Lesson Explainer: Cloning DNA Sequences Biology

In this explainer, we will learn how to describe the process of creating recombinant DNA using vectors and outline how reverse transcriptase can be used to clone sections of DNA.

One of the most significant scientific achievements since the 1970s is the development of recombinant DNA technology. Recombinant DNA is DNA composed of genetic material taken from two or more sources. Recombinant DNA allows us to study genes in more detail and to create interesting combinations of genes that can benefit humans.

Definition: Recombinant DNA

Recombinant DNA is the combination of DNA from at least two different sources to form new genetic information not previously found in the genome.

Insulin is a great example of the power of this technology. Insulin is a protein needed to regulate glucose uptake by the cells and is a necessary medication for people with diabetes. Before the development of recombinant DNA technology, insulin was mostly extracted and purified from animals (cows or pigs). However, this created problems, such as immune rejection, and incurred high costs. By piecing together the DNA for insulin and bacterial DNA, it became possible to introduce this recombinant DNA into bacteria and to grow it to make many copies. By doing this, we were able to produce human insulin in bacteria in greater quantities at a lower cost.

Definition: DNA (Deoxyribonucleic Acid)

DNA is the molecule that carries the genetic instructions for life. It is composed of two strands that coil around each other to form a double helix.

Example 1: Understanding the Definition of Recombinant DNA

What term is given to a DNA molecule formed from genetic material taken from two or more different sources?

  1. Reconstructed
  2. Synthetic
  3. Manufactured
  4. Recombinant
  5. Reversed

Answer

Recombinant DNA is DNA composed of genetic material taken from two or more sources. It involves taking two sections of DNA and combining them, or recombining them, to form a new sequence. Insulin is a great example of this. Before the development of recombinant DNA technology, insulin was primarily extracted from cows or pigs. By piecing together the DNA for insulin and bacterial DNA, it became possible to synthesize insulin inside bacteria in great quantities at a lower cost.

Therefore, the only possible answer to this question is option D, recombinant.

Recombinant DNA in itself is a powerful idea, but for it to be useful, we need to have a way to make many copies of it. This is where a laboratory technique called DNA cloning comes in. A clone is a genetic copy of another organism, or a copy of the DNA itself.

Definition: Clone

A clone is a genetically identical copy of an organism or DNA sequence.

DNA cloning is a technique that allows us to clone, or make many copies of, a DNA sequence or a gene of interest. To do this, the desired DNA sequence is isolated and combined with carrier DNA and then transferred into suitable host cells (usually bacteria). Inside these bacteria, this recombinant DNA replicates to make many copies of itself. This allows studying the sequence in more detail or manufacturing proteins, as in the case of insulin.

Key Term: DNA Cloning

DNA cloning is the process of making more copies of a particular segment of DNA.

In order to clone DNA into bacteria, we first need to place the DNA into specialized carrier DNA. This is called a vector and is a piece of DNA that acts as a vehicle to transport DNA into a cell. A type of vector that we use to do this is called a plasmid.

Plasmids were originally discovered in bacteria and are extrachromosomal circular pieces of DNA that carry antibiotic resistance genes. They can be used by molecular biologists to introduce our DNA of interest into bacterial cells.

Definition: Plasmid

A plasmid is often a small, extrachromosomal, circular DNA molecule that may contain additional genes not found in the bacterial chromosome, such as genes for antibiotic resistance. They can be used to transport DNA into bacterial cells for DNA cloning.

Example 2: Understanding the Origins of Plasmids in DNA Cloning

Using plasmids to form recombinant DNA is a crucial part of cloning DNA.

In which microorganisms were plasmids originally discovered?

  1. Viruses
  2. Fungi
  3. Protists
  4. Bacteria
  5. Algae

Answer

Recombinant DNA is DNA composed of genetic material taken from two or more sources.

For us to effectively use recombinant DNA, we need to make lots of it. This can be accomplished using a technique called DNA cloning, which allows us to make many copies of a desired sequence of DNA. First, the desired DNA sequence needs to be isolated and combined with a carrier DNA sequence called a plasmid. Plasmids are extrachromosomal, circular pieces of DNA, some of which may contain antibiotic resistance genes. They were originally discovered in bacteria.

Plasmids allow transporting DNA into bacteria. By making recombinant DNA with the desired DNA sequence and the plasmid DNA, it is possible to transfer this DNA into bacteria to clone the desired DNA sequence.

Therefore, the only possible answer to this question is option D, bacteria.

Plasmids can be cut using restriction enzymes to insert a DNA sequence of interest. Cutting the DNA sequence and plasmid using the same restriction enzyme produces compatible sticky ends that can come together. You may recall that sticky ends are produced when DNA is cut with a restriction enzyme to leave an overhang of unpaired bases that have an affinity for each other based on complementary base pairing rules. You can see this in Figure 1.

Figure 1: An illustration showing how using the same restriction enzyme (BamHI in this example) can result in compatible sticky ends.

In Figure 1, we can see two complementary sticky ends, which can come together because of their complementary base pairs. However, this is only a weak association based on the hydrogen bonding of the nucleotides, so we need something more permanent.

An enzyme called DNA ligase can bond these two sticky ends together, in a process called ligation. This enzyme can reform the phosphodiester bond that was broken by the restriction enzyme, to reform the gaps of the sugar–phosphate backbone. This process is shown in Figure 2.

Definition: DNA Ligase

DNA ligase is an enzyme that can join the gaps between the sugar–phosphate backbones of two pieces of DNA by forming a phosphodiester bond.

Figure 2: An illustration showing how two compatible sticky ends can be ligated using DNA ligase.

Figures 1 and 2 show one side of the DNA of interest and the plasmid. The same steps occur on the other side so that the entire fragment of the desired DNA can be ligated into a plasmid. The overall process is summarized in Figure 3.

Figure 3: An illustration showing how recombinant DNA can be prepared using restriction enzymes and how it can be ligated using DNA ligase. For simplicity, only one side of this reaction is shown.

Example 3: Understanding the Importance of Complementary Sticky Ends in DNA Cloning

In the process of forming recombinant DNA, why is it important that both the desired section of DNA and the bacterial plasmid are cut using the same restriction enzyme?

  1. To leave complementary sticky ends
  2. To reduce the likelihood of the bacterial cell rejecting the DNA
  3. To leave noncomplementary blunt ends
  4. To ensure the DNA and plasmid are the same size
  5. To save money

Answer

Recombinant DNA is DNA composed of genetic material taken from two or more sources.

For us to effectively use recombinant DNA, we need to make lots of it. This can be accomplished using a technique called DNA cloning, which allows us to make many copies of a desired sequence of DNA. First, the desired DNA sequence needs to be isolated and combined with a carrier vector DNA sequence called a plasmid.

To combine the plasmid with the desired DNA sequence, restriction enzymes are used. These are enzymes that can cut DNA at specific recognition sequences in order to leave either sticky or blunt ends. Sticky ends are sticky because they have an affinity for one another due to complementary base pairing rules, and so they are only sticky for one another if the same restriction enzyme is used. Let’s take a look at the following example of a section of DNA being cut using the restriction enzyme BamHI (which recognizes the sequence GGATCC).

In this example, we can see that, by cutting both sequences with BamHI, we can generate compatible sticky ends that can be joined together. If we use different restriction enzymes, different sticky ends, which are not compatible, will be generated.

By making recombinant DNA with the desired DNA sequence and the plasmid DNA, it is possible to transfer this DNA inside bacteria to clone the desired DNA sequence.

Therefore, the correct answer is option A, to leave complementary sticky ends.

Once the recombinant DNA is prepared, it can then be inserted into bacterial cells, in a process called transformation. Here, bacterial cells are made permeable by exposing them to calcium ions and increased temperatures, which allows the recombinant DNA to pass through the cell membrane into the cytoplasm of the bacterium.

Definition: Transformation

Transformation is a lab technique used to transfer DNA into a bacterial cell.

Example 4: Understanding the Role of Transformation in DNA Cloning

Which of the following best describes the process of transformation in cloning DNA sequences?

  1. Generating a double-stranded molecule of DNA from a section of mRNA
  2. Incorporating a genetically modified plasmid into a bacterial cell
  3. Selecting and removing a section of DNA using restriction enzymes
  4. Combining sections of DNA from two different sources

Answer

DNA cloning is a technique that allows us to make many copies of a desired DNA sequence. First, this sequence must be isolated and then combined with a vector DNA sequence called a plasmid. The resulting recombinant DNA can then be transferred inside bacteria, where it can be copied many times to make more of the desired DNA or the associated protein.

The process of transferring DNA into bacterial cells is called transformation. Here, bacterial cells are made permeable by exposing them to calcium ions and increased temperatures, which allows the recombinant DNA to pass through the cell membrane into the cytoplasm of the bacterium.

Let’s take a look at the answers to the question. Option A is not a good choice because it does not describe the process of transformation in cloning DNA sequences. Option B is a good choice because it describes the process of transformation. Option C is not a good choice because it is describing a method of isolating a section of DNA. Option D is not a good choice because it is describing the definition for recombinant DNA.

Therefore, the answer is option B, incorporating a genetically modified plasmid into a bacterial cell.

Once the recombinant DNA is inside the bacterial cell, it is replicated to make more copies. It can also be transcribed and translated into the associated protein. For example, if the gene for insulin is cloned into a plasmid and then transformed into bacteria, we can harvest the insulin protein from the bacterial cells, as shown in Figure 4. DNA cloning revolutionized the way insulin is produced.

Figure 4: An illustration showing that recombinant DNA can be transformed into bacteria, where the resulting protein can be manufactured for medical purposes.

There are numerous other applications of DNA cloning.

To fight off viruses, researchers have isolated and cloned the genes for interferons. These are special proteins that can interfere with the replication of viruses (particularly viruses with RNA genomes, like influenza). Inside the body, virus-infected cells release interferons in order to protect themselves and their neighboring cells. In the 1970s, interferon was extracted from human cells for medical purposes. However, this process was very expensive. In the 1980s, 15 different genes for interferons were cloned into bacteria to manufacture interferons for a much more reasonable cost. Interferons can also be used to treat cancer.

In agriculture, researchers have cloned genes for pest resistance into crops. This allowed the farmers to grow food without spraying expensive pesticides while also protecting the environment from these potentially toxic substances. The genes that allow plants to house nitrogen-fixing bacteria in their roots are also desirable, because this eliminates the need for nitrogen fertilizers.

The DNA of fruit flies has been manipulated to show that these genetic modifications can be passed on to the offspring. In one experiment, a gene for red eye color was inserted into fruit fly embryos, whose offspring also inherited the red eye color gene.

Genes have also been cloned into mammals. In mice, researchers cloned the human gene for growth hormone, which allowed the mice to grow twice their sizes.

The world of DNA cloning is very exciting, but people are nevertheless worried about it. Suppose a strain of bacteria that produces a deadly toxin was released into the world. These kinds of fears are not realistic, however, since many precautions are taken and the bacteria themselves are unable to survive outside the special laboratory conditions.

Now, let’s discuss how to isolate our DNA of interest so we can clone it into a plasmid. You may recall that mRNA can be transcribed from DNA, which can then be translated into a protein.

Definition: mRNA (Messenger RNA)

mRNA is a message that is transcribed from the DNA of a gene and can be translated to produce the corresponding protein.

We can isolate cells that produce a lot of a particular protein, for example, the beta cells of the pancreas that produce insulin, and then isolate the mRNA for this protein from these cells. But the mRNA itself must first be converted into DNA if we want to clone it into bacterial cells (since restriction enzymes recognize only double-stranded DNA).

The process of converting mRNA back into DNA is called reverse transcription, and the enzyme responsible for this conversion is called reverse transcriptase.

This enzyme uses mRNA as a template to make a complementary strand of DNA called cDNA. cDNA is a single strand of DNA. The formation of cDNA is based on complementary base pairing rules (where A pairs with T and G pairs with C), except that we need to consider uracil (U) in place of thymine (T) in RNA, which pairs with adenine (A). You can see how cDNA is synthesized in Figure 5.

Figure 5: An illustration showing how cDNA can be produced by reverse transcription. Note that the mRNA strand is degraded after the reverse transcription step, leaving just the cDNA.

Definition: Reverse Transcriptase

Reverse transcriptase is an enzyme used to make cDNA from mRNA.

Definition: cDNA (Complementary DNA)

cDNA is the complementary DNA that is produced from a specific sequence of RNA.

Key Term: Complementary Base Pairing

DNA bases generally base-pair according to specific rules, where adenine (A) binds to thymine (T) and guanine (G) binds to cytosine (C). In RNA, uracil (U) is substituted for thymine (T). These rules of complementary base pairing are critical for DNA replication and transcription.

Making cDNA, however, is not enough. Because it is a single-stranded molecule, we need a way to convert this into a double-stranded DNA molecule. The enzyme that can do this is called DNA polymerase. This is an enzyme that converts single-stranded DNA into double-stranded DNA and is also the enzyme responsible for replicating DNA in our cells. You can see this in Figure 6.

Figure 6: An illustration showing how double-stranded DNA can be produced from cDNA using DNA polymerase.

Key Term: DNA Polymerase

DNA polymerase is the enzyme that synthesizes a second strand of DNA to convert single-stranded DNA into double-stranded DNA.

Example 5: Understanding the Role of Reverse Transcriptase and DNA Polymerase in DNA Cloning

The diagram provided shows the basic outline of how the enzyme reverse transcriptase can be used to clone a section of genetic material. What is the role of DNA polymerase in this process?

  1. To form a strand of DNA that is complementary to the cDNA
  2. To provide the site for the synthesis of the cDNA strand
  3. To join gaps in the sugar–phosphate backbone of the mRNA and the cDNA molecules
  4. To form a strand of cDNA that is complementary to the mRNA
  5. To break hydrogen bonds between complementary base pairs

Answer

DNA cloning is a technique that allows us to clone, or make many copies of, a DNA sequence or a gene of interest. To do this, the desired DNA sequence is isolated and combined with carrier vector DNA and then transferred into suitable host cells (usually bacteria). Inside these bacteria, this recombinant DNA replicates to make many copies of itself. This allows studying the sequence in more detail or manufacturing proteins.

One way to isolate a DNA sequence for cloning is to convert its corresponding mRNA back into DNA. You may recall that mRNA can be transcribed from DNA, which can then be translated into a protein. The mRNA itself must first be converted into DNA if we want to clone it into bacterial cells. The process of converting mRNA back into DNA is called reverse transcription. The enzyme responsible for this conversion is called reverse transcriptase.

This enzyme uses mRNA as a template to make a complementary strand of DNA called cDNA. cDNA is a single strand of DNA and must be converted into double-stranded DNA for DNA cloning. The enzyme that can do this is DNA polymerase, which can use the cDNA as a template to form another complementary strand of DNA to produce a double-stranded DNA molecule.

Let’s take a look at the possible answers. Option A looks like a good choice because this is what DNA polymerase does. Option B does not describe what DNA polymerase does. Option C describes the activity of DNA ligase and not DNA polymerase. Option D describes the activity of reverse transcriptase and not DNA polymerase. Option E does not describe what DNA polymerase does.

Therefore, the correct answer is option A, to form a strand of DNA that is complementary to the cDNA.

Once the cDNA is converted into double-stranded DNA, it can be processed by a restriction enzyme, which only recognizes double-stranded DNA and is why we had to go through all this trouble. After this step, the DNA becomes ready to be cloned.

Let’s recap the process of converting mRNA into double-stranded DNA:

  1. The mRNA for a gene is isolated.
  2. The mRNA is converted into cDNA by reverse transcriptase.
  3. The cDNA is converted into double-stranded DNA by DNA polymerase.
  4. The double-stranded DNA can be used for DNA cloning.

Figure 7 provides an overview of the whole process.

Figure 7: An illustration showing how the gene for insulin can be cloned from mRNA to ultimately manufacture insulin protein.

There are other ways to isolate a gene of interest. One of them is to use the polymerase chain reaction (PCR), which is a specialized technique used to amplify specific DNA sequences using an enzyme called Taq polymerase. While cloning requires propagation of bacteria to contain the recombinant DNA, PCR can be performed in a laboratory tube without using bacteria. PCR can do this by making multiple copies of, or amplifying, a target DNA sequence.

Key Term: Polymerase Chain Reaction (PCR)

PCR is a technique used to amplify sequences of DNA using the enzyme Taq polymerase.

Let’s recap some of the key points that we have covered in this explainer.

Key Points

  • DNA cloning is a multistep process used to make more copies of a desired DNA sequence.
  • Restriction enzymes can be used to cut the desired DNA and plasmid DNA to form compatible sticky ends so that they can be joined together using DNA ligase.
  • Bacterial cells can be transformed with recombinant DNA so that they can produce more copies of the DNA and protein.
  • Reverse transcriptase can be used to convert mRNA into cDNA, which can then be converted into double-stranded DNA using DNA polymerase, and this can be used in cloning.
  • PCR (polymerase chain reaction) can be used to make many copies of a target DNA sequence.

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