The discovery and characterization of restriction enzymes first took place in the late 1960’s and early 1970’s. The scientists responsible for the discovery were molecular biologists Werner Arber, Hamilton Smith and Daniel Nathans. In the late 60’s Arber observed a sharp change in the bacteriophage DNA he had been working with after it invaded resistant strains of bacteria. It had been cut into pieces and degraded. He hypothesized that bacteria could express two different enzymes: one that recognizes and destroys foreign DNA, the restriction enzyme, and one that modifies bacterial DNA to protect it from the former, a modification enzyme. A short time later he, along with Stuart Linn, confirmed his second hypothesis that both enzymes act on the same specific sequence of DNA, the recognition sequence. In 1970 Hamilton Smith both verified and elaborated on Arber and Linn’s hypothesis and initial discovery using a …show more content…
When the enzyme finds one of these sequences, it severes the DNA by catalyzing the hydrolysis of the bonds holding adjacent nucleotides together in a process called enzyme digestion. If the DNA is double stranded the sequence is on both strands and runs in opposite directions, allowing the restriction enzyme to cut both strands. There are two possibilities when it comes to cutting double stranded DNA: a blunt cut, where the ends severed are even, or a sticky end, where one end is longer than the other and has a string of nucleotides dangling over the other strand. Bacteria protect themselves from restriction enzymes by disguising their recognition sequences through the methylation of the sequence’s Adenine or Cytosine bases. In molecular biology, restriction enzymes can be used to manipulate DNA fragments and are extremely important tools for recombinant DNA technology and genetic
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
Show your understanding of the structure of nucleic acids by describing the similarities and differences between DNA, mRNA and tRNA. Your descriptions should include drawings with labels of the nucleotide structures and the overall structures of each where applicable.
This experiment was very successful as a credible restriction map for the unknown plasmid could be constructed. Within this experiment, both single digest and double digests consisting of three restriction endonucleases were used in order to map out the restriction sites of the enzymes making up an unknown plasmid. In order to separate the DNA fragments by their distinct number of base pairs, it was necessary to run an agarose gel electrophoresis. Within the gel electrophoresis, it is necessary to run a 1kB ladder in the first well. This ladder contains numerous known lengths of base pairs, and is run next to and unknown product in order to approximate the sizes of unknown fragments simply by comparing the unknown fragments to the coinciding fragments of the known ladder. This ladder gives us the ability to precisely and accurately draw conclusions about the results derived from the gel electrophoresis as it serves as an essential reference point. Because of the known fragments in the ladder, we were able to create a standard curve. Within the standard curve, the distance the fragments traveled was plotted against the length of the known base pairs within the ladder. Once the points were plotted, a line of best fit was constructed and an equation of the line was electronically derived. By plugging in the measured distance of how far the fragments traveled, shown by “x”, into the equation for the line of best fit, the lengths of the base pairs created by the restriction enzymes was able to be calculated.
The purpose of this experiment is to identify an unknown insert DNA by using plasmid DNA as a vector to duplicate the unknown insert DNA. The bacteria will then be transformed by having it take in the plasmid DNA, which will allow us to identify our unknown insert as either the cat gene or the kan gene.
The origins of DNA were first discovered during 1857 by Gregor Mendel the "Father of Genetics”, whom was performing an experiment of genetics with pea plants, and would provide a basic foundation towards DNA and Genetics. Friedrich Miescher and Richard Altmann in 1869 were also part of the first people to discover DNA. While testing some sperm of a salmon, they discover a strange substance that they would name as "nuclein", which is known as DNA. This new form of "nuclein" (DNA) would be found to only exist in chromosomes. Frederick Griffith, a researcher, found the basis on DNA, from a molecule inheritance experiment involving mice and two types of pneumonia. His findings were that, when virulent disease is heated up (to kill) and is injected into a mouse, the mouse survives. Unlike the second mouse that has been injected with non-virulent disease and virulent disease (that had been heated and killed) is killed. This would be caused by an inheritance of molecule (transformation) of virulent bacteria passing on a characteristic to the non-virulent. DNA findings would continue to be tested and tried to better understand how DNA works.
strands which make up the letters of a genetic code. In certain regions of a DNA strand
A human DNA, in which biologists have identified and isolated the gene of interest using probes or antibodies, will then be chosen. This gene of interest is incorporated into the plasmid cuts. These new plasmids are mixed with, and taken up by bacterial cells under suitable conditions. As these bacterial cells reproduce, the plasmids containing the gene of interest will be copied, and transferred to the bacterial progenies. Genes are segments of chromosomes that code for specific polypeptide or RNA molecules. Plasmids are small loops of DNA separated from bacterial chromosomes, or viral vectors. Restriction enzymes are enzymes that cut DNA at highly specific areas that always contains the same sequence of
Tsou, J. A., Hagen, J. A., Carpenter, C. L., & Laird-Offringa, I. A. (2002, August 05). DNA
Gene therapy is a method used by doctors that ‘uses genes as medicine’. It repairs faulty or damaged gene copies by transferring a therapeutic or working gene into specific parts of faulty gene copy. This means that gene therapy can be used to replace a faulty gene or to introduce a new gene whose function is to modify the clinical course of a condition or to cure a problem in the gene (Refer to image 2). Image 1 shows how gene therapy is completed using an adenovirus vector while image 2 explains the method of gene therapy and shows how cells are removed, altered and injected back into the patient. Gene therapy has potential to treat and cure many medical conditions such as various types of cancer, degenerative diseases and viral infections such as AIDS. Research into gene therapy still needs to be done before this approach to the treatment of certain conditions will realise its full potential. Trials are being conducted in the United States and Europe and a modest number initiated in other countries, including Australia. The majority of these trials are focussing on treating acquired conditions such as cancer (European Society of Gene & Cell therapy, 2011).
a) The V, D, or J double-stranded DNA sequence is flanked by an RSS site. b) Rag-1 and Rag-2 bind simultaneously on the RSS joints. c) The Rag complex initiates a single stranded nick between the RSS and the coding region. d) Through an attack by the 3’ OH end, the DNA forms a hairpin leaving the RSS site with a double stranded break. e) With the help of accessory proteins, the newly cleaved coding region is joined to a separate coding region with added nucleotides in between, that were added by a DNA transferase. Note: Rag-1 and Rag-2 interact with both strands of DNA.
Gene cloning works by first isolating the desired gene and ‘cutting’ it from the original chromosome using restriction enzymes. The piece of DNA is ‘pasted’ into a vector and the ends of the DNA are joined to the vector DNA by ligation. The vector is introduced into a host cell, often a bacteria or yeast, by a process called transformation. The host cells copy the vector DNA along with their own DNA, creating multiple copies of the inserted DNA. The vector DNA is separated from the host cells’ DNA and purified.
The birth of genetic engineering and recombinant DNA began in Stanford University, in the year 1970 (Hein). Biochemistry and medicine researchers were pursuing separate research pathways, yet these pathways converged to form what is now known as biotechnology (Hein). The biochemistry department was, at the time, focusing on an animal virus, and found a method of slicing DNA so cleanly that it would reform and go on to infect other cells. (Hein) The medical department focused on bacteria and developed a microscopic molecular messenger, that could not only carry a foreign “blueprint”, or message, but could also get the bacteria to read and copy the information. (Hein) One concept is needed to understand what happened at Stanford: how a bacterial “factory” turns “on” or “off”. (Hein) When a cell is dividing or producing a protein, it uses promoters (“on switches”) to start the process and terminators (“off switches”) to stop the process. (Hein) To form proteins, promoters and terminators are used to tell where the protein begins and where it ends. (Hein) In 1972 Herbert Boyer, a biochemist, provided Stanford with a bacterial enzyme called Eco R1. (Hein) This enzyme is used by bacteria to defend themselves against bacteriophages, or bacterial viruses. (Hein) The biochemistry department used this enzyme as a “molecular scalpel”, to cut a monkey virus called SV40. (Hein) What the Stanford researchers observed was that, when they did this, the virus reformed at the cleaved site in a circular manner. It later went on to infect other cells as if nothing had happened. (Hein) This proved that EcoR1 could cut the bonding sites on two different DNA strands, which could be combined using the “sticky ends” at the sites. (Hein). The contribution towards genetic engineering from the biochemistry department was the observations of EcoR1’s cleavage of
Secondly the gene has to be cut from its DNA chain. Controlling this process are many restriction endonucleases (restriction enzymes). Each of these enzymes cut DNA at a different base sequence called a recognition sequence. The recognition sequence is 6 base pairs long. The restriction enzymes PstI cuts DNA horizontally and vertically to produce sticky ends.
What are the principle, ethical issues and experimental procedures used in genetic engineering and cloning? Should Cloning be allowed to continue?