WO2007016929A1

WO2007016929A1 - Method for preparation of dna ladder using pcr and its optimization by numerical modeling thereof

Info

Publication number: WO2007016929A1
Application number: PCT/EG2006/000015
Authority: WO
Inventors: 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: 2005-08-09
Filing date: 2006-04-03
Publication date: 2007-02-15
Also published as: EG24237A

Abstract

The patent pending describes an easy method for preparing DNA ladder using polymerase chain reaction and, consequently, applying numerical modeling for optimization of PCR yield. In this method (11) oligonucleotide primers have been design to amplify (10) DNA fragments from plasmid DNA with known lengths. Based on the required fragment lengths, three PCR programs have been implemented to give the best amplification results. In order to optimize the PCR yield, numerical modeling methodology has been applied by studying (6) significant variables simultaneously. The optimized PCR yield reached (5) times the basal conditions. In addition, a mathematical equation has been described to correlate the relationship between variables and the PCR yield which saves time and fine chemicals consumption.

Description

TITLE OF INVENTION

Method for preparation of DNA ladder using PCR and its optimization by numerical modeling thereof

Technical field

The patent pending is in the field of molecular biology. In particular, 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. Moreover, optimization of the PCR yield by applying numerical modeling.

Background Art

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. Typically, 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.

One established method for manufacturing a DNA ladder is by partial restriction digestion of a special plasmid. 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.

Another method for DNA ladder preparation is the restriction digestion of λ phage viruse with an appropriate restriction enzyme to create DNA fragments with specified lengthes depending on the restriction enzyme used.

In the first method there are disadvantages refererring to the tedious cloning experiments which must be done to construct the vector with several tandom repeats beside the unreproducibility of restriction digestion resulting in variable yield each time. In the second method the ladder fragments sizes are not fully controlled as it relies on the used restriction enzyme. Disclosure of the Invention

A method for preparing a DNA Ladder using polymerase chain reaction (PCR) and the optimization of the amplification reaction therof by applying numerical modeling experimental design.

For the amplification of DNA ladder fragments a set of oligonucleotides, representing one forward and ten reverse primers, was designed based on the sequence of the template DNA used (plasmid DNA). The sequence of the primers is represented as follows:

F1 : AGAGGCCCCAAGGfiGTTAT R1 : GGCCGCTCGAGCAGATC R2: ACAGCAAATGGGTCGGGAT R3: CGAAATTAATACGACTCACTAT R4: GGCAACCCCGCCAGCCTA R5: ACCGAAGACCATTCATGTTGT R6: GAGAGAGGATGCTCACGAT R7: TGTCAGAGGTTTTCACCGTC R8: TGTCGGGTTTCGCCACCT R9: CTGCGCGTAATCTGCTGCT R10: TGTAACTCGCCTTGATCGTT

Fig. 1 illustrates the DNA sequence of the plasmid used as target DNA along with the location of primers annealing. By applying simple PCR 10 DNA fragments with different sizes were amplified, where different operation programs for PCR were constructed based on the required fragments' length. PCR operation programs are listed as follows:

1. For fragment lengths 100-500 base

T = 94^°C 5 minutes

30 cycles as follows:

T = 94°C 45 second

T = 53^°C 45 second

T = 72^°C 45 second Then Hold 4°C

2. For fragment lengths 750-1500 base T = 94⁰C 5 minutes

30 cycles as follows: T = 94⁰C 1 minute

T = 53°C 1 minute

T = 72⁰C 105 second

Then Hold 4°C

3. For fragment lengths 2000-2500 base T = 94°C 5 minutes

30 cycles as follows: T = 94°C 1 minute

T = 53°C 1 minute

T = 72⁰C 150 second

Then Hold 4°C

Preliminary amplification reactions (50 μl) were done as follows : 25 μl 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). For example, 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). In order to describe the nature of the response surface and the interactions between variables under experimental constraints, 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).

For predicting the optimal point, within experimental constrains, a second-order polynomial function was fitted to the experimental results (linear optimization algorithm) of PCR yield:

Ypc_R yield = 189.3+2.5*primer+26.44*cycles+18*Taq+5.2*Mg+3.9*Annealing-

5.1 *Extension-0.25*primer*cycles-17.84*primers*Taq-10.14*cycles*Taq-

10.58*primers*Mg+7.8*cycles*Mg-24.85*Taq*Mg+8.4*primers*Extension-

11.96*cycles^*Annealing+9.4*Taq*Annealing-

3.14^*Mg*Annealing+0.48*primers*Extension+0.05*cycles*Extension-

13.4^*Taq*Extension-20.64*Mg^*Extension-8.7^*Annealing*Extension-

0.18^*primers^*primers-7.18^*cycles*cycles-10.77*Taq*Taq-

29.9^*Mg*Mg+5.86*Anealing*Anealing-28*Extension*Extension

Where:

Primers = primer concentration

Cycles = number of PCR cycles

Taq = enzyme concentration

Mg = MgCb concentration

Annealing = primer annealing temperature

Extension = extension time

Fig. 2 illustrates the three dimensional surface response showing the correlation between studied variables and the PCR yield (response). On mathematical calculation of the previously mentioned polynomial equation ( within the experimental constraints) maximum PCR yield for amplifying the 2 Kb fragment ( more than 5 folds the initial conditions) was attained under the following conditions: Primer concentration 10 pmole Taq concentration 2.4 U/50μl reaction

MgCb concentration 2.2 mM

Number of cycles 30 cycle

Annealing temperature 53°C

Extension time 1.4 minutes

Fig. 3-B illustrates the prepared DNA marker after optimization on running in electrophoresis 2% agarose gel with different concentrations (400-2000 ng).

Figures and Table legends

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.

Table 1. Real values of experimental variables used in the Box-Behnken design matrix for optimization of PCR yield.

Claims

1. A method for making a DNA ladder, by PCR amplification of plasmid DNA with known sequence using a set of eleven primers, whereby:

(a) Only one forward primer (F1) is used and the other ten primers are reverse (R1-R10);

(b) the length between each pair of primers is an integer multiple of a minimal length for fragments 100-500 base, said minimal length is the length between the start of the forward primer and the end of the reverse primer;

(c) the PCR amplification generates a DNA ladder with ten differently sized DNA fragments; and

(d) said DNA ladder has the properties that (i) all DNA fragments of the ladder have lengths which are integer multiples of the minimal length (100 base which results from PCR amplification using F1and R1 primers); (ii) the smallest DNA fragment length is the minimal amplification length; and (iii) the largest DNA fragment is the length of the entire plasmid;

2. A method according to claim 1 , for optimization of the PCR yield of the 2 Kb fragment, which was obtained weak in comparison with other fragments, by applying response surface methodology and numerical modeling, whereby:

(a) Six variables were tested namely: primers concentration, number of cycles, Taq concentration, magnesium concentration, annealing temperature, and extension time;

(b) A 46 trials design matrix was carried out and the intensity of the fragment was considered as response;

(c) A standard regression analysis of the matrix was applied to calculate the regression coefficients of the structured polynomial model; (d) Three folds PCR yield was achieved after solving the polynomial within the experimental constraints.