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One of the most common ways these days is from cDNA. RNA is extracted from human cells, purified, and then treated with an enzyme called reverse transcriptase which is able to make DNA from RNA templates (this DNA made from RNA is called cDNA). The advantage of using cDNA is that in the genome human genes are typically distributed across multiple exons spread over tens or even hundreds of thousands of basepairs of DNA. Such a massive segment of DNA is extremely hard to manipulate and far too large to insert into a plasmid. However, in cDNA, all the introns have been spliced out (because cDNA is made from mature mRNA).
To isolate a particular gene from cDNA, PCR is often used to selectively amplify one gene's cDNA using specific primers. To insert the amplified cDNA into a plasmid, the traditional approach was to use restriction enzymes - enzymes that cut precise DNA sequences. The great thing about many restriction enzymes is that they cut DNA but leave behind "sticky ends". Thus if you cut both your cDNA and a plasmid with a particular restriction enzyme, the resulting sticky ends will allow the human cDNA to be taken up by the plasmid (the sticky ends will mesh). The sticky ends will have to be sealed by an enzyme called DNA ligase.
However, there are other ways these days - often involving recombination to insert the PCR product directly into a plasmid without resorting to restriction enzymes and ligations.
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In the process of human gene cloning using recombinant plasmids what is the bacterial plasmid?
functions as a vector
The principal problem with inserting an unmodified mammalian gene into the bacterial chromosome and then getting that gene expressed is that?
bacteria cannot remove eukaryotic introns; bacterial dna does not contain introns like eukaryotic genes do so they had to be removed before being added to the plasmid.
What is a prophage gene?
A prophage gene is a gene of a bacteriophage (virus that targets bacteria) that is inserted and integrated into the circular bacterial DNA chromosome or plasmid. Bacteriophages reproduce by inserting their genome into that of a bacterium and thus getting their genes read and viral proteins produced.
What are the genes on the pVIB plasmid?
It contains a gene for luciferase, a Lux gene (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). This causes bacterial cells to glow!
Describe the use of plasmids as vectors in biotechnology?
Plasmids are often used as expression vectors in biotechnology. Plasmids are small, circular or linear pieces of DNA containing non-essential genes that are found in all life, although much more common in prokaryotes, especially bacteria. These genes confer abilities such as metabolizing a previously unusable compound, building an amino acid previously unbuildable, or even antibiotic resistance. Plasmids are used in research to induce the expression of a gene usually not found in the given organism. For example, you can construct a plasmid with a bacterial promoter connected to the gene for a human protein through a process called 'cloning'. The plasmid with the human gene can then be introduced into bacteria by transforming a competent gram-negative with the plasmid. Usually the plasmid also has an antibiotic resistance gene in addition to the target gene. This antibiotic resistance can be used to select for bacteria containing the plasmid. For example, the most common resistance gene is ampicillin resistance gene. If you grow the transformed bacteria in a culture containing ampicillin, only bacteria containing the antibiotic resistance, and therefore containing the plasmid, can survive. This will ensure that what you have is a pure culture of bacteria containing the plasmid. After selection, these bacteria can be cultured in suitable media to increase their numbers to a point that their production of the human protein becomes appreciable. Then these bacteria are usually lysed (killed) to extract the protein. Sometimes, however, these bacteria can also be made to secrete the protein into the medium.
In the process of human gene cloning using recombinant plasmids what is the bacterial plasmid?
functions as a vector
How does a human insulin genes become part of a plasmid?
1. Scientists remove plasmids, small rings of DNA, from bacterial cells. 2. An enzyme cuts open the plasmid DNA. The same enzyme removes the human insulin gene from its chromosome. 3. The human insulin gene attaches the open ends of the plasmid to form a closed ring. 4. Some bacterial cells take up the plasmids that have the insulin gene. 5. When cells reproduce, the news cells will contain copies of the engineered plasmid. The foreign gene directs the cell to produce human insulin.
Why is ampicillin added to the pound medium?
Ampicillin is an antibiotic that is usually used as a reporter gene in cloning. A plasmid containing the ampicillin resistance gene (as well as another target gene within the plasmid) is introduced into the bacterial host. If the bacterium has taken up the plasmid and is expressing the plasmid, it will be resistant to ampicillin. LB is used as a growth medium and ampicillin to verify the plasmid is within the bactrium. No growth means no plasmid in the bacterial host...
Can genetic engineering techniques treat cystic fibrosis?
Yes, there are two similar techniques in which i am aware of.AdenovirusesFirstly adenoviruses are made harmless by interefering with a gene involved in replication. A healthy from of the CFTR gene is extracted and cut with restriction endonucleases, the same enzyme is used to cut a bacterial plasmid. The gene and plasmid are mixed together along with DNA ligase to anneal the phosphosugar framework of the DNA fragment and bacterial plasmid. The plasmid is then mixed with epithelial cells. The plasmid is then isolated and purified and places into adenoviruses. These are then placed onto the nostrils of individuals with cystic fibrosis. The viruses find their way to epithelial cells in the airways and injected their DNA. The DNA contains the functional CFTR gene, the cells can then produce fucntional CFTR proteins.LiposomesA healthy gene is extracted from a human. This gene is then inserted into a bacterial plasmid, in a similar manner as discussed above. The bacterial plasmids are then inserted into bacteria. These are allowed to grow and divide, producing large quantities of the plasmid, with the required gene. These plasmids are then extracted and coated in a lipid soluble substance. They are then put into nasal sprays and sprayed onto the nostrils of effected individuals.
What does the bacterial cell reproduce that the human gene codes for?
the bacterial cell reproduces the bacterial chromosome that the human gene codes for.
What is one way to determine whether a bacterial culture has received a recombinant plasmid?
The plasmid have a "reporter gene" inside it, generally resistance to specific antibiotic. the plasmid is transformed into bacteria that don't have resistance to that specific antibiotic drug, and than the cultured on a petri-dish that contain the antibiotic drug. Only bacteria that had receive the plasmid will have resistance and grow, all the other will die.
What is the function of the plasmid?
Plasmids are extra circular genetic material that can be passed from bacteria to bacteria, which basically is their function; in bacterial conjugation. But, in biotechnology it is often used in recombination work. Some other organisms gene is inserted into the bacterial plasmid and then the bacteria multiply and transcribe this inserted gene into many useful products.
A plasmid that contains a gene for human growth hormone is and example of what?
recombinant dna
When is a plasmid considered a recombinant plasmid?
When the original function of the gene in the plasmid is altered or another gene is inserted in the non- coding region of the plasmid is called the recombinant plasmid.
When pharmaceutical companies use recombinant bacteria to make insulin what do they insert the insulin gene into?
The human insulin gene, which is located on the top of the short arm of chromosome 11 in human DNA, is cut from the DNA strand using restriction enzymes (genetics scissors). A plasmid (floating circular disks of DNA in bacteria) is extracted from a bacteria and cut open with another restriction enzyme, and the gene for human insulin is taken up by the plasmid. Another enzyme, ligase, is used to permanently seal the exposed nucleotides (ends of the DNA strands) together (like genetic glue). the plasmid is then put back into the bacterial cell, and the bacteria will then manufacture insulin. its offspring will also have the genetic data for human insulin.
How can large quantities of protein be produced from a bacterial colony containing the gene of interest?
In Gene clonning copy no of gene increse and translation of each gene produce more no of protein so one can increas production of protein
Explain how viruses might be used to copy the gene for producing human insulin?
the human insulin gene, which is located on the top of the short arm of chromosome 11 in human DNA, is cut from the DNA strand using restriction enzymes (genetics scissors). a plasmid (floating circular disks of DNA in bacteria) is extracted from a bacteria and cut open with another restriction enzyme, and the gene for human insulin is taken up by the plasmid. Another enzyme, ligase, is used to permanently seal the exposed nucleotides (ends of the DNA strands) together (like genetic glue). the plasmid is then put back into the bacterial cell, and the bacteria will then manufacture insulin. its offspring will also have the genetic data for human insulin.
