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Gene Technology

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Biology

Do you know what genetic engineering involves? Have you heard of GMOs? How about the applications of genetic engineering? This article discusses genetic engineering and its application. It also compares the different methods used to create DNA fragments in genetic engineering (aka the gene technology).

What is gene technology?

Genetic engineering is the process of using advanced DNA technology to modify the genetic content of an organism. Scientists can use genetic engineering to isolate genes and then combine them with the genome of the recipient DNA. The DNA created from this combination is called recombinant DNA.

The organism that contains recombinant DNA is a transgenic or a genetically modified organism (GMO), which can be as simple as bacteria or as complex as plants and animals.

Modifying the genetic makeup of organisms is a complex process that involves utilising genetic engineering techniques in multiple steps.

Genetic engineering aims to develop GMOs with more desirable outcomes. Crops, such as corn, soybeans, and cotton can be genetically modified to resist pesticides. This helps farmers have a better yield since they can use pesticides to eradicate pests without damaging the crops themselves. Bacteria can also be genetically modified. Scientists can introduce a gene that encodes a valuable protein (a drug or a hormone such as insulin) into a bacterium. By growing the new bacterial colonies in bulk, they can collect and purify the desired protein and use it for pharmaceutical or industrial purposes.

The genetic code is universal, meaning it is the same in all organisms. Therefore, the DNA is transcribed and translated within the transgenic organisms the same way as in the donor organism, producing the same protein.

Here are some benefits of genetic engineering:

  • Allows for creating more nutritious foods to eradicate malnutrition around the world.
  • Disease and drought resistant plants allow for the use of fewer environmental resources.
  • Increased food supply with lower cost.
  • Increased shelf life of certain rootstock.
  • Greater yields in farming crops for making biofuels.
  • Faster growing crops and animals.

Gene Technology [+] benefits of genetic engineering [+] StudySmarter

Figure 1. Benefits of genetic engineering. Source: Finty Royle - StudySmarter Originals

Genetic engineering is not all benefits. There are some implications and disadvantages that come with it. These include:

  • The food produced from GMO animals can have less nutritional value due to a faster rate of development.
  • Introducing new genetic material into nature can result in new resistant pathogens that are stronger and pose a risk to health concerns.
  • There can be adverse side effects that are unknown and unexpected.
  • Many companies place copyright over the GMOs they develop. It can have costly consequences for the farmers since they would have to pay greater GMO seeds.
  • Abuse of genetic engineering technology and knowledge on animals and humans can lead to ethically questionable outcomes.
Gene Technology [+]drawbacks detriments genetic engineering ethical issues side effects resistant pathogens [+] StudySmarterFigure 2. Drawbacks and ethical implications of genetic engineering. Source: Finty Royle - StudySmarter Originals.

We can break down the process of creating recombinant DNA and transferring it successfully to create a GMO into five main steps:

  1. The gene that encodes the desired product (e.g., protein) must be isolated. This process involves producing DNA fragments that contain the desirable gene.
  2. The gene then needs to be inserted into a vector.

    Vectors are carriers for delivering foreign genetic material, such as a functional gene, directly into a cell.

  3. The vector delivers the gene to the recipient cells. This delivery process is called transformation.

    Transformation describes the cell's genetic modification via direct uptake and inclusion of foreign genetic materials from its surroundings.

  4. The recombinant cells that have successfully transformed and now contain the recombinant DNA need to be identified. Marker genes, such as antibiotic resistance genes, assist in this step.
  5. Following identification, the recombinant cells are cloned to produce a large population of transgenic cells.

Producing DNA fragments via gene technology

Identifying and isolating a specific gene that is a few hundred bases in length amongst millions of bases in eukaryotic DNA is quite challenging. There are three main techniques that scientists use to create DNA fragments:

  1. Using reverse transcriptase to convert mRNA (messenger RNA) to cDNA (complementary DNA).
  2. Using restriction endonuclease to cut the DNA molecule at a specific sequence.
  3. Using a gene machine to create a gene-based on a protein with a known structure and composition.

Reverse transcription

This process uses a particular enzyme called reverse transcriptase. This enzyme naturally occurs in retroviruses such as HIV, and it creates DNA strands based on mRNA molecules.

  • mRNA in cells correspond to the genetic sequence, and ribosomes read them to synthesise polypeptide molecules (proteins). This process is known as translation.
  • A cell that naturally produces a protein should contain large amounts of that protein's mRNA, which can be isolated. For example, the beta cells in the islets of Langerhans in the pancreas secrete insulin and should contain high levels of insulin mRNA. So, the insulin mRNA can be extracted from beta cells.
  • Following extraction of the desired mRNA, they can be treated with the reverse transcriptase enzyme, which polymerises deoxyribonucleotides and creates single-stranded cDNA molecules that have a complementary base sequence to that of the mRNA.

Complementary DNA or cDNA is synthesised via the reverse transcription procedure based on a single-stranded RNA molecule such as mRNA.

  • The obtained cDNA molecules are subsequently treated with DNA polymerase in the polymerase chain reaction (PCR) process. The DNA polymerase synthesises a complementary DNA strand based on the cDNA, creating a double-stranded DNA. It also replicates the DNA molecules in repeated cycles that amplifies the number of DNA fragments.
  • The main advantage of the reverse transcription process in isolating genes is that the product created at the end is a cDNA that does not contain any non-coding DNA (introns). This is important for inserting genes into prokaryotes since prokaryotic systems cannot remove introns.

Restriction endonucleases

Restriction endonucleases (RE) are enzymes that are naturally part of the bacteria's defence mechanism. They act by cutting the foreign DNA molecules.

There are various types of REs with active sites complementary to specific bases nucleotide sequences. These sequences are called recognitions sites since each RE creates incisions in the DNA specifically at these locations.

We can classify REs into two main groups based on how they cut the DNA:

  1. REs that incise the DNA at the exact location on both strands creates two blunt ends.
  2. REs that cut both DNA strands at positions a few bases apart from each other lead to the creation of staggered ends with exposed DNA bases (bases without complementary pairs). These unpaired DNA bases can join to a DNA with complementary bases. These ends are also called sticky ends since they can easily join other DNA samples with sticky ends. The recognition sites for these types of REs have a palindromic sequence, meaning they are read the same on the forwards and reverse strands.

Gene Technology [+] Creating DNA fragments with sticky ends using restriction enzymes [+] StudySmarter

Figure 3. Creating DNA fragments with sticky ends using restriction enzymes. Source: Created with Biorender.com

The gene machine

The gene machine is the most modern technique where scientists create DNA fragments in a laboratory using computers and special devices.

They first identify the protein of interest and examine its constituting amino acid sequence. Then, scientists can determine the mRNA and DNA sequences that could encode that protein by using the genetic code in reverse.

The obtained DNA sequence can then be entered into the computer. The computer checks the DNA fragments for biosafety and biosecurity, ensuring that it follows the various ethical regulations and does not violate international regulations. The computer then creates a series of small single-stranded nucleotides with overlapping sequences in an automated process. These strands are called oligonucleotides and can be assembled to generate the DNA sequence of the desired gene. Lastly, the engineered DNA strand is amplified and converted to the double-stranded DNA molecule using PCR.

Oligonucleotides are polynucleotides that contain relatively small numbers of nucleotides.

The gene machine technique is accurate and can be conducted in as little as ten days. It also creates DNA strands that do not contain any non-coding regions (introns) and can be transcribed and translated by prokaryotic systems.

Table 1. Advantages and disadvantages of different gene technologies summarised.

Advantages

Disadvantages

Reverse transcriptase technique

  • Large amounts of mRNA in a cell that is actively transcribing the gene. The mRNA can be extracted to make cDNA.
  • cDNA produced is intron free.
  • More steps are involved.
  • Time-consuming.
  • Requires more technical expertise.

Restriction endonuclease technique

  • Sticky staggered ends on DNA fragments make DNA ligation and recombinant DNA production easier.
  • Contains introns.

Gene machines

  • The exact DNA fragment can be designed with custom sequences.
  • The fragments can be designed to contain markers and sticky ends.
  • Requires the amino acid sequence of the desired protein.

Gene Technology - Key takeaways

  • Genetic engineering is the process of using advanced DNA technology to modify the genetic content of an organism.
  • The organism that contains this recombinant DNA is a transgenic or a genetically modified organism (GMO), which can be as simple as bacteria or as complex as plants and animals.
  • Application of GMO:
    • Production of valuable proteins in bulk, such as insulin.
    • Creation of pesticide- and herbicide-resistant crops.
  • Five main steps of transferring recombinant DNA to create a GMO:
    • Isolation of the desired gene.
    • Insertion of the gene into a vector.
    • Transformation.
    • Identification of transformed cells.
    • Cloning of the amplified cells.
  • Three main techniques for creating DNA fragments:
    • Reverse transcription.
    • Usage of restriction endonucleases.
    • Gene machine.

Gene Technology

Genetic technologies in areas of stem cells, cloning and gene therapy. It can also be used to create plant GMOs, such as soybean and corn, resistant to pesticides and herbicides.

Gene technology is used to transfer a gene of interest, created in the lab or isolated from an organism to another organism.

Recombinant DNA technology combines two DNA molecules from different origins together. This technology introduces genes of value to organisms and creates GMO applications in medicine, science, agriculture, and industry.

Recombinant DNA technology has been proven to be beneficial in producing vaccines, producing hormones, such as insulin, for hormone therapies, and creating crops that give a better yield and are resistant to pesticides and herbicides.

Recombinant DNA is formed by artifically combining two or more DNA molecules. This process involves five steps:

  1. The gene encoding the desired needs to be isolated, which involves producing DNA fragments that contain the desirable gene.
  2. The gene then needs to be inserted into a vector. Vector then carries the gene to the recipient cell.
  3. The vector delivers the gene to the recipient cells. This delivery process is called transformation. 
  4. The recombinant cells that have successfully transformed and now contain the recombinant DNA must be identified. Marker genes, such as genes that confer antibiotic resistance, assist this step. 
  5. Following identification, the recombinant cells are cloned to produce a large population of transgenic cells. 

Recombinant DNA technology has proven helpful in producing vaccines, hormones for hormone therapies, clotting factors for treating haemophilia and creating crops that give a better yield and are resistant to pesticides and herbicides.

Final Gene Technology Quiz

Question

What is recombinant DNA?

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Answer

DNA is created by combining two or more DNA molecules from different origins.

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Question

What is genetic engineering?

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Answer

Genetic engineering involves modifying the genetic material of an organism to manipulate a characteristic in the host organism. It is achieved by deleting, substituting or inserting DNA fragments at specific locations in the host organism’s genome.

Show question

Question

What are the main stages of genetic engineering?


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Answer

  1. The gene that encodes the desired product (e.g., protein) must be isolated. This process involves producing DNA fragments that contain the desirable gene.

  2. The gene then needs to be inserted into a vector. Vectors are carriers for delivering foreign genetic material, such as a functional gene, directly into a cell.

  3. The vector delivers the gene to the recipient cells. This delivery process is called transformation.

    Transformation describes the cell's genetic modification via direct uptake and inclusion of foreign genetic materials from its surroundings.

  4. The recombinant cells that have successfully transformed and now contain the recombinant DNA need to be identified. Marker genes, such as antibiotic resistance genes, assist in this step.

  5. Following identification, the recombinant cells are cloned to produce a large population of transgenic cells.

Show question

Question

What are the three ways genes can be generated for genetic engineering?

Show answer

Answer

  1. Using reverse transcriptase to convert mRNA to cDNA (coding DNA). 
  2. Using restriction endonuclease to cut the DNA molecule at a specific sequence.
  3. Using a gene machine to create a gene based on a protein with known structure and composition.

Show question

Question

What is PCR?


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Answer

PCR is an in vitro method of copying and amplifying a DNA segment. It is also used for creating many double-stranded copies of a single-stranded DNA fragment.

Show question

Question

What are the advantages of the reverse transcriptase technique for generating DNA fragments?


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Answer

Large amounts of mRNA in a cell that is actively transcribing the gene. The mRNA can be extracted to make cDNA. cDNA produced is intron free.

Show question

Question

What are the disadvantages of the reverse transcriptase technique for generating DNA fragments?


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Answer

More steps are involved. It is time-consuming. It requires more technical expertise.

Show question

Question

What are the advantages of the restriction endonuclease technique for generating DNA fragments?


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Answer

Sticky staggered ends on DNA fragments make DNA ligation and recombinant DNA production easier.

Show question

Question

What are the disadvantages of the restriction endonuclease technique for generating DNA fragments?


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Answer

The fragments created may contain introns and thus may not be usable in prokaryotic systems.

Show question

Question

What are the advantages of the gene machine technique for generating DNA fragments?

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Answer

The exact DNA fragment can be designed with custom sequences. The fragments can be designed to contain markers and sticky ends.

Show question

Question

The recognition sites for restriction endonucleases that create sticky ends have a _________ sequence.


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Answer

Palindromic

Show question

Question

Restriction endonucleases naturally occur in _________ and they create ______ based on _____ molecules.

Show answer

Answer

retroviruses such as HIV; DNA strands; mRNA

Show question

Question

What is a GMO?


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Answer

The organism that contains this recombinant DNA is a transgenic or a genetically modified organism (GMO) which can be as simple as bacteria or as complex as plants and animals.

Show question

Question

What are the advantages of genetic engineering?


Show answer

Answer

  • Allows for creating more nutritious foods to eradicate malnutrition around the world.
  • Disease and drought resistant plants allow for the use of fewer environmental resources.
  • Increased food supply with lower cost.
  • Increased shelf life of certain rootstock.
  • Greater yields in farming crops for making biofuels.
  • Faster growing crops and animals.

Show question

Question

What are the disadvantages of genetic engineering?


Show answer

Answer

  • The food produced from GMO animals can have less nutritional value due to a faster rate of development.
  • Introducing new genetic material into nature can result in new resistant pathogens that are stronger and pose a health risk.
  • There can be adverse side effects that are unknown and unexpected.
  • Many companies place copyright over the GMOs they develop. It can have costly consequences for the farmers since they would have to pay greater GMO seeds.
  • Abuse of genetic engineering technology and knowledge on animals and humans can lead to ethically questionable outcomes.

Show question

Question

What are the advantages of the gene machine technique for generating DNA fragments?


Show answer

Answer

Requires the amino acid sequence of the desired protein.

Show question

Question

What is in vivo gene cloning?

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Answer

In vivo is Latin for ‘within the living.’ It involves transferring the gene into a host cell using a vector to make many copies of the gene.

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Question

What is in vitro gene cloning?

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Answer

In vitro is Latin for ‘in the glass.’ It involves using the polymerase chain reaction (PCR) in the lab.

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Question

What is the name of the site where the restriction endonucleases cut the DNA?


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Answer

Restriction sites

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Question

What is the characteristic of restriction sites?


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Answer

They have a palindromic sequence.

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Question

What are sticky ends?


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Answer

The single-stranded part of any of the DNA fragments can join the single-stranded part of any other DNA fragment hence causing the ends to be sticky.

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Question

What is the promoter?


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Answer

In both eukaryotic and prokaryotic systems, RNA polymerase initially binds to regulatory regions of the DNA called the promoter which is adjacent to the gene.

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What is the terminator region?


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Answer

The region of DNA after the gene where the RNA polymerase dissociates.

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What is a vector?


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Answer

A vector is any carrier, usually a virus or plasmid, that is used to transport a desired piece of DNA into a host cell.

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Question

What is the transformation of host cells and what conditions does it require?


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Answer

Transformation involves the introduction of recombinant DNA into the host cells. It requires calcium ions and a change in temperature.

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Question

Why does transformation have a low yield?


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Answer

1. Not all bacterial cells successfully uptake the recombinant plasmids.

2. Some of the plasmids may have been closed up without the incorporation of the desired gene.

3. The sticky ends of some of the desired DNA fragments may have also paired up with each other without being incorporated into a plasmid.

Show question

Question

The R-plasmid contains genes for resistance against _________ and _________.


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Answer

Ampicillin and tetracycline

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Question

What are the three common types of marker genes?


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Answer

Antibiotic resistance genes, fluorescence markers, and enzyme markers.

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Question

What is GFP?


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Answer

Green fluorescent protein is a fluorescence marker first identified in jellyfish.

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What is the name of the enzyme that permanently joins two DNA fragments?


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Answer

DNA ligase

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What is the characteristic of the lactase marker?


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Answer

Lactase converts a colourless substrate to blue.

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What does PCR mean?

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Answer

polymerase chain reaction

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What does PCR do?

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Answer

It makes copies of a specific DNA fragment outside of living systems (in vitro).

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What is the main enzyme used in PCR?


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Answer

Taq DNA polymerase

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Why is Taq DNA polymerase best suited for PCR?


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Answer

Taq DNA polymerase works best at relatively high temperatures such as 72C. This allows for faster replication of DNA.

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What are primers?


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Answer

Primers are fragments composed of 18-30 nucleotides that are complement the beginnings of each of the two DNA strands at either end.

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What do primers do?


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Answer

 Primers bind to the beginnings of the two DNA strands and allow the recruitment of Taq polymerase.

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Question

What is a thermocycler?


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Answer

An instrument that automatically increases and lowers the temperature of samples in a closed container according to a program.

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Question

True or False: PCR is very rapid and has a high throughput.


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Answer

True

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True or False: PCR replicates the DNA of interest and any contaminating DNA from the host or other sources.


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False

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True or False: PCR does not require any living systems. This lowers the costs, time, and technical skills required for using PCR relative to in vivo processes.


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True

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True or False: PCR can be used to create double-stranded DNA molecules from single DNA strands


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Answer

True

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True or False: PCR can be used to produce mRNA and proteins in addition to DNA replication.


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Answer

False

Show question

Question

Name two genetic illnesses that can be treated with gene therapy.


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Answer

Cystic fibrosis and severe combined immunodeficiency (SCID).

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Question

What are some of the benefits of gene editing?


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Answer

  • Bacteria may be genetically engineered to generate a variety of compounds such as antibiotics, hormones, and enzymes used to treat illnesses and disorders.
  • Genetically modified crops can also be made tolerant against pesticides and herbicides. This increases the cultivation yield and hence reduces the price of food.
  • Certain genetic illnesses, such as cystic fibrosis (CF) and severe combined immunodeficiency (SCID), may be cured by replacing faulty genes. This treatment is called gene therapy.
  • Microorganisms can be utilised to reduce pollution. They can be modified to break down and digest oil slicks to remove toxic gases emitted by companies.
  • Transgenic crops can be designed to be more resistant to environmental extremes, such as drought, cold, heat, salt, or contaminated soils. This allows crops to be cultivated economically in areas where they do not naturally grow.

Show question

Question

What are some of the risks of gene editing?


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Answer

  • Antibiotic resistance marker genes are often introduced to genetically engineered bacteria which are used for identifying successfully transformed recombinant cells. These bacteria have the potential to transfer their antibiotic resistance genes to dangerous pathogenic microbes.
  • We are uncertain of what long term consequences gene editing and introducing new genetic material into nature can have on the ecosystem both on a micro and macro level.
  • The ability to manipulate and exchange faulty genes brings on ethical questions on where the line should be drawn. Treating cystic fibrosis by gene editing is acceptable but what about changing one’s features, such as eye colour, muscle tone or intelligence?

Show question

Question

What is a DNA probe?

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Answer

DNA probes are short pieces of single stranded DNA that are labelled so that they are easily identifiable.

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Question

What is DNA hybridisation?

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Answer

DNA hybridisation describes the combination of a section of DNA or RNA with a single stranded piece of DNA probe that contains complementary sequences and is labelled to ease its detection.

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Question

Name two different types of DNA probe labelling?


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Answer

Radiolabelling with radioactive phosphorous or labelling with a fluorescent dye

Show question

Question

Why are donor DNA molecules heated before DNA hybridisation?


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Answer

To break the hydrogen bonds between strands and separate the DNA strands from each other.

Show question

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