BISC 130 - Chapter 17: Biotechnology

Chapter 17 - Biotechnology

Molecular Cloning

• Molecular Cloning is frequently used in Molecular Biology Labs to get large quantities of pure Protein.

• Example: Alzheimer's Disease - Tau Protein is only found in the human brain.

The Process:

1. We need a Bacterium to take and hold our Gene of interest.

-However, it's likely to just break down the Gene for spare parts, so we need a way to get the Bacterium to keep the Gene intact.

1. Put the Gene of interest into a Plasmid so the Bacteria recognizes it & receives it.

   • Plasmid: A small optional circular DNA molecule. Bacteria are familiar with these things, but humans don't have these.

-Now that the Bacterium has the Plasmid with the Gene of interest, every time it multiplies, it multiplies the Gene with it.

-This way we can grow, nurture, and "harvest" these Genes of interest. This by nature produces our Proteins of interest, too.

-The Bacteria are making lots of other Proteins, so we have to isolate (Purify) our Protein of interest.

-But this yield is very low…

-So, we need to artificially amplify DNA.

Simplified Steps:

1. Get Gene for Protein of interest.

-Need high concentration of that DNA.

1. Put Gene of interest into a Vector/Plasmid.

1. Put Plasmid into Bacteria.

-Commonly, the Bacterium used is E. Coli because it's easy to keep alive

1. Let the Bacteria grow.

-They will express the Gene & make the Protein of interest.

1. Break open the Bacteria, and Purify Protein of interest from other Proteins.

******How to get high concentrations of Gene of interest?******

-Get whole Genome DNA from source organism cells.

• Problem: Low yield of DNA.

  • Just due to the nature of the cell, as only a fraction of the cell is made up of DNA.

• Solution: Process that can take DNA and copy it… DNA Replication!

  • Artificially amplify Gene of interest.

    • Similar to DNA Replication, but takes place within a test tube and only Replicates ONE Gene.

This Process is called Polymerase Chain Reaction (PCR).

-PCR has many applications beyond Molecular Cloning.

LECTURE STOPPED: 10:15 -  (5/22)

-First, what's inside the test tube? PCR Starting Reagents:

-DNA template (source DNA)

-Can be low concentration.

-Primers (Nucleotides)

-DNA Polymerase (the ONLY Enzyme present)

-dNDPs (DNA Primers)

-Complimentary to Sequences on either end of the Gene of interest.

-Buffer/Cofactors (Like Distilled Water)

STEP 1: Heat to unwind double-stranded DNA.

-DNA Polymerase needs a single-stranded template.

-Now the DNA Primers can bind to our Gene of interest at either end of the Gene…

STEP 2: Cool to allow DNA Primers to bind.

STEP 3: Heat slightly to allow DNA Polymerase to extend to the Primers, 5' --> 3', Replicating DNA.

After ONE process of STEPS 1-3, the Gene of interest* has been doubled, but because of the very small scale, the process must be repeated over and over again, dozens of times.

*All of this takes place in a Thermal Cycler Instrument which heats and cools the DNA according to the STEPS.

*But wait, there's a PROBLEM: STEP 1! When heating the DNA in STEP 1, the Protein in the tube (DNA Polymerase) is Denatured (unfolded). Because of this, it can no longer do its job - it's no longer "Active". So, "fresh" DNA Polymerase would need to be added to the tube every time the process starts over. That's way too tedious, though. So, we created a solution… stealing from Nature:

*Anything living in a part of the world that gets near-boiling temperatures must possess something that renders their Enzymes as heat-stable. So, we went to Hot Springs and stole Thermus Aquaticus and extracted its DNA…

*SOLUTION: Use DNA Polymerase from an organism that lives in very hot environments. These organisms will have evolved Enzymes that function at high temperatures. These (as stated above) are Hot Springs bacteria: Thermus Aquaticus. Thermus Aquaticus (TAQ) Polymerase is what we use.