"The technology entailing all processes of varying the genetic material of a cell to make it competent of performing the preferred functions, such as producing novel substances." (http://www.biology-online.org/dictionary/Genetic_engineering). A clone consists of asexual progeny of a single individual or cell this procedure of producing a clone is known as cloning. The genes which are cloned always have the same genotype and hence when a single gene produces a clone all the clones which are produced will be identical. A recombinant DNA molecule is produced by fusing 2 or more DNA fragments. The steps involved in production of rDNA molecules are 1) isolation of the gene which requires isolation of the required gene, nucleic acids and proteins are use molecular probes, DNA sequencing, gene synthesis. 2) Splicing of the foreign DNA into the coning vector (i.e) vectors, plasmids, cosmids, phages, and splicing techniques. 3) Cloning (i.e) introduction of foreign DNA into host cells and its amplification. pUC19 is a plasmid cloning vector, it has a base pair of 2686 base pairs in length with 54 base pairs multiple cloning site. It is a plasmid isolated from the E.coli, amicillin is the best marker used for pUC19. It has 3 open reading frames. In the experiment the size of pUC19 was to be found with the distance travelled from the wells to the markers but as there were no bands seen in the lane the size of it was not been calculated if we assume the results the size of the plasmid could be found out by drawing a slope for the bands which could have been seen. As there were no bands seen in lane 4 we wait for the results of the transformation process which could have shown blue or white colonies but as there were no colonies seen its clear that the experiment was unsuccessful and so the process of restriction isolation ligation transformation might have gone wrong or might be the experiment was unsuccessful due to some mistakes like no proper time maintenance." DH5 (alpha) is a bacterial strain, its taxonomy ID:-668369.Enterobacteria is its inherited blast name. Its genetic code is translation table 11(bacterial archeal and plant plastid)" (medicine.net, taxonomic virology). "EcoR1 i.e Escherichia coli RY13 recognition sequences and cleavage sites from 5' to 3' are G * A A T T C, the enzyme EcoR1 normally cuts at the sequence -GAATTC- but in the presence of glycerol". Alan.E.H.Emery. (1985). An introduction to recombinant DNA.London:Portsmouth,Avon. Thus the purpose of the experiment is to produce more clones from which an exact and a perfect clone can be isolated. The deoxyribose nucleic acid was first proposed by Watson and crick in the year 1953. It is a double stranded structure. One strand runs from 5' to 3' and the other is antiparallel. Rdna technology is very important in day to day life it is used in agriculture, prevention of diseases like cystic fibrosis, sickle cell anemia. And thus it's been clear that the future in rDNA technology has much in store.
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A recombinant DNA molecule is produced by joining together two or more DNA segments.DNA is deoxyribonucleic acid. But usually a recombinant DNA is produced by joining DNA originating from different organisms. For example a plasmid, phage, or virus into which a desired DNA fragment will be inserted to enable its cloning in an appropriate host. Specific enzymes are used for cutting the DNA, these enzymes are called restriction enzymes and also for joining together appropriate fragments which is called as ligation. Thus a recombinant DNA (rDNA) molecule is produced which contains a gene from one organism joined to a regulatory sequence from another organism, such a gene is called chimeric gene. Therefore this rDNA molecule has given an opportunity to create novel gene combination to suit specific needs.
Deoxyribonucleic acid: - It is a double helical structure which was proposed by "WATSON" and "CRICK" in the year 1953.It results from the coiling around a common axis of the two right handed helical polynucleotide strands. There are two chains, one chain runs in 3' to 5' direction while the other is antiparallel and runs from 5' to 3' direction. There are adjacent residues which are rotated by 36Â° relative to each other. For every 10 residues the DNA double helix completes one turn. Each base pair advances the double helix by 3.4AÂ° along the axis. The DNA molecule has a diameter of 20AÂ°, it is a polymer (the DNA molecule) consisting of several thousand pairs of nucleotide monomers, each nucleotide contains pentose sugar deoxy-ribose, a phosphate group and a nitrogenase base which is either a purine or a pyramidine. The two purines adenine and guanine are double ringed and the pyramidines thymine and cytosine are single ringed.
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(Mathew kuure-kinsey and Beth Mc Cooey for Biochemical Engineering Fall 2000)www.rpi.edu/dept/chem-eng/Biotech-Enivorn/Projectsoo/rdna.html.
Nucleic acids were first discovered by "Fedrich Meischer". There are 2 various nucleotides they are DNA which is found in the nucleus and RNA which is found in the cytoplasm. Nucleic acids are the polymers of nucleotides i.e polynucleotides.
PENTOSE SUGAR: There are two types of sugars involved in nucleic acids they are ribose in RNA and deoxy-ribose in DNA. These sugars are called pentose sugars because they contain 5 carbon atoms of which 4 are carbon atoms and 5th atom is the oxygen atom which is present outside the ring and thus forms a part of the CH2 group. The sugars of one strand of DNA are directed to one end i.e the strand has polarity.
PHOSPHATE GROUP AND PHOSPHO-DIESTER BONDS: Phosphate groups are the ionized derivatives of orthophosphoric acid (H3PO4). Two of the three hydroxyl groups of phosphoric acid are esterified by the "OH" groups on the pentose sugar. The acid character and the name of the nucleic acid is due to the proton donating character of the phosphate group. The phosphodiester bond is analogous to the peptide bond formation of proteins.
NITROGENOUS BASES: These are hetro-cyclic rings which are of two types' purines and pyramidines. Most bases have ionisable groups, such as the NH2 group which undergoes ionization.
Uracil and thymine do not have NH2 groups. Purines and pyramidines are said to be bases not because of the NH2 groups but because of the nitrogen atoms of the nitrogen atoms of the ring. These bases have hydrophobic properties.
The absence of hydroxyl groups at 2' carbon makes cyclic phosphate formation impossible due to which the DNA does not get hydrolysed by alkali which is possible in RNA, this technique is used to separate DNA from RNA.
REPLICATION OF DNA: The duplication of genomic or plasmid DNA is called replication of DNA. The most important properties of DNA is that it can make exact copies of itself.
DNA LIGASE: It catalyses the formation of phosphodiester linkage between 5' phosphoryl group of one nucleotide and 3' (OH) group of its immediate neighbour. DNA ligase is similar to DNA polymerase but the difference is that ligase seals the nicks left behind but it cannot fill the gaps. They function in discontinuity repair by joining single stranded breaks.
ENDONUCLEASES: These help in both DNA replication and repair. The endonuclease produces an internal cut in a DNA molecule whereas a restriction endonuclease induces a nick in the vicinity of DNA damage or cut those sites which have specific nucleotide sequences.
Restriction endonuclease/Restriction enzymes:-Restriction enzymes are a class of nucleases which can cleave double stranded DNAin a "precise manner" at a "limited number of sites" which have a unique base sequence. The ATP provides energy for the enzyme to move along the DNA molecule from the recognition site to the cleavage site. These enzymes have both restriction (cutting) and modification (methylation) activity. Large number of enzymes is required in DNA manipulation for Rdna technology.
Nucleases: - These depolymerise the nucleic acids by breaking the phosphodiester bonds which link the nucleotides in the DNA strand. Mostly nucleases act specifically on DNA and are called as DNases or on RNA called as RNases. These are of two types: exonucleases which remove nucleotides one at a time from the end of a DNA molecule and endonucleases are the ones which act within the DNA molecule by breaking internal phosphodiester bonds. Restriction endonucleases cut the double stranded DNA only at a limited number of specific recognition sites.
POLYMERASES: These are used to make copies of DNA or RNA molecule. DNA polymerase is used in catalyzing polymerization of new nucleotide chain along the existing template using monomeric units called deoxy nucleotide phosphate (i.e) dTTP dATP, dGTP, dCTP/dUTP. DNA polymerase I, Klenow polymerase and reverse transcriptase are the polymerases used in rDNA technology. The functions of DNA polymerase I are polymerization and degradation. The enzyme either attaches to a short single stranded region or nicks in double stranded DNA molecules, then a short strand is synthesized to fill in the gap of nick region and then the existing nucleotides are replaced by DNA replication.
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Klenow polymerase is a part of DNA polymerase enzyme which has a polymerase activity but lacks nuclease activity. On a single stranded template a complimentary DNA strand is synthesized by filling the nicks. But it cannot degrade nucleotide and so doesn't replace existing nucleotides.
Reverse transcriptase synthesis a new complimentary strand on RNA template is used for complimentary DNA cloning.
GEL ELECTROPHORESIS: The migration of ions in an electric field at a definite pH is known as electrophoresis. It is based on the principle that in an electric field the proteins migrate but not at the pH of their isoelectric point (i.e) the point at which the molecule has net zero charge. Agarose gels are the most commonly used means of isolating and purifying fragments of DNA. A dye known as ethidium bromide is used to facilitate visualization of DNA after electrophoresis. It is a fluorescent dye that intercalates between bases of nucleic acids and allows clear detection of DNA fragments in gels. The buffers are used for the electrophoresis of DNA. The buffers provide ions to support conductivity, they also establish a pH.
The electrophoretic rate of migration depends on four factors like the net charge on the molecule, molecule size, electric field strength and pH of the medium. Migration of a charged ion or molecule takes place when it is placed in an electric field. The equation which represents the rate of migration is:
Ï… = Ïµq/f
Ï… = velocity of migration of the molecule
Ïµ =electric field in volts cm-1
q =net charge
f =frictional co-efficient (function of the mass and shape of the molecules)
The velocity per unit field is the movement of a charged molecule in an electric field which is expressed in terms of Î¼:
Î¼ = Ï…/ Ïµ = Ïµ.q/fÃ- Ïµ (since Ï…= Ïµq/f)
TRANSFORMATION: The vectors which contain DNA segments which are to be cloned known as DNA inserts or chimeric vectors are then introduced into a suitable organism such as a bacterium. Such an organism is called a "host" while the process is called as transformation. These transformed host cells are selected and then cloned. Then transformation of a suitable host with a chimeric vector and cloning the transformant cells is called DNA cloning or gene cloning. The genome present in members of a single clone is identical which applies rDNA technology. Thus large number of copies of the gene or the DNA is cloned by DNA cloning.
pUC19:- It is a high copy number cloning vector. A linearised version can be generated by PCR. The screening of blue and white colony screening, replication of pUC origin and also the propogation and selection in Escherichia coli (E.coli) which is carried out by ampicillin resistance gene. To cut a plasmid pUC19 and phage Î» a restriction enzyme Hind III is used for which the recognition sequence is A.AGCTT which occurs once in pUC19 creating one linear piece of DNA and seven sites are separated in Î» creating 8 fragments. These fragments are separated and analyzed by using gel electrophoresis. pUC19 is a ampicillin resistance gene, it is part of the lac-operon (i.e) Lac Z gene which codes for Î²-galactosidase.
puc19 by koshiks.
LAC OPERON-AN INDUCIBLE OPERON: Jacob and Monad proposed the operon model. A continuous unit which includes the structural, operator and the promoter is called as an operon. A promoter an operator and 3 structural genes (i.e) z,y,a which code for Î²-galactosidase,Î²-galactoside permease and Î²-galactoside transacetylase respectively,constitute the lac operon. Lactose is cleaved into glucose and galactose by Î²-galactosidase, lactose is pumped into the cell by Î²-galactiside permease.
rDNA molecule production is carried out to obtain a large number of copies of specific DNA fragment, to recover large quantities of protein produced by the concerned genes or to integrate the gene in question into the chromosome of a target organism where it expresses itself. To achieve the above objectives a large number of copies of a concerned gene should be obtained for which the DNA segments are integrated into a self replicating DNA molecule which is called a vector such as bacterial plasmids or DNA viruses. Thus bringing together DNA segments of various origins and placing them into a suitable vector constitutes rDNA technology.
MATERIALS AND METHODS:
Follow practical booklet from pages 1 to 16
GEL-ELECTROPHORESIS: The 7 wells were loaded with DH5Î± extract, DH5Î±[pUC19] extract, #1 pUC19+EcoR1, #2 pUC19-EcoR1, #3 Î»+EcoR1, Î»+pUC19-ligase, Î»+pUC19+ligase in the order of 1 to 7 respectively.
According to the figure 3 below, there are one band each in the lanes 1 and 2 with smears at the lower end of the 2nd lane. There are five very clear bands in lane 3 and three bands seen in 4th lane and no bands are shown in lane 5 whereas lane 6 and 7 show similar bands in the similar mobilities of each other. There are four bands seen in them which are not that clear.
Bacterial transformation:- There are 6 plates prepared for the transformation process in order to calculate the efficiency of transformation. In each of the 6 plates 6 different components were used that is DH5Î± (-ve control), pUC19, pUC19+Î»-ligase, pUC19+Î»+ligase, 1 in 10 dilution, 1 in 10 dilution in the plates 1 to 6 respectively.
There are no colonies seen in any of the plates (i.e) there are zero blue and white colonies observed on the plates.
Gel Electrophoresis Picture.jpg
Figure 3:- In the above figure the 7 wells were loaded with the following samples DH5Î± extract, DH5Î±[pUC19] extract, #1 pUC19 +EcoR1, #2 pUC19-EcoR1, #3 Î»+EcoR1, Î»+pUc19-ligase, Î»+pUC19+ligase
TOTAL NUMBER OF BANDS OBSERVED
DISTANCE TRAVELLED FROM THE WELL TO THE BAND
Table 1:-The table shows the distance travelled by the bands from the well 3 which was loaded with #1 pUC19+EcoR1
Graph 1:- In the above graph the molecular weights of the bands are taken and plotted on the X-axis and the distance travelled by the bands from the 3rd well are plotted on the y axis. This plotted graph shows that a straight line passes through 2 points.
Total number of colonies
DH5Î± (-ve control)
One in 10 dilution
One in 10 dilution
Table 2:- The above table shows the number of colonies seen on the 6 plates which were prepared for transformation process.
Transformation efficiency is calculated by the following formula:-
Transformation efficiency=total number of colonies on all the plates/amount of DNA plated (in Î¼g/ml)
There are different bands which are appeared in the lanes, some bands are supercoiled, some samples formed smears due to lower molecular weights, some formed anomalies and so the bands appeared in this might be due to various reasons such as inadequate amounts of sample, to avoid this, stopping mixture can be added to the samples so that the samples are adequate enough to load in the wells, and in some lanes there were no bands seen this might be due to insufficient loading of the sample. There are other reasons which can be assumed like contaminated or degraded or insufficient DNA might be the samples were not properly electrophorosed for ample time which the gel needs. There is a band in lane 2 which is not visible to the naked eye this might be due to improper W light source. In lane 3 five visible bands are present whereas 6 visible bands should appear. But looking the figure 3 it shows that one band is larger in its width when compared to the other four bands, hence it can be assumed that the 3rd and the fourth bands in lane 3 are merged together and so to plot a restriction graph, the average of the 3rd and the fourth bands log valus is taken to plot the graph. This might be due to the supercoiling of the DNA. In lane 4 the bands appeared in the mobility of such a region where it shouldn't have appeared, this shows that the bands appeared are anomalies. There were no bands in lane 5 but according to the interpretation of the result there should be a band through which the size of the plasmid DNA is calculated and the slope is found. This might be due to insufficient DNA sample. As such the bands in lane 5 and lane 6 should be identical but might be due to the above reasons it didn't happen but the bands in lane 6 and 7 appeared to be very identical.
There are no colonies seen on the plates this might be due to various reasons like the quantity of the DNA, the insert is needed in more quantity than the vector. The important point to be watched is that whether the plasmid used is a high copy number plasmid or a low copy number plasmid, the optimum density of DH5Î± must be maintained. X-gal a chemical analogue which will be added to the growth media instead of lactose might for the growth of the colonies. This analog produces a visible blue product when exposed to Î²-galactosidase. Thus the transformed colonies can be selected as they grow on ampicillin and produce blue colonies. Transformants grow slowly on LB agar. The other problems in transformation might be due to failure of ligation reaction, loss of DNA during precipitation, insufficient or degraded DNA, might be the spreader was too hot which killed the organism.
Interpretation of the colonies on plates 2 and 4 can be as follows:
Plate 2 which has a component pUC19 should mostly contain satellite colonies that are the break through colonies. Plate 4 might contain either blue or white colonies. If a white colony appears separately and not around the blue colonies is known as a recombinant.Thus above assumptions might be the cause for the failure in transformation process.
The aim of the experiment was not achieved perfectly.