Polymerase Chain Reaction Essay

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

There were several steps used to acquire the colony necessary for the PCR. First a student forearm was swabbed using a cotton swab, the cells were then placed in an agar plate. DNA was then extracted from the cultured bacteria by using a technique to lyse the cells and solubilize the DNA, then enzymes were used to remove contaminating proteins. The DNA extraction consisted of a lysis buffer that contained high concentrations of salt for denaturing. Binding with the use of ethanol and a washing step to purify the DNA. The final step for the DNA extraction was elution where the pure DNA was release. Proceeding the extraction of DNA the results of the 16s gene amplification were examined through gel electrophoresis it was analyzed by estimating the size of the PCR bands with marker bands. After measuring the success of the extraction, a technique called TA cloning was started. Cloning of PCR products was done by using partially purified amplified products with

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

Numerous attempts are being made in the hopes of finding an answer to the problem of the millennium. The concept of cloning has caught scientists attention throughout this past century. "The world has to come to grips that the cloning technology is almost here,” a representative for a European research group, Panos Zavos. Clones are life forms that have identical genetic coding. A clone is an organisms replica. Originating in the late 1880’s; the first record of synthetic embryo

First, PCR technique was used to amplify the SpHTS gene. PCR is a molecular biology technique for replicating DNA without using a living organism, such as E. coli or yeast (1). The PCR process was carried in a thermal cycler which denatures the double DNA strands. Then, primers was annealed to the single DNA strand. Then, 4 primers were used from a given DNA sequence in the experiment.

Cloning has the potential to bring desired changes in the genetic makeup of the individual. Through cloning, the genetic nature of both can be promoted while the negative properties can be eliminated. Cloning can also be applied to the plants to

Cloning has been one of the most widely discussed and controversial scientific topics in the past decade. Cloning has been applied in many fields including the creation of new breeds of plants and animals. However, the cloning of plants has been practiced for hundreds of years. Cloning can be done on a larger level in animals and has potential benefits. The general process of cloning is to take one cell from the parent making the offspring and parent genetically identical (Tsunoda and Kato, 158-161). In addition by making completely identical cells, we can apply different treatments to different clones to see if they react in different ways. The potential benefits of cloning out way the consequences of cloning. In this paper I will first elaborate on microorganisms. Then I will talk about plants, animal and human cloning, and the controversies in cloning.

Cloning is a process that involves removing the chromosomes from an animal’s egg cell and replacing them with chromosomes taken from a cell belonging to a different adult animal. The copied material, which has the same genetic makeup as the original, is referred to as a clone. There are three different types of artificial cloning: gene cloning, reproductive cloning and therapeutic cloning. Cloning is a very delicate process because many cloned animals resemble animals that are born prematurely. The problem seems to found in the genes because certain genes get turned on or off when a cell becomes specialized during development.

There were two parts of this lab and part 1 was; Transforming E. coli with the pGreen plasmid and Part 2; PCR and Electrophoresis. For this lab, a genetic transformation procedure was performed to introduce a plasmid to another cell and when the cell reproduces it will make a new copy of the plasmid. And genetic transformation is a process whereby genetic materials that are carried by individual cells are changed adding foreign DNA into its genome. Also, a“Plasmids are pieces of double-stranded DNA that can be replicated independently of chromosomal DNA, and normally

2. McClean, Phillip. (1997). Cloning and molecular analysis of genes: Polymerase chain reaction (or PCR). Retrieved on December 6, 2014, from http://www.ndsu.edu/pubweb/~mcclean/plsc431/cloning/ clone9.htm. 3.

In addition, one PCR condition will be varied to determine its affect on amplification, and which conditions are optimal for PCR amplification [1]. This particular part of the experiment brings into light the optimal conditions for PCR, as well as, strengthening understanding of amplification with PCR.

A future experiment for site directed mutagenesis/PCR might involve a slightly increased amount of template DNA and primers in the PCR reaction or increased amount of PCR product that is being transformed, in case if the transformation of the plasmid did not work. Another possible change could be done to the PCR thermal cycles, by altering the denaturation and annealing temperatures to a reasonable degree. This is to ensure amplification of the desired plasmid DNA incorporating the desired mutation with accuracy at the appropriate locations within the DNA template. A future experiment for protein expression and purification should be done with further precision. And more care needs to be taken as to include all necessary antibiotics during transformation and expression, depending on the type of plasmids used in the experiment. This is essential to obtain the desired protein containing the appropriate genes to procced with further

PCR is a tool used by those looking to amplify small amounts of DNA for identification purposes. Thermus Aquaticus’ main use is within this DNA amplification process (PCR) is its reproduction enzyme. The bacterium’s polymerase, called taq polymerase (name after the bacterium), is used to reproduce

The aim of this experiment was to utilise the Polymerase Chain Reaction (PCR) to amplify eight genomic DNA samples, before using both P20 and P200 micropipettes to accurately load the made-up PCR samples and a control PCR into the wells of an agarose gel. Having carried out the electrophoresis of these PCR samples for an hour, the agarose gel was placed under an ultraviolet light in order to image the separation of the samples into their constituent DNA fragments. Subsequently, the agarose gel image could be analysed to evaluate through chromosome inheritance whether the shared DNA fragments between a murder victim, the potential murder weapon and various relatives of a suspect (referred to as Mr X) could be used to implicate Mr X in the theorised

The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.