Biotechnology Tools and Applications: Insulin, Gel Electrophoresis, and DNA Fingerprinting

Context: This lecture follows the study of DNA structure and function. Understanding the basic structure of DNA allows for the application of biological principles to develop technology.
Scope: Biotechnology involves using biological systems and organisms to develop or make products. The field can be categorized into the tools (the "tech" part) and the specific applications of those tools.
Selected Examples: Key topics include synthetic insulin, DNA fingerprinting, Genetically Modified Organisms (GMOs), and animal cloning.

Biotechnology Tools: These are the technical methods used to manipulate or analyze DNA.
- Restriction Enzymes: Used to cut DNA at specific sequences.
- Gel Electrophoresis: Used to separate DNA fragments by size.
- PCR (Polymerase Chain Reaction): Used to amplify/copy specific DNA sequences.
Applications and their Required Tools:
- Synthetic Insulin: Primarily uses restriction enzymes; it may also involve PCR.
- DNA Fingerprinting: Utilizes restriction enzymes, gel electrophoresis, and often PCR to obtain samples.
- Genetically Modified Organisms (GMOs): Uses restriction enzymes and PCR, though usually does not require gel electrophoresis during the final application.
- Animal Cloning: This procedure does not utilize restriction enzymes, gels, or PCR, yet it remains a prominent example of biotechnology.

Foundations: The field of biotechnology began in the $1960s$ and $1970s$ with the discovery of restriction enzymes in bacteria.
Natural Biological Function:
- Bacteria use restriction enzymes as a defense mechanism against viruses.
- When a virus infects a bacterium, the restriction enzymes cut the viral DNA into pieces, rendering it non-infectious.
- Bacteria possess a protection mechanism to prevent the enzyme from cutting their own cellular DNA.
Scientific Utility: Scientists "borrow" these enzymes to cut DNA from various sources (human, plant, etc.) to isolate specific genes or create fragments for analysis, such as in DNA fingerprinting.

Definition of Plasmids: Small, circular loops of DNA found in prokaryotes (bacteria) and some eukaryotes (e.g., yeast, which is a fungus).
Extra-chromosomal DNA: Plasmids are separate from the main bacterial chromosome and are not where the primary genes for cell control and protein synthesis are located.
The Insertion Process:
- A circular plasmid is cut open using a restriction enzyme.
- A "gene of interest" (e.g., the human insulin gene) is inserted into the opened plasmid.
- The plasmid is sealed back up to create Recombinant DNA (recombined DNA consisting of original bacterial/yeast DNA and inserted foreign DNA).
Biological Expression: Because the genetic code is universal, the host organism (bacteria or yeast) will treat the inserted DNA as its own. It uses its internal machinery—ribosomes and amino acids—to synthesize the protein coded by the inserted gene.

Historical Significance: Creating synthetic insulin was one of the earliest successful applications of biotechnology to help the millions of people with diabetes.
Structure of Insulin DNA: The insulin molecule is relatively small. The transcript highlights the DNA sequence for one of the chains of insulin.
Genetic Landmarks in the Sequence:
- Start Codon: The DNA sequence $ATG$ serves as the start of the gene. When transcribed into messenger RNA (mRNA), this becomes the $AUG$ start codon.
- Codons: The sequence of bases following the start codon is read three at a time during translation.
- Stop Codon: The DNA sequence $TAG$ serves as the termination point for the gene.
Manufacturing: The gene is cut from a chromosome, spliced into a plasmid, and the host bacterium or yeast is induced to produce the human insulin protein.

Purpose: To sort DNA fragments by their size.
The Medium: An agarose gel, which is derived from a sugar ( $agarose$ ). It is poured as a liquid into a mold and solidifies at room temperature, creating a porous internal structure.
The Mechanism of Movement:
- DNA is loaded into wells at one end of the gel.
- A negative electrode is attached to the well side, and a positive electrode is attached to the opposite far end.
- Charge Dynamics: DNA molecules have a negative charge and are consequently repelled by the negative end and drawn toward the positive end.
Sorting by Size:
- DNA fragments "snake" through the pores of the agarose.
- Shorter Fragments: Move faster and further through the gel.
- Longer Fragments: Lag behind because they have more difficulty passing through the pores.
Visualization: The sorted fragments appear as a series of bands in "lanes," creating a visual pattern similar to a barcode.

The Concept of Variability: Humans share very similar genes, but $0.1\%$ of the genome is highly variable. This variation occurs primarily in the "non-gene" or non-coding regions of the DNA.
Short Tandem Repeats (STRs):
- Definition: Specific regions where short sequences of bases (e.g., $3$ to $4$ bases) repeat side-by-side (in tandem) multiple times.
- Example: A sequence like $AATG$ may repeat several times in a row.
- Inheritance: Individuals inherit STRs from their parents. For example, a person might inherit $7$ repeats from their mother and $8$ repeats from their father on a set of homologous chromosomes.
- Allele Analogies: Though the term "allele" usually refers to variations of a gene, it is often applied to these variations in repeat numbers, even though STRs do not code for proteins.
Statistical Probability: Looking at a single STR site is insufficient for identification. Forensics labs typically look at a large number of sites (e.g., $20$ different STR locations across various chromosomes) to create a unique gene profile.

Applications: Identification of criminals, missing persons, or family members (paternity testing).
CODIS (Combined DNA Index System): A DNA database maintained for law enforcement. It stores the DNA profiles of individuals convicted of crimes.
The Profile: A CODIS profile looks similar to a karyotype but focus on specific STR locations. For example:
- Chromosome $1$ : Contains a specific variable STR site.
- Chromosome $2$ : May contain multiple sites (e.g., $3$ standard STR locations).
Banding Pattern Comparison: Investigators compare the DNA banding pattern from a crime scene against suspect patterns. While a match indicates the DNA is identical, it is one piece of evidence in determining guilt.

Scenario: A practice problem involving a bloodstain found at a crime scene and four suspects: Bob, Sue, John, and Lisa.
Question: "Whose blood is it?"
Analysis:
- Bob: Matches some bands but not all.
- Sue: Matches some bands but not all.
- John: His DNA profile matches every single band found in the bloodstain sample.
- Lisa: Does not have all matching bands.
Conclusion: Based on the banding pattern, the bloodstain belongs to John.