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a means of transport to get DNA from one organism to a host cell.

types of vectors and the methods are
Mechanical
Methods: Micropipette and gene gun
Biological
Methods: viruses and plasmids (Circular pieces of DNA found in bacteria)

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Definition: The process in which a gene of interest is located and copied from DNA extracted from an organism

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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 a 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.

Introduction

When you hear the word "cloning," you may think of the cloning of whole organisms, such as Dolly the sheep. However, all it means to clone something is to make a genetically exact copy of it. In a molecular biology lab, what's most often cloned is a gene or other small piece of DNA.

If your friend the molecular biologist says that her "cloning" isn't working, she's almost certainly talking about copying bits of DNA, not making the next Dolly!

Overview of DNA cloning

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 insertion is done using enzymes that "cut and paste" DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources.

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. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as "factories" to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.

Steps of DNA cloning

DNA cloning is used for many purposes. 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:

1. Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).

2. Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid.

3. 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.

1. Cutting and pasting DNA

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. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.

Our goal in cloning is to insert a target gene (e.g., for human insulin) into a plasmid. 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.

2. Bacterial transformation and selection

Plasmids and other DNA can be introduced into bacteria, such as the harmless E. coli used in labs, in a process called transformation. During transformation, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.

A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each surviving bacterium will give rise to a small, dot-like group, or colony, of identical bacteria that all carry the same plasmid.

Not all colonies will necessarily contain the right plasmid. That's because, during a ligation, DNA fragments don't always get "pasted" in exactly the way we intend. Instead, we must collect DNA from several colonies and see whether each one contain the right plasmid. Methods like restriction enzyme digestion and PCR are commonly used to check the plasmids.

3. Protein production

Once we have found a bacterial colony with the right plasmid, we can grow a large culture of plasmid-bearing bacteria. Then, we give the bacteria a chemical signal that instructs them to make the target protein.

Once the protein has been produced, the bacterial cells can be split open to release it. There are many other proteins and macromolecules floating around in bacteria besides the target protein (e.g., insulin). Because of this, the target protein must be purified, or separated from the other contents of the cells by biochemical techniques. The purified protein can be used for experiments or, in the case of insulin, administered to patients.

Uses of DNA cloning

DNA molecules built through cloning techniques are used for many purposes in molecular biology. A short list of examples includes:

Biopharmaceuticals. DNA cloning can be used to make human proteins with biomedical applications, such as the insulin mentioned above. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to synthesize the hormone, and tissue plasminogen activator (tPA), which is used to treat strokes and prevent blood clots. Recombinant proteins like these are often made in bacteria.

Gene therapy. In some genetic disorders, patients lack the functional form of a particular gene. Gene therapy attempts to provide a normal copy of the gene to the cells of a patient's body. For example, DNA cloning was used to build plasmids containing a normal version of the gene that's nonfunctional in cystic fibrosis. When the plasmids were delivered to the lungs of cystic fibrosis patients, lung function deteriorated less quickly.

Gene analysis. In basic research labs, biologists often use DNA cloning to build artificial, recombinant versions of genes that help them understand how normal genes in an organism function.

These are just a few examples of how DNA cloning is used in biology today. DNA cloning is a very common technique that is used in a huge variety of molecular biology applications.

Explore outside of Khan Academy

Do you want to learn more about bacterial transformation? Check out this simulation from LabXchange.

Do you want to learn more about the selection of transformed bacteria? Check out this simulation from LabXchange.

LabXchange is a free online science education platform created at Harvard's Faculty of Arts and Sciences and supported by the Amgen Foundation.

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Overview: DNA cloning (article) | Khan Academy

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Learn for free about math, art, computer programming, economics, physics, chemistry, biology, medicine, finance, history, and more. Khan Academy is a nonprofit with the mission of providing a free, world-class education for anyone, anywhere.

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-DNA fingerprinting only requires a tiny bit of blood to allow it to succeed
https://youtu.be/7onjVBsQwQ8

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DNA fingerprint :: CSHL DNA Learning Center

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We've adapted our programming to accomodate schools, homeschool groups, education pods, and the public looking for virtual and on-site options.

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Even as few as five genetic markers (bands formed from restriction enzymes) from an individual can be used to create a DNA fingerprint
https://youtu.be/7onjVBsQwQ8

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Gel electrophoresis separates DNA fragments on the basis of their rate of movement through a gel in an electrical field. The rate of movement depends on the size, electrical charge, and other physical properties of DNA.

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definition:
A protein produced by bacteria that cuts DNA at specific sites along the molecule
Examples of where this tool is used:
DNA fingerprinting
Links to further information and visuals:

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“Gel Electrophoresis” Biology Animation Library - CSHL DNA Learning Center

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In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix.

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-creating a desirable organism used for certain things.
-sometimes sterile, although some transgenic animals can pass their new DNA onto their offspring

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Any organism can use foreign DNA as its own because DNA is universal

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A type of more controllable enzyme that can edit DNA fixing issues like health problems and stop them from being passed down.

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CRISPR is a powerful tool for editing genomes, meaning it allows researchers to easily alter DNA sequences and modify gene function. It has many potential applications, including correcting genetic defects, treating and preventing the spread of diseases, and improving the growth and resilience of crops. However, despite its promise, the technology also raises ethical concerns.

In popular usage, "CRISPR" (pronounced "crisper") is shorthand for "CRISPR-Cas9." CRISPRs are specialized stretches of , and the protein Cas9 - where Cas stands for "CRISPR-associated" - is an that acts like a pair of molecular scissors, capable of cutting strands of DNA.

CRISPR technology was adapted from the natural defense mechanisms of and archaea, a domain of relatively simple single-celled microorganisms. These organisms use CRISPR-derived , a molecular cousin to DNA, and various Cas proteins to foil attacks by . To foil attacks, the organisms chop up the DNA of viruses and then stow bits of that DNA in their own genome, to be used as a weapon against the foreign invaders should those viruses attack again.

When the components of CRISPR are transferred into other, more complex, organisms, those components can then manipulate genes, a process called "gene editing." No one really knew what this process looked like until 2017, when a team of researchers led by Mikihiro Shibata of Kanazawa University in Japan and Hiroshi Nishimasu of the University of Tokyo showed, for the very first time, what it looks like when a CRISPR is in action, Live Science previously reported.

Related: Genetics by the numbers: 10 tantalizing tales

Key components of CRISPR

CRISPRs: The term "CRISPR" stands for "clusters of regularly interspaced short palindromic repeats" and describes a region of DNA made up of short, repeated sequences with so-called "spacers" sandwiched between each repeat.

When we talk about repeats in the genetic code, we're talking about the ordering of rungs within the spiral ladder of a DNA molecule. Each rung contains two chemical bases bound together: A base called adenine (A) links up to another called thymine (T), and the base guanine (G) pairs with cytosine (C).

In a CRISPR region, these bases appear in the same order several times, and in these repeated segments, they form what's known as "palindromic" sequences, according to the Max Planck Institute. A palindrome, like the word "racecar," reads the same forward as it does backward; similarly, in a palindromic sequence, bases on one side of the DNA ladder match those on the opposing side when you read them in opposite directions.

For example, a super simple palindromic sequence might look like this:

Short palindromic repeats appear throughout CRISPR regions of DNA, with each repeat bookended by "spacers." Bacteria swipe such spacers from viruses that have attacked them, meaning they incorporate a bit of viral DNA into their own genome. These spacers serve as a bank of memories, which enables the bacteria to recognize the viruses if they should ever attack again. You can also think of spacers like "Wanted" posters, providing a snapshot of the bad guys so they can be easily spotted and brought to justice.

Related: Going viral: 6 new findings about viruses

Rodolphe Barrangou and a team of researchers at Danisco, a food ingredients company, first demonstrated this process experimentally. In a 2007 paper published in the journal Science, the researchers used Streptococcus thermophilus bacteria, which are commonly found in yogurt and other dairy cultures, as their model, according to the Joint Genome Institute, part of the U.S. Department of Energy. They observed that after a viral attack, the bacteria incorporated new spacers into their CRISPR regions. Moreover, the DNA sequence of these spacers was identical to parts of the virus genome.

The team also manipulated the spacers by removing them and inserting new viral DNA sequences in their place. In this way, the researchers were able to alter the bacteria's resistance to an attack by a specific virus, confirming CRISPRs' role in regulating bacterial immunity.

CRISPR RNA (crRNA): CRISPR regions of DNA act as a kind of bank of viral memories; but for that stored information to be useful elsewhere in the cell, it must be copied, or "transcribed," into a different genetic molecule called RNA. Unlike DNA sequences, which remain lodged inside the DNA molecule, this CRISPR RNA (crRNA) can roam about the cell and team up with proteins - namely the molecular scissors that snip viruses to bits.

RNA also differs from DNA in that it's only one strand, rather than two, meaning it looks like just a half of a ladder. To build an RNA molecule, one part of the CRISPR acts as a template and proteins called polymerases swoop in to construct an RNA molecule that is "complementary" to that template, meaning the two strands' bases would fit together like puzzle pieces. For example, a G in the DNA molecule would get transcribed as a C in the RNA.

Each snippet of CRISPR RNA contains a copy of a repeat and a spacer from a CRISPR region of DNA, according to a 2014 review by Jennifer Doudna and Emmanuelle Charpentier, published in the journal Science. The crRNA interacts with the Cas9 protein and another kind of RNA, called "trans-activating crRNA" or tracrRNA, in order to help bacteria fend off viruses.

Cas9: The Cas9 protein is an enzyme that cuts foreign DNA. The protein binds to crRNA and tracrRNA, which together guide Cas9 to a target site on the virus's DNA strand where the protein will make its cut. The target DNA that the Cas9 will cut through is complementary to a 20-nucleotide stretch of the crRNA, where a "nucleotide" is a building block of DNA that contains one base.

Using two separate regions or "domains" on its structure, Cas9 cuts both strands of the DNA double helix, making what is known as a "double-stranded break," according to the 2014 Science article.

There is a built-in safety mechanism that ensures that Cas9 doesn't just cut just anywhere in a genome. Short DNA sequences known as "protospacer adjacent motifs," or PAMs, serve as tags and sit adjacent to the target DNA sequence. If the Cas9 complex doesn't see a PAM next to its target DNA sequence, it won't cut. This is one possible reason that Cas9 doesn't ever attack the CRISPR region in bacteria, according to a 2014 review published in Nature Biotechnology.

How CRISPR works as a genome-editing tool

Genomes encode a series of messages and instructions within their DNA sequences, and genome editing involves changing those sequences, thereby changing the messages they contain. This can be done by inserting a cut or break in the DNA and tricking a cell's natural DNA repair mechanisms into introducing the targeted changes. CRISPR-Cas9 provides a means to do so.

In 2012, two pivotal research papers were published in the journals Science and , describing how the bacterial CRISPR-Cas9 could be used to chop up any DNA, not just that of viruses. In this way, the natural CRISPR system could be transformed into a simple, programmable genome-editing tool.

To direct Cas9 to snip a specific region of DNA, scientists can simply change the sequence of the crRNA, which binds to a complementary sequence in the target DNA, the studies concluded.In the 2012 Science article, Martin Jinek and his colleagues further simplified the system by fusing crRNA and tracrRNA to create a single "guide RNA." Thus, genome editing requires only two components: a guide RNA and the Cas9 protein.

"Operationally, you design a stretch of 20 base pairs that match a gene that you want to edit," and from there, one can figure out what the complementary crRNA sequence would be, George Church, a professor of genetics at Harvard Medical School, told Live Science. Church emphasized the importance of making sure that the nucleotide sequence is found only in the target gene and nowhere else in the genome.

"Then the RNA plus the protein [Cas9] will cut - like a pair of scissors - the DNA at that site, and ideally nowhere else," Church explained. Once the DNA is cut, the cell's natural repair mechanisms kick in and work to piece the DNA back together, and at this point, edits can be made to the genome. There are two ways this can happen:

According to the Huntington's Outreach Project at Stanford University, one repair method involves gluing the two cuts back together. This method, known as "non-homologous end joining," tends to introduce errors where nucleotides are accidentally inserted or deleted, resulting in that could disrupt a gene.

In the second method, the break is fixed by filling in the gap with a sequence of nucleotides. In order to do so, the cell uses a short strand of DNA as a template. Scientists can supply the DNA template of their choosing, thereby writing-in any gene they want, or correcting a mutation.

Who discovered CRISPR?

Scientists originally discovered the CRISPRs in bacteria in 1987, but they didn't initially understand the biological significance of the DNA sequences, and they didn't yet call them "CRISPRs," according to Quanta Magazine. Yoshizumi Ishino and colleagues at Osaka University in Japan first found the characteristic nucleotide repeats and spacers in the gut microbe , and as the technology for genetic analysis improved in the 1990s, other researchers found CRISPRs in many other microbes.

Francisco Mojica, a scientist at the University of Alicante in Spain, was the first to describe the distinct characteristics of CRISPRs and found the sequences in 20 different microbes, according to a 2016 report in the journal Cell. At one point, he dubbed the sequences "short regularly spaced repeats" (SRSRs), but he later suggested that they be called CRISPRs instead. The term CRISPR first appeared in a 2002 report, published in the journal Molecular Microbiology and authored by Ruud Jansen of Utrecht University, with whom Mojica had been in correspondence.

In the following years, scientists also discovered Cas genes and the function of Cas enzymes, and they figured out that the spacers in CRISPRs came from invasive viruses, Quanta reported.

Among these pioneering researchers was Jennifer Doudna, a professor of biochemistry, biophysics and structural biology at the University of California, Berkeley, who went on to share the 2020 Nobel Prize in chemistry with Emmanuelle Charpentier, director of the Max Planck Unit for the Science of Pathogens. The two scientists are credited with adapting the bacterial CRISPR/Cas system into a handy gene-editing tool, Live Science previously reported.

Charpentier initially discovered tracrRNA while studying the bacteria Streptococcus pyogenes, which causes a range of diseases from tonsillitis to sepsis. Having uncovered tracrRNA as a previously unknown component of the CRISPR/Cas system, Charpentier began collaborating with Doudna to recreate that system in a test tube. In 2012, the team published their seminal work in the journal Science, announcing that they'd successfully simplified the molecular scissors into a gene-editing tool.

Some thought that biochemist Feng Zhang of the Broad Institute might also earn the Nobel for his own, separate work with the CRISPR system, Science Magazine reported. Zhang demonstrated that the CRISPR system works in mammalian cells, and based on this work, the Broad Institute earned the first patent for the use of CRISPR gene-editing technology in eukaryotes, or complex cells with nuclei to hold their DNA.

How has CRISPR been used?

(Image credit: KEITH CHAMBERS/SCIENCE PHOTO LIBRARY via Getty Images)

In 2013, researchers in the labs of Church and Zhang published the first reports describing the use of CRISPR-Cas9 to edit human cells in an experimental setting. Studies conducted in lab dish and animal models of human disease have demonstrated that the technology can effectively correct genetic defects. Examples of such diseases include cystic fibrosis, cataracts and Fanconi anemia, according to a 2016 review article published in the journal Nature Biotechnology. These studies have paved the way for therapeutic applications in humans.

In the realm of medicine, CRISPR has been tested in early-stage clinical trials as cancer therapy and as a treatment for an inherited disorder that causes blindness. It's also been investigated as a strategy for preventing the spread of Lyme disease and malaria from viral vectors to people, and it's also been studied in animal models of HIV as a way to rid infected cells of the virus, Live Science previously reported. One research team in China attempted to treat a human patient's HIV using CRISPR, and while the treatment wasn't successful in curing the infection, the gene therapy also didn't cause any harmful effects, Live Science reported.

"I think the public perception of CRISPR is very focused on the idea of using gene editing clinically to cure disease," said Neville Sanjana of the New York Genome Center and an assistant professor of biology, neuroscience and physiology at New York University. "This is no doubt an exciting possibility, but this is only one small piece."
Related: 10 amazing things scientists just did with CRISPR
CRISPR technology has also been applied in the food and agricultural industries to engineer probiotic cultures and to vaccinate industrial cultures (yogurt, for example) against viruses. It is also being used in crops to improve yield, drought tolerance and nutritional properties.

One other potential application is to create gene drives, a genetic engineering technique that increases the chances of a particular trait passing on from parent to offspring; this kind of genetic engineering derives from a natural phenomenon, where specific versions of genes are more likely to be inherited. Eventually, over the course of generations, the trait spreads through entire populations, according to the Wyss Institute. Gene drives could be used for various applications, such as eradicating invasive species or reversing pesticide and herbicide resistance in crops, according to a 2014 report published in the journal .

During the COVID-19 pandemic, the CRISPR-Cas9 system has been used to develop various diagnostic tests for the viral infection, BBC News reported.

In April 2017, a team of researchers released research in the journal Science that they had programmed a CRISPR molecule to find strains of viruses, such as Zika, in blood serum, urine and saliva.

On Aug. 2, 2017, scientists revealed in the journal Nature that they had removed a heart disease defect in an embryo successfully using CRISPR.

On Jan. 2, 2018, researchers announced that they may be able to stop fungi and other problems that threaten chocolate production using CRISPR to make the plants more resistant to disease.

On April 16, 2018, researchers upgraded CRISPR to edit thousands of genes at once, according to research published by the journal BioNews.

In addition, CRISPR has recently been used in the following ways:

However, despite its wide range of uses, the tool is not without its drawbacks.

"I think the biggest limitation of CRISPR is it is not a hundred percent efficient," Church told Live Science. That means, in a given experiment, CRISPR may successfully edit only a percentage of the targeted DNA. According to the 2014 Science article by Doudna and Charpentier, in a study conducted in rice, gene editing occurred in nearly 50% of the cells that received the Cas9-RNA complex. Meanwhile, other analyses have shown that depending on the target, editing efficiencies can reach as high as 80% or more.

The technology can also create "off-target effects" when DNA is cut at sites other than the intended target. This can lead to the introduction of unintended mutations. Furthermore, Church noted, even when the system cuts on target, there is a chance of not getting a precise edit. He called this "genome vandalism."

Potential risks and ethical concerns of using CRISPR

The many potential applications of CRISPR technology raise questions about the ethical merits and consequences of tampering with genomes. And in particular, a slew of ethical debates flared up in 2018 when He Jiankui, formerly a biophysicist at the Southern University of Science and Technology in Shenzhen, announced that his team had edited DNA in human embryos and thus created the world's first gene-edited babies.

He was subsequently sentenced to three years in prison and fined 3 million yuan ($560,000) for practicing medicine without a license, violating Chinese regulations on human-assisted reproductive technology and fabricating ethical review documents, Live Science previously reported. But even after his sentencing, He's experiments raised questions about how the use of CRISPR should be regulated going forward, especially given that the technology is still fairly new.

Illegal experimentation in human embryos represents an extreme misuse of CRISPR, of course, but even seemingly ethical uses of the technology could carry risks, scientists say.

In general, making genetic modifications to human embryos and reproductive cells such as sperm and eggs is known as germline editing. Since changes to these cells can be passed on to subsequent generations, using CRISPR technology to make germline edits has raised a number of ethical concerns.

Variable efficacy, off-target effects and imprecise edits all pose safety risks. In addition, there is much that is still unknown to the scientific community. In a 2015 article published in Science, David Baltimore and a group of scientists, ethicists and legal experts note that germline editing raises the possibility of unintended consequences for future generations "because there are limits to our knowledge of human genetics, gene-environment interactions, and the pathways of disease (including the interplay between one disease and other conditions or diseases in the same patient)."

In the 2014 Science article, Oye and colleagues point to the potential ecological impact of using gene drives. An introduced trait could spread beyond the target population to other organisms through crossbreeding. Gene drives could also reduce the genetic diversity of the target population, potentially hampering its ability to survive.

Other ethical concerns are more nuanced. Should we make changes that could fundamentally affect future generations without having their consent? What if the use of germline editing veers from being a therapeutic tool to an enhancement tool for various human characteristics?

To address these concerns, the National Academies of Sciences, Engineering and Medicine put together a comprehensive report with guidelines and recommendations for genome editing.

Although the National Academies urge caution in pursuing germline editing, they emphasize "caution does not mean prohibition." They recommend that germline editing be done only on genes that lead to serious diseases and only when there are no other reasonable treatment alternatives. Among other criteria, they stress the need to collect data on the health risks and benefits and to maintain continuous oversight during clinical trials. They also recommend that, after a trial concludes, trial organizers should follow up with the participants' families for multiple generations to see what changes persist in the genome over time.
This article includes additional reporting by Alina Bradford, Live Science contributor. Originally published on Live Science.
Additional resources

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CRISPR is a powerful tool for editing genomes, meaning it allows researchers to easily alter DNA sequences and modify gene function. It has many potential applications, including correcting genetic defects, treating and preventing the spread of diseases, and improving the growth and resilience of crops. However, despite its promise, the technology also raises ethical concerns.

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DNA has a negative charge so it will go towards the positive end of the gel during gel electrophoresis