Natural cloning[ edit ] Cloning is a natural form of reproduction that has allowed life forms to spread for hundreds of millions of years.
Definition, purpose, and basic steps of DNA cloning. DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.
In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid. The plasmid is introduced into bacteria via process called transformation, and bacteria carrying the plasmid are selected using antibiotics.
Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein. However, all it means to clone something is to make a genetically exact copy of it. In a typical DNA cloning procedure, the gene or other DNA fragment of interest perhaps a gene for a medically important human protein is first inserted into a circular piece of DNA called a plasmid.
Diagram showing the construction of a recombinant DNA molecule. A circular piece of plasmid DNA has overhangs on its ends that match those of a gene fragment. The plasmid and gene fragment are joined together to produce a gene-containing plasmid. Next, the recombinant plasmid is introduced into bacteria.
Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.
What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. For instance, the human insulin gene is expressed in E.
It travels through the bloodstream and binds to cells of the body, allowing them to take up the sugar glucose. In a person with type I diabetes, the cells of the pancreas that produce insulin are damaged or destroyed.
People with type I diabetes must regularly inject themselves with insulin from another source to prevent dangerously high levels of blood sugar and to allow sugar to enter the cells of the body as fuel. For many years, all of the insulin used by type I diabetics came from the pancreases of pigs and cows that had been slaughtered for meat.
The insulin molecule is similar between humans, pigs, and cows, so pig or cow insulin can substitute for human insulin in the bodies of type I diabetics. Starting in the s, however, scientists developed new techniques to make human insulin using Escherichia coli E.
Basically, they turned the bacteria into tiny insulin-producing factories. This recombinant insulin, or insulin made by combining DNA from multiple sources humans and bacteriais now the primary treatment used by type I diabetics.
As an example, let's see how DNA cloning can be used to synthesize a protein such as human insulin in bacteria. The basic steps are: Cut open the plasmid and "paste" in the gene.
Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid. Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it. Let's take a closer look at each step.
A common method uses two types of enzymes: A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs.
If two molecules have matching overhangs, they can base-pair and stick together. Our goal in cloning is to insert a target gene e. Using a carefully chosen restriction enzyme, we digest: The plasmid, which has a single cut site The target gene fragment, which has a cut site near each end Then, we combine the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene.
Diagram depicting restriction digestion and ligation in a simplified schematic. We start with a circular bacterial plasmid and a target gene.Publisher Summary. This chapter on deoxyribonucleic acid (DNA) provides information on its structure and its replication.
In the s, researchers wanted to understand the structure of the DNA to find out about the molecular functions in the hereditary process. and this is a cell’s genotypic expression. The genotype is the genetic com- position of an organism in terms of the forms of specific genes, i.e.
alleles, that it contains, and its . Cloning .
Cloning is a method of recombining genes in order to take advantage of a bacteria's native ability to recreate plasmids. Engineered plasmids can be used to clone genetic material of up to 10, base pairs, the amount of genetic material is limited by the size of the plasmid. DNA cloning is the process of making multiple, identical copies of a particular piece of DNA.
In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid.
The discovery of the structure of DNA also revealed the principle that makes this copying possible: because each strand of DNA contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand, each strand can act as a template, or mold, for the synthesis of a new complementary strand.
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