Biotechnology Unit 4

Discovery of restriction enzymes

• First discovered in the 1950s-1960s by Werner Arber, Hamilton Smith, and Daniel Nathan

• Studied how bacteria defended themselves against bacteriophages

• Werner Arber discovered the phenomenon of host-controlled DNA modification and proposed the existence of restriction enzymes in bacteria

• Hamilton Smith isolated and characterized the first type II restriction enzyme (HINDIII)

• Daniel Nathan used restriction enzymes as a tool for DNA Analysis. He applied them to study the genetics of SV40 Virus (induces brain and bone cancers)

Restriction Enzymes

• Are proteins that cut DNA at specific nucleotide sequences

• They are naturally in bacteria as a defense mechanism against viruses

• They act like molecular scissors cutting apart viral DNA

How Do They Work?

• The enzyme binds to the DNA at their recognition sequence

  • recognize specific DNA sequences that are usually between 4-8 base pairs long

  • They are often palindrome sequences (the same forwards as they are backwards)

  • Example: EcoRi recognizes GAATTC

• It will scan DNA by sliding along the molecule

• When it reaches its specific sequence, it will stop and cut

Blunt Ends vs. Sticky Ends

• can create two types of cuts in DNA

  • Blunt Ends: straight cut across both strands

  • Sticky Ends: Staggered cut leaving single-stranded overhangs

• Sticky ends are easier to recombine. However, blunt ends are used when the DNA fragments may not be compatible

Applications in Genetic Engineering

• Restriction enzymes cut DNA at specific sites for analysis or manipulation

• They are essential for creating recombinant DNA

• Allow for the insertion of genes into plasmids for cloning

• Ethical Issues:

  • Possibility of creating harmful organisms

  • Unequal access to genetic engineering technology

  • Potential for designer babies

  • Allows for modification of human genomes

DNA fingerprinting

• Restriction enzymes are cut into fragments of varying lengths

• These fragments create unique patterns for individuals

• Used in forensic sciences and paternity testing

Mapping Genomes

• These patterns created by restriction enzymes can be used to map gene locations

• crucial in the human genome project

Future of Restriction enzymes

• continued use in genetic engineering and biotechnology

• development of engineered restriction enzymes

• Integration with other gene-editing technologies

Polymerase Chain Reaction (PCR)

• PCR is a technique used to make millions of copies of a specific DNA segment

• The key components are:

  • Templete DNA: The segment you want to copy

  • Primers: Short DNA sequences that mark the target region

  • DNA polymerase: builds new DNA strands

  • Nucleotides: Building blocks of DNA

Steps of PCR:

• Typically done using a thermal cycler

  • Step 1: Denaturation

    • heating to seperate strands

  • Step 2: Annealing

    • Cooling to allow primers to attach

  • Step 3: Extension

    • DNA polymerase builds new strands

Applications of PCR

• Forensics

  • Amplifies small amounts of DNA from crime scenes

  • can identify individuals from hair, blood, or skin cells

  • used in solving cold cases and exonerating wrongly convicted people

• Genetics

  • helps diagnose genetic disorders

  • can detect mutations associated with diseases

  • used in prenatal testing and cancer screening

PCR and Covid-19

• PCR tests are used to detect the presence of the presence of the SARS-CoV-2 virus

• Can identify infections even with small amounts of viral RNA

• Results are typically available within 24 hours

Restriction Enzymes and Recombinant DNA

• Recombinant DNA is a molecule containing genetic material from multiple sources

• It is created by combining DNA from different organisms in a lab

Process

• Step1: Choose the gene you want to transfer

• Step 2: Select a suitable vector (usually a plasmid) to carry the gene

• Step 3: Use restriction enzymes to cut both the insert and vector DNA

• Step 4: Join the insert and vector using DNA ligase

• Step 5: Intrduce the recombinant DNA into host cells for replication

Leading Up to the Discovery of Recombinant DNA

• 1920s-1940s: Griffith laid the groundwork for the idea that recombinant DNA could be introduced into cells with Bacterial Transformation experiment

• 1950s-1960s: Development of Enzyme systems like DNA polymerase and ligase enabled the manipulation of DNA in the lab (biochemistry)

• 1970s: Discovery of Restriction Enzymes (genetics)

• 1970s: Scientists like Paul Berg demonstrated that foreign DNA could be inserted into plasmids and replicated with bacterial cells

Discovery of Recombinant DNA

• emerged in the early 1970s and allowed scientists to combine DNA from different sources

• Hubert Boyer and Stanley Cohen

  • together they completed the first recombinant DNA experiment-inserted a gene from an african clawed frog into a plasmid, that was then inserted into e.coli

From Discovery to Application

• 1975: Asilomar conference - landmark conference where scientists established guidelines for safely working with recombinant DNA technology, promoting the responsible use of these new technologies

  • discussed biohazards and regulation

• 1978: synthetic insulin production

  • revolutionized diabetes treatment

Example of Recombinant DNA: Gene therapy

• Restriction enzymes help in creating gene constructs that can be used to replace faulty genes

• First FDA approved gene therapy was in 2023 for patients with sickle cell

  • patients blood stem cells are modified by genome editing using CRISPR/Cas9

  • two therapies: Casgevy and Lyfgenia

  • There are now 22 FDA approved Gene therapy treatments