in electrophoresis 2% agarose gel with different concentrations (400-2000 ng).
  • Fig. 1 Tamplate DNA plasmid sequence representing target amplification sites (highlighted).
  • F1 represents the forward primer
  • R1-R10 representing the corresponding reverse primers for the ten PCR reactions.
  • Fig. 2 Three-dimensional surface plots representing the correlation between independent variables and the PCR yield.
  • Fig. 3A 1 % agarose gel representing basal PCR amplification of the tempelate plasmid uding the primer set to prepare the 100 base DNA ladder .
  • Fig. 3B 1% agarose gel representing different concentrations of final product 100 base DNA ladder.

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  • 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

Le présent brevet concerne un procédé facile de préparation d’une échelle d'ADN en utilisant une amplification en chaîne par polymérase (RCP) et, par conséquent, en appliquant une modélisation numérique pour l'optimisation du rendement de l’ACP. Dans ce procédé des amorceurs d’oligonucléotide (11) ont été conçus pour amplifier (10) des fragments d'ADN à partir d'ADN plasmidique de longueurs connues. Sur la base des longueurs de fragment souhaitées, trois programmes d’ACP ont été mis en application pour donner les meilleurs résultats d'amplification. Afin d'optimiser le rendement d’ACP, une méthodologie de modélisation numérique a été appliquée en étudiant (6) des variables significatives simultanément. Le rendement optimisé d’ACP a atteint 5 fois les conditions de base. En outre, une équation mathématique a été décrite de façon à corréler la relation entre les variables et le rendement d’ACP ce qui économise du temps et la consommation en produits de chimie fine.
PCT/EG2006/000015 2005-08-09 2006-04-03 Procédé de préparation d’une échelle d'adn en utilisant une acp et son optimisation par sa modelisation numérique WO2007016929A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EG2005080359A EG24237A (en) 2005-08-09 2005-08-09 Method for preparation of dna ladder using pcr andits optimization by numerical modeling thereof
EG2005080359 2005-08-09

Publications (1)

Publication Number Publication Date
WO2007016929A1 true WO2007016929A1 (fr) 2007-02-15

Family

ID=43857853

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EG2006/000015 WO2007016929A1 (fr) 2005-08-09 2006-04-03 Procédé de préparation d’une échelle d'adn en utilisant une acp et son optimisation par sa modelisation numérique

Country Status (2)

Country Link
EG (1) EG24237A (fr)
WO (1) WO2007016929A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011971A1 (fr) * 1993-10-28 1995-05-04 Life Technologies, Inc. Echelle de reference d'acide nucleique pour l'estimation de la masse
US5824787A (en) * 1993-12-03 1998-10-20 Gensura Laboratories, Inc. Polynucleotide sizing reagent
WO1999003872A1 (fr) * 1997-07-15 1999-01-28 Life Technologies, Inc. Echelles d'acides nucleiques
WO2004063322A2 (fr) * 2003-01-13 2004-07-29 Seegene, Inc. Marqueurs de taille d'adn et methode de preparation de ces derniers

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011971A1 (fr) * 1993-10-28 1995-05-04 Life Technologies, Inc. Echelle de reference d'acide nucleique pour l'estimation de la masse
US5824787A (en) * 1993-12-03 1998-10-20 Gensura Laboratories, Inc. Polynucleotide sizing reagent
WO1999003872A1 (fr) * 1997-07-15 1999-01-28 Life Technologies, Inc. Echelles d'acides nucleiques
WO2004063322A2 (fr) * 2003-01-13 2004-07-29 Seegene, Inc. Marqueurs de taille d'adn et methode de preparation de ces derniers

Also Published As

Publication number Publication date
EG24237A (en) 2008-11-11

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