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Biotechnology! By: Naman Patel

Naman Patel English

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Restriction Endonuclease/Enzymes
- What are restriction enzymes?
  - Restriction enzymes act like molecular scissors, they cut double stranded DNA at certain recognition sites; unique for every restriction enzyme
    - They recognize palindromic stretches of DNA that are 4-8 base pairs in length. Palindromic stretches are the recognition sequences where the enzymes cut the DNA
      - The restriction enzyme cuts through both strands of DNA, creating fragments with one of two types of DNA ends: “blunt” or “sticky”
- How are restriction Enzymes used?
  - For recombinant purposes
    - Plasmid/DNA from two organisms are cut by using the same restriction enzymes. This produces complementary single stranded ends.
      - DNA Ligase is then used to join the complementary ends from the two organisms, and the foreign DNA is mixed into the organism.
        This forms recombinant DNA in which the foreign DNA transferred into the host cell begins to clone.
  - In bacteria
    - Bacteria produce restriction enzymes as a method of self defense against infections & bacteriophage
      - If a bacteriophage inserts its DNA into the bacteria, the restriction enzymes within the bacteria are able to attack the foreign DNA.
        To protect the bacteria's own DNA, a methyl group is added to the restriction enzymes which allows the restriction enzymes to recognize the host DNA and protect the host's DNA.
- Applications
  - In forensic sciences, restriction enzymes can be used to cut DNA samples into small fragments and placed into a gel electrophoresis to identify the criminal by comparing the fragments!!
  - Restriction enzymes can be used in recombinant DNA technology to take alter the DNA of a plasmid by cutting our a specific gene and inserting it with one that is desired.
  - Restriction enzymes can also be used to create a restriction map, a diagram or map of a DNA molecule within an organism which indicates all the restriction sites.
    - Restriction maps are used to identify fragments of DNA that contain specific genes.
Gel Electrophoresis
- What is Gel Electrophoresis?
  - Gel Electrophoresis is a method that utilizes the charges on macromolecules: proteins, polypeptides, DNA or RNA fragments, and causes them to migrate in an electrical field
    - Since nucleic acids are negatively charged due to their phosphate groups, they migrate through the gel towards the positive end.
      - Throughout the migration, DNA is separated by sizes, as larger macromolecules travel much slower in the gel compared to smaller ones
        This ensures accurate measurements of the molecular weights of the fragments and when the Gel is stained, all the different fragments are visible with their locations and number
- How is Gel Electrophoresis used?
  - An electrical field is set up in a gel material that is usually composed of agarose or polyacrylamide, and is then poured on a thin slab of glass.
    - Once the gel has solidified, small wells are made on one side of the gel usually the one towards the negative side. DNA samples are then inserted into the small wells.
      - An electrical current is applied, and the fragments of DNA travel from the negative pole towards the positive, where the smaller the fragement, the farther it travels away from the well.
        The gel is removed from the field and is stained with ethidium bromide, a dye which binds to the DNA and allows it to fluoresce under UV light.
- Applications
  - Gel Electrophoresis can be used in labs, for the process of separating DNA fragments in DNA fingerprinting to investigate crime scenes.
  - Gel electrophoresis can also be used to analyze the results of a polymerase chain reaction, or PCR.
  - Can be used in paternity tests by using DNA fingerprinting.
  - It can be used to test the purity of an antibiotic. This can be done by applying electrophoresis to a solution containing the antibiotic, and researchers can then differentiate between the antibiotic along with any impurities
    - This can also be used to determine the concentrations of antibiotics which is useful when prescribing dosages.
Plasmids and Cloning Vectors
- What are plasmids?
  - Plasmids are circular pieces of DNA that exist outside of the bacterial chromosome, and they carry their own genes for special functions.
    - Plasmids contain a few genes, but they make a big difference.
      - They contain genes that are not essential for a bacteria's day-to-day survival, but those that help the cell overcome occasional stress. Due to this, bacteria with plasmids tend to survive longer and reproduce, whereas those that dont have a plasmid are less likely to survive.
- How are plasmids used?
  - Plasmid and DNA from another organism are cut using the same restriction enzymes. This produces molecules with complementary single stranded ends.
    - The two types of molecules are mixed together so that their sticky end pairs combine, and DNA ligase is than used to form covalent bonds at their junctions.
      - The plasmid now has foreign genes incorporated within its DNA sequence and can be inserted into a bacterial cell.
- Applications
  - Plasmids play a significant role in gene therapy.
    - In gene therapy they are used as vectors, where therapeutic genes are inserted into the plasmid and then into the human body to fight against diseases.
      - Also used in the production of many hormones outside of the human body (See application section under recombinant DNA technology).
  - Plasmids are also used for cloning!
    - By inserting a gene into a plasmid and then the modified plasmid into a bacteria, the desired gene can be replicated over and over again making more quantities of it! Connects hand in hand with gene therapy!
- What are cloning vectors?
  - A cloning vector is a DNA molecule in which foreign DNA can be inserted into and can replicate within a host cell to produce more copies of the recombinant DNA. A plasmid is an example of a cloning vector.
    - A foreign gene can be inserted into a plasmid and then the plasmid can be introduced to a bacterial cell by a method called transformation.
      - The plasmid is then replicated and distributed to daughter cells when mitosis occurs. In cell division, the foreign DNA in the recombinant plasmid is also replicated.
Recombinant DNA Technology
- What is Recombinant DNA Technology?
  - Recombinant DNA is DNA that has been formed artificially by combining DNA from different organisms.
  - This technology has made it possible for researchers to identify the nucleotide sequences of a specific gene, mutate it in highly specific ways. The mutated gene can then be reinserted into a living organism and display the modified characterististics.
- How does Recombinant DNA Technology work?
  - Strands of DNA and plasmid from two organisms are treated with the same restriction enzymes which cuts our the target gene and creates complementary ends.
    - Then the DNA from one organism is attached to the Plasmid, and DNA Ligase is used to stick the complementary ends together.
      - The modified plasmid or recombinant DNA strand is then inserted into a bacteria cell through a process called heat shocking.
        The bacteria with the new plasmid is then left to replicate on agarose gel into visible colonies
        Once the plasmid is inside the bacteria, the foreign gene can be expressed and the bacterial cell has transformed
        In a scenario where an antibiotic resistance gene has been inserted into the bacterial cell, the transformation is confirmed by testing if the bacteria are able to survive in the presence of that particular antibiotic. If the bacteria survives it has successfully transformed. If the bacteria die, that means they lack the plasmid.
- Applications
  - Recombinant DNA technology is extremely useful due to its important role in gene therapy.
    - Recombinant DNA technology can be used to grow certain hormones which can help treat many diseases, an important example being the production of lab grown insulin.
      - Scientists have used recombinant technology to artifically insert genes that code for insulin onto a plasmid which through transformation is inserted into a bacteria. The bacteria replicates and produce insulin, which is then given to patients with type 1 diabetes.
  - Recombinant DNA technology is also heavily used in the agriculture field.
    - Many crops are being genetically modified. The DNA of crops are being altered to either make them grow in larger yields, mature faster, become resistant to pests and many more.
  - Recombinant DNA technology can also be used to diagnose diseases!
    - The technology has provides a variety of tools, primarily the development of probes.
      - Probes are short and single strands of DNA that are attached to a radioactive or flourescent marker which are then used to identify infectious agents.
CRISPR
- What is CRISPR?
  - Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is the main characteristic of a bacterial immune system.
    - CRISPRs consist of short repetitions of base sequences and are interrupted by "spacers" sequences.
      - "Spacers" sequences contain remnants of genetic code from past invaders and serve as a genetic memory which is used by the cell to detect invaders (bacteriophages) and destroy them.
  - Apart from being known as the immune system of bacteria, CRISPR refers to systems and technology that is able to edit genes/DNA at precise locations.
    - These systems are able to modify genes in living organisms and cells.
      - How does the CRISPR system work?
        CRISPR "spacer" sequences are transcribed into short RNA sequences called, crRNA or CRISPR RNA.
        The short RNA sequences are capable of guiding the system to matching sequences of DNA.
        When the DNA sequence is found an enzyme produced by the system called Cas9, binds to the DNA and cuts it which results in the termination of the targeted gene.
        Modified versions of the Cas9 enzyme allow the DNA sequences to be turned on rather than cut. This allows the researchers to study the gene's function.
- Applications
  - The applications to the CRISPR technolog is limitless! The DNA sequence of the host can be altered for any type of situation.
    - The DNA of pigs can be genetically modified so that their organs are more complementary for humans.
    - CRISPR can be used to fight various diseases and viruses, for example, HIV or cancer.
      - At Temple University in Philadelphia, Pennsylvania, a research team was able to eliminate the HIV - 1 DNA from human cultures. The team also tried to re-infect the human cells with HIV, however, the victims were not re-infected. This shows promise for a long term treatment for a very common virus.
    - CRISPR also works hand in hand with Recombant DNA technology as it can be used to genetically modify plants (Explained in the application section under Recombinant DNA technology).
    - CRISPR can also be used for genetically modifying humans, animals or other type of species.
      - Humans can be genetically modifed to be immune against certain diseases and viruses, or possibly in the future, be used to select personal characteristics.
      - There was a case at Harvard University, where a research team were trying to revive an extinct creature known as the Wooly Mammoth. They were trying to use the DNA found in the fossils along with elephant genes and combine them to possibly create hybrid embryos in artifical wombs invitro.
Sanger Method
- What is Sanger?
  - The Sanger method is a process in which the sequence of nucleotide bases in a sequence of DNA can be determined.
    - It is also known as the chain termination method and works in union with PCR
  - The Sanger Method requires:
    - DNA polymerase
    - RNA primer
    - 4 DNA nucleotides (dATP, dTTP, dCTP, dGTP)
    - A template DNA to be sequenced
    - Dideoxy, or chain terminating, versions of all 4 nucleotides (ddATP, ddTTP, ddCTP, ddGTP), each labeled with a different colour of dye
      - Dideoxy nucleotides are similar to regular deoxy nucleotides, however, they lack a hydroxyl group on the 3' carbon of the sugar ring. The lack of the hydroxyl group means that no other bases can be added as DNA is synthesized in 5' to 3' direction.
        This means that the chain will end in a specific dideoxy nucleotide base, and the base can be identified by the colour of its dye, as it will be different for each base.
- How is the Sanger Method used?
  - The sample of DNA is first denatured to uncoil the helical structure into its two strands, and then the sample is cooled, which allows the primer to bind to the single stranded template.
    - The temperature is raised which allows the DNA polymerase to begin synthesizing the new strand of DNA
      - The DNA polymerase will continue synthesizing the new DNA strand until a dideoxy base is used instead of a normal deoxy base where the synthesis stops.
        The process up until now is continued until it is ensured that a dideoxy nucleotide has been incorporated on every position of the sample DNA.
        This will create different sizes fragments, ending at each of the nucleotide positions of the original DNA.
        The different sized fragments are then run in a gel electrophoresis process
        Based on the position of each fragment, the original sequence of the DNA can be determined one at a time.
- Applications
  - As the Sanger method is a process to determine the sequence of nucleotide bases in a sequence of DNA, it can possibly be used to detect unknown mutations, specifically when using PCR.
PCR (Polymerase Chain Reaction)
- What is PCR?
  - PCR is a technique which allows researchers to amplify a tiny sample of DNA without cloning in a cell.
    - With the use of nucleotides, primers and Taq polymerases, DNA can be synthesized in vitro (Outside of a living organism
- How does PCR work?
  - A sample of DNA is denatures which breaks apart its helical structure forming two single strands.
    - An enzyme known as Taq polymerase is used to synthesize DNA from the two template strands. This results in the duplication of the original strands of DNA
      - This process is repeated and more copies of the original DNA are synthesized.
- Applications
  - PCR is a very quick way to amplify DNA. This can be very useful when dealing with a really small sample of DNA and more is needed, for example, blood form a crime scene or even DNA from fossils.
  - PCR can be used for the diagnosis of genetic diseases and in various situations!
    - Can be used for genetic counselling where patients are screened to detect any genetic diseases before having children.
    - Can be used to screen for genetic diseases before implanting an embryo. This is called a preimplantation diagnosis.
    - Can be used to screen for genetic diseases before giving birth by using samples of tissues!
  - The applications of PCR allow an analysis of a very small sample of DNA or damaged samples of DNA.
  - PCR can be used in the agriculture industry as a replacement for traditional detection methods for diseases.
  - PCR can be used to screen donated blood for infections