Molecular Biology

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ABSTRACT

The experiment was carried out to introduce EZH2 gene into a plasmid. We had used only one restriction enzyme to split the pbluescript. EcoR1 was used as restriction enzymes as it allows self-ligation by leaving cohesive ends after splitting the plasmid at specific palindromic gene sequence of interest. After insertion the EZH2 gene was allowed to be amplified through polymerase chain reaction technique. EZH2 gene holds great importance as it’s over expression responsible for the suppression of gene expression resulting in adverse effects in cellular functionalities.

Through polymerase chain reaction, we were able to produce nine multiple samples to be interpreted via gel electrophoresis. We had done electrophoresis twice in order to confirm the rate of success of our experiment. The electrophoresis results comparatively exhibited positive results located at 0.5 kilobases and 3 kilobases exhibiting insert and plasmid respectively. The results indicated of successful gene insertion.

Introduction

With the help of advanced cloning techniques, we have been able to improve the researches and determine the causative agents of many incurable diseases at genetic levels. Recombinant DNA techniques has provided a platform to study the effects of selected micro-molecules from a new diagnostic perspective. Polymerase chain reaction (PCR) has allowed us to unwind the most complexed systems by simply amplifying their specific components.

In past decades, the advancement in the analytical study through molecular techniques has provided an ease towards the determination of inhibiting, regulating, suppressing and replacing certain genes for welfare of mankind. Through this experiment we aim to study (Enhancers of Zeste Homolog 2) EZH2 gene, which might be relevant to multiple cancers.

 EZH2 overexpression is linked to the advanced stages of prostate cancer as it is known for maintaining memory by regulating CD8 cells. It also has been found to have strong linkages with the progressive breast cancer as well). Male deaths are found to be relevant to prostate tumor, thus the study of over expressed EZH2 gene holds great importance towards the health care system and cancer researches. EZH2 also promises to be a strong targeted therapeutic inhibitor in pharmacology. EZH2 overexpression is relevant to many variable tumors. As an oncogene it can play important prognostics role for breast and prostate tumors. EZH2 gene can also suppresses particular genes by inducing methylation in histones which causes suppressed defective transcription and translations. (Roles of the EZH2 histone methyltransferase in cancer epigenetics)

 

EcoR1 (Restriction enzyme) was specifically used for the cleavage of pbluescript in this experiment due to its ability to leave sticky ends. The cohesive or sticky ends would enable EZH2 gene to anneal without causing any frameshift mutation. pbluescript was used as a vector phagemid in this experiment as it provides immediate DNA mapping results regarding the gene inserted. pbluescript also known to provide restriction sites that flank the inserted gene into the plasmid DNA. Histidine tags were also added to the pbluescript for the purified extraction and identification of EZH2. Through DNA recombination technology six histidine tags were added to the insert gene as this is a highly effective way for the purification step of recombinant molecules. The plasmid tagging method is useful and recommended because it can be performed in denaturing environment that occurs later while performing P.C.R (Jason L Scragg, 2004)) Through the process of recombination of plasmid DNA, EZH2 gene was inserted into a phagemid (pbluescript) by the help of restriction endonucleases (EcoR1). Recombination technique involves only one restriction enzyme for digestion and ligation of plasmid DNA.

(Polymerase chain reaction) P.C.R involves denaturing, annealing and extending that are to be repeated for the amplification of gene insert. The two samples newly formed plasmid and insert were amplified using P.C.R. P.C.R was required so we could do a comparative study between the gene insert and plasmid D.N.A via Gel electrophoresis. ((p 908-911, n.d.) Gel electrophoresis is the technique through which proteins, nucleotides, DNA and gene sequences are separated under the influence of electric charge on the basis of molecular weights and sizes respectively at certain p.H. Small sized proteins show quicker movement as they have less resistance moving through the gel bed whereas comparatively the large sized proteins move slower due to more resistance while travelling through the gel. This difference in the distance covered at various velocities by different proteinaceous molecules is recorded. It shows the difference in proteins that were loaded in the sample rack of gel with respect to control and standard.

For our experiment, gel electrophoresis was done twice over our experiment’s samples. First time gel electrophoresis was done in mid-way, whereas second time after completion of experiment to determine the accuracy of our expected results. Gel electrophoresis technique was used to compare the difference and determine the success rate of our recombinant DNA cloning experiment.

 

Methodology

FORMATION OF RECOMBINANT DNA AND AMPLIFICATION VIA P.C.R (polymerase chain reaction)

  • PROTOCOL FOR DIGESTION OF VECTOR PLASMID

Pbluescript of 2 micro-grams concentration was digested into a 25 micro-liters of enzymatic substrate solution with 2 units of restriction enzyme (table 1).

This dissolved solution was incubated for 60 minutes at temperature of 37C.

 

 

 

 

 

 

 

MATERIALS Initial data Volumetric concentrations (micro-Liters)
DNA ( pBS) Sample phagemid conc.= 250 gm/µL                                          08
Restriction Enzyme Buffer                                 10:1

Restriction enzyme concentration: dilution concentration

                                      2.50
Diluted H2O Biological water                                     13.50
B.S.A Enzyme stabilizer concentration = 1                                          0.0
Restriction Enzyme                                          01
Summed up volumes                                        25.0

Table 1: “volumes of the substrates used for the digestion of pBluescript”

 

 

  • C.R STANDARD SOLUTION AND THERMOCYCLER PROTOCOL

 

Given below is the standard volumetric concentrations of substrates to be used in P.C.R. (table: 2). Thermocycler’s optimized temperature and time duration per cycle for P.C.R has also been provided (figure1).

 

 

 

 

 

Materials Concentration (micro-Liters)
EZH2 PHAGEMID (TEMPLATE)                        30
FORWARD TEMPLATE PRIMER (10µM)                       3.0
REVERSE TEMPLATE PRIMER  (10µM)                     3.0
RESTRICTION ENZYME + BUFFER + HISTAMINE TAGS                        40
DILUTED WATER                       4.0
TOTAL VOLUMES                        80

 

Table 2: “P.C.R solution’s standard substrate volumes”

 

FIGURE 1: “optimized thermocycling conditions for P.C.R”

  • VECTOR’S PLASMID DE-PHOSPHORYLATION

Plasmid de-phosphorylation is a process required to prevent re-ligation of vectors to avoid transfection. As we have had used only one restriction enzyme to cut yhe pbluescript.

  • PLASMID PURIFICATION

Purification of plasmid is a process that separates the plasmid through isolation and purification of plasmid DNA from genomic DNA. Purification comprises of three stages growth, harvesting and lysis of bacterial cell culture. Purification of plasmid is necessary prior to digestion of PCR product in order to eliminate the active thermophilic DNA polymerases. If not removed, DNA polymerases may alter the ends of cleaved DNA and cause reduction in the efficient yield of DNA ligation…..