Polymerase Chain Reaction: History, Mechanics, and Applications

Historical Context and Development of Polymerase Chain Reaction (PCR)

• Original Nomenclature: The transcript begins by mentioning the "pulmonary chain reaction," which is immediately clarified as the polymerase chain reaction (PCR).

• Inventor: PCR was developed by Carey Mollis.

  • Carey Mollis passed away during the COVID time block, though reportedly not due to COVID itself.

  • He was an interesting and controversial figure who authored an autobiography about his life and scientific journey.

  • He was a Nobel Prize laureate, an honor that includes a $1,000,000 cash prize. This award is typically reserved for those who achieve profound scientific advancements.

• Early Development: Mollis developed the technology in the Bay Area while working at a company called CITUS (also referred to as CETIS).

  • The initial process involved purified DNA polymerase from bacteria and a DNA template.

  • Mollis used water baths to manually denature the template, then added enzymes and primers.

  • This early process was extremely tedious because he lacked a thermocycler (a device for automated heating and cooling) and was using a DNA polymerase that was not heat-stable. It would denature every time it was heated, necessitating the addition of fresh enzyme for every cycle.

• Impact on Science: PCR revolutionized molecular biology by allowing researchers to start with very low concentrations of a gene or DNA fragment and amplify it into billions of copies within a few hours.

  • Metaphor for Amplification: The speaker compares this to starting with $1 and returning 2 to 4 hours later with $1,000,000,000.

  • The technology bypassed the initial heavy reliance on plasmids for copying DNA in vitro.

The O. J. Simpson Case and Forensic Application

• Application in Forensics: PCR spilled over into forensic science and crime scene investigation, allowing DNA from samples to be amplified and compared against suspects.

• The O. J. Simpson Trial Connection:

  • O. J. Simpson was a famous football running back for San Francisco and the Buffalo Bills who lived in LA.

  • In the early 1990s, he became the prime suspect in a murder case involving his female partner.

  • After a high-profile white Bronco chase and his arrest, he hired a "dream team" of expensive attorneys.

  • PCR was used to match samples from the crime scene to Simpson's DNA.

  • Carey Mollis was almost brought in as a witness for the defense to testify that PCR can produce false positives.

  • Mollis was reportedly interested in participating because he found the prosecutor, Marsha Clark, attractive.

  • The defense team ultimately decided not to put Mollis on the stand. This was due to a prior conviction at Stanford, where he was caught synthesizing cocaine in an organic chemistry laboratory.

Professional Controversies and COVID-19 Perspectives

• Citus/Cetis Dispute: Mollis was going through a divorce during the development of PCR. He felt the company CITUS railroaded him, took his findings without compensation, and made billions while never even sending him a birthday card.

• Personality and Anecdotes: Mollis was known as an eccentric character. While in Europe to receive his Nobel Prize, he was nearly arrested for shining a laser pointer from his hotel room at a police officer's head.

• COVID-19 Skepticism: Before his death, Mollis released a video arguing that PCR should not be used as a diagnostic tool for being COVID positive.

  • Argument: The PCR assay detects the presence of viral DNA/RNA segments but does not determine if the virus is "alive" or if the person is infectious. A positive result can occur even if the virus is "dead."

  • The speaker notes that Mollis, as a Nobel laureate, had a significant platform ("the noble laureate tag") that challenged the synchronized global response to the pandemic.

The Mechanics and Mathematics of PCR

• Logarithmic Amplification: The formula for DNA amplification is given as $2^n$, where $n$ represents the number of cycles performed.

  • Standard Cycle Count: Most people run $30$ cycles of PCR.

  • Product Yield: Starting from a single molecule, $30$ cycles can produce billions of copies in a few hours.

• The PCR Cycle Checklist: A single cycle consists of three primary steps:

  • 1. Denaturation:

    • Performed at approximately $94^{\circ}C$.

    • Heat breaks the hydrogen bonds between complementary bases, turning double-stranded DNA into single strands.

    • DNA is very stable, but excessive heating/cooling (beyond 40 cycles) can begin to break covalent bonds.

  • 2. Annealing:

    • This is the most critical parameter where synthetic primers dock to the target sequence.

    • Melting Temperature ($T_m$): Defined as the temperature where $50\%$ of the primer is docked to the target.

    • Goldilocks Principle: If the temperature is too high, primers won't stick; if it is too low, they stick non-specifically to wrong locations.

    • GC Content: Primers with more Guanine and Cytosine have a higher $T_m$ because they form 3 hydrogen bonds compared to the 2 bonds in Adenine-Thymine pairs.

  • 3. Extension (Synthesis):

    • Performed at approximately $72^{\circ}C$.

    • DNA polymerase adds nucleotides to the primers to synthesize new strands.

Essential Components and Reagents

• Template DNA: Requires only nanogram amounts ($ng$).

• Primers: Short synthetic strands, typically $15$ to $20$ nucleotides long (can be up to $30$ to $40$).

  • Restriction Sites: Researchers can add specific restriction sites to the $5'$ end of primers. These are incorporated into the amplified product and can be used to create "sticky ends" for cloning.

• Building Blocks: Deoxynucleotide triphosphates (DNTPs).

• Thermocycler: An automated instrument that ramps temperatures up and down. High-quality machines use efficient metals for faster temperature transitions.

• DNA Polymerases:

  • TAC (Taq): Derived from Thermus aquaticus, a microbe from hot springs. It lack proofreading capabilities, making a mistake roughly every $1$ out of $2,000$ nucleotides. It also adds a single A (Adenine) overhang on the $3'$ end, preventing it from being truly blunt.

  • q five (Q5): A high-fidelity polymerase with proofreading functions that leaves blunt ends.

  • Practical Caveat: TAC is hard to kill with heat. If you use it to add restriction sites, you must perform a gel extraction before digestion; otherwise, the remaining TAC will fill the sticky ends back in during the digestion phase.

Variants: RT-PCR vs. RT-qPCR

• RT-PCR (Reverse Transcription PCR):

  • Purpose: Primarily used to clone a gene or cDNA quickly.

  • Mechanism: Involves mRNA as a starting point, which is converted to cDNA via reverse transcription (using an oligo dt primer) before being amplified by PCR.

  • Can bypass the weeks-long process of building a library; a cDNA can be obtained in a single afternoon.

• RT-qPCR (Quantitative PCR):

  • Purpose: Used for quantitation (counting) of mRNA levels rather than cloning.

  • Detection: Uses fluorescence instead of radioactivity.

  • Methods: Includes the TACman probe method and cyber green (SYBR Green).

Narrative Example: Cloning MyoD cDNA

• Target: MyoD is a master regulator transcription factor found in muscle tissue. It is powerful enough to convert fat cells into muscle by activating muscle-specific circuitry.

• Experimental Design:

  • Tissue Selection: To obtain MyoD mRNA, you must use muscle tissue (not pancreas or kidney) because it is only expressed there.

  • Process: Lyse muscle tissue → isolate mRNA → use reverse transcriptase to convert to cDNA → amplify via PCR → insert into a cloning vector.

Questions & Discussion

• Question: How many of you have heard of Carey Mullis?

• Response: Nobody in the audience responded affirmatively.

• Question: How many of you have heard of O. J. Simpson?

• Response: Most or all of the audience responded affirmatively.

• Question: Can you add restriction sites to the ends of your primers, specifically the five prime end?

• Response: Yes, this allows for the rapid incorporation of these sites for sticking into cloning vectors.

• Question: What happens if you run your annealing temperatures too high?

• Response: The primers won't stick to the target because there isn't enough stabilization from base complementarity.

• Question: What happens if you run them too low?

• Response: The primers will stick non-specifically to the wrong locations because the standards for docking are too low.