1. Introduction: Gene Cloning Before PCR.

In a previous tutorial, we explored how plasmids (letter “c” below) can be used to copy a gene. Making identical copies, in this context, is known as cloning. In the diagram below, the gene of interest (f) is inserted into a plasmid, creating recombinant DNA. This engineered plasmid is then inserted into a bacterial cell (h). As the cell reproduces (i), it also replicates the recombinant plasmid, cloning the gene.

Figure 1: Gene Cloning Using Plasmids

In 1983, Kary Mullis developed a technique for in vitro cloning of genes (or any other small section of DNA). In vitro means “in glass,” but in this context it just means “outside of cells.” The technique is called the Polymerase Chain Reaction, or PCR. and Mullis was awarded the Nobel Prize for this work in 1993. The technique is widely used, both in forensics (amplifying DNA from a crime scene for analysis), and in medical/biological research.

2. How PCR Works

Figure 2: PCR (adapted from Wikipedia)

In the diagram above, letter “a” represents the gene that you’re interested in cloning. Along with the gene, you’ll need

  • primers: short single stranded segments of DNA that bind with known sequences at the start of the DNA you want to clone. These are shown at “c.” We’ll need primers that bind to the 3′ end of both strands of our target DNA.
  • Nucleotide triphosphates (shown at “b”). These are the As, Ts, Cs, and Gs that make up DNA.

    Figure 3: Nucleotide triphosphate
  • Heat resistant DNA Polymerase (not shown). DNA polymerase is the enzyme that replicates DNA by adding new nucleotides to the 3′ end of a growing strand. Because PCR involves repeated cycles of heating and cooling, we’ll need DNA polymerase that won’t become denatured at high temperatures. Nature has provided this in the form of Taq polymerase. This is DNA polymerase derived from Thermus aquaticus , a bacterium that lives in hot springs and hydrothermal vents.

Once we’ve assembled our ingredients, we can begin PCR. There are three steps.

  1. Denaturation: Heat the DNA to 94°C to 96° C. This breaks the hydrogen bonds holding the strands together, rendering the DNA into two single strands (shown at “d.”)
  2. Annealing: Let the mixture cool to about 68°C. This allows the primers to bind with the single strands of DNA (shown at “e.”)
  3. Elongation: Once the primer is in place, DNA polymerase will elongate each strand, adding new nucleotides that are complementary to the DNA templates. This is shown at “f.”

Each cycle doubles the amount of DNA. Repeat this a dozen times, and you’ll have 212 more DNA than you started with. That’s a 4096-fold increase. If you repeat 30 times, you’ll have amplified the DNA by 230, which is over a billion-fold.

3. PCR: Checking Understanding

Complete the following quiz.

[qwiz random = “true” style=”width: 600px !important;” qrecord_id=”sciencemusicvideosMeister1961-PCR Quiz (M17)”] [h]

PCR Quiz

[i]

[q] In the diagram below, which number or letter indicates the target gene that’s about to be amplified?

[textentry single_char=”true”]

[c*] a

[f] Excellent! Letter “a” is the target DNA.

[c] *

[f] No. Just identify what’s being copied, and you’ll have your answer.

[c] Enter letter

[f] No, that’s not correct.

[q] In the diagram below, which number or letter indicates free nucleotides?

[textentry single_char=”true”]

[c*] b

[f] Excellent! Letter “b” is a free nucleotide. 

[c] *

[f] No. Look for the monomers, or units, that are going to be used to build new DNA.

[q] In the diagram below, which number or letter indicates the primer before it binds with the template DNA?

[textentry single_char=”true”]

[c*] c

[f] Correct! Letter “c” represents an unbound primer.

[c] *

[f] No. Look for a short string of nucleotides that will bind at the start of the target DNA.

[q] In the diagram below, which number or letter indicates the DNA after it’s been denatured?

[textentry single_char=”true”]

[c*] d

[f] Correct! Letter “d” represents denatured DNA (now two single strands). 

[c] *

[f] No. Denaturation breaks the hydrogen bonds that hold the double helix together. Once those bonds are broken, what will the DNA look like?

[q] In the diagram below, which letter shows a primer binding with the template DNA?

[textentry single_char=”true”]

[c*] e

[f] Correct! Letter “e” shows a primer binding with the template DNA.

[c] *

[f] No. Look for a small sequence of nucleotides that’s complementary with nucleotides at the 3′ end of the template strand.

[q] In the diagram below, which number or letter indicates newly synthesized DNA?

[textentry single_char=”true”]

[c*] f

[f] Correct! “F” shows newly synthesized DNA.

[c] *

[f] No. Look for assembly of the individual nucleotides (shown at “b”) into a connected strand.

[q multiple_choice=”true”] In the diagram below, phase 1 is

[c*] denaturation

[f] Excellent! Denaturation breaks the hydrogen bonds holding DNA together, breaking the DNA into two single strands.

[c] annealing

[f] No. “Annealing” means sticking to.” In phase 1, nothing’s sticking. Rather bonds are being broken.

[c] elongation

[f] No. Elongation is what’s happening in step 3.

[q multiple_choice=”true”] In the diagram below, phase 2 is

[c] denaturation

[f] No. Denaturation breaks the hydrogen bonds holding DNA together, breaking the DNA into two single strands. What you see in phase two are two things sticking together.

[c*] annealing

[f] Excellent. “Annealing” means sticking to.” In phase 2, the primer is sticking to the template strands.

[c] elongation

[f] No. Elongation is what’s happening in step 3.

[q multiple_choice=”true”] In the diagram below, phase 3 is

[c] denaturation

[f] No. Denaturation breaks the hydrogen bonds holding DNA together, breaking the DNA into two single strands. What you see in phase three involves DNA polymerase adding new nucleotides the DNA molecule.

[c] annealing

[f] No. “Annealing” means sticking to,” and it refers to the primer sticking to the template strand.

[c*] elongation

[f] Way to Go! Elongation is what’s happening in step 3.

[q] In the diagram below, which letter indicates annealing?

[textentry single_char=”true”]

[c*] c

[f]Yes! Letter “c” represents annealing.

[c] Enter word

[f] No.

[c] *

[f] No. “Annealing” means “sticking to.” Find where the primer is sticking to the DNA template.

[q] In the diagram below, which letter indicates elongation?

[textentry single_char=”true”]

[c*] d

[f]Yes! Letter “d” represents elongation.

[c] Enter word

[f] No, that’s not correct.

[c] *

[f] No. Find where DNA polymerase is creating an increasingly long stand of complementary DNA.

[q] In the diagram below, which number is pointing to a primer?

[textentry single_char=”true”]

[c*] 2

[f]Nice job. “2” represents a primer.

[c] Enter word

[f] Sorry, that’s not correct.

[c] *

[f] No. The primer is a short sequence of single stranded DNA that binds with the 3′ end of the template DNA.

[q] In the diagram below, which number is pointing to free nucleotides?

[textentry single_char=”true”]

[c*] 3

[f]Yes! “3” represents free nucleotides.

[c] Enter word

[f] No.

[c] *

[f] No. The free nucleotides will be used during the elongation phase to create new strands of DNA.

[q] In the diagram below, which number is pointing to newly synthesized DNA?

[textentry single_char=”true”]

[c*] 4

[f]Excellent. “4” represents newly synthesized DNA.

[c] Enter word

[f] No.

[c] *

[f] No. The new DNA is what’s being synthesized during the elongation phase.

[q] In the diagram below, which number indicates DNA polymerase?

[textentry single_char=”true”]

[c*] 5

[f]Way to go. Number “5” shows DNA polymerase.

[c] Enter word

[f] Sorry, that’s not correct.

[c] *

[f] No. The DNA polymerase is the enzyme that’s synthesizing new DNA during the elongation phase.

[q] The image below shows a closeup view of what’s happening during PCR. The primer must be at number

[textentry single_char=”true”]

[c*] 1

[f] Excellent! The primer is at “1.”

[c] Enter word

[f] No.

[c] *

[f] No. The primer is a short strand of DNA that binds with the template strand.

[q] The image below shows a closeup view of what’s happening during PCR. Newly synthesized DNA must be at number

[textentry single_char=”true”]

[c*] 3

[f] Great! Number “3” is the newly synthesized DNA.

[c] Enter word

[f] No, that’s not correct.

[c] *

[f] No. Here’s a hint: the template DNA is at “2.”

[q] The image below shows a closeup view of what’s happening during PCR. DNA polymerase must be at number

[textentry single_char=”true”]

[c*] 5

[f] Good! DNA polymerase is at number 5. 

[c] Enter word

[f] No.

[c] *

[f] No. DNA polymerase is the enzyme that’s connecting free nucleotides to create new DNA.

[q] PCR stands for[hangman] chain[hangman]

[c] polymerase

[f] Great!

[c] reaction

[f] Correct!

[q] PCR stands for the polymerase[hangman] reaction

[c] chain

[f] Great!

[q] The image below shows a closeup view of what’s happening during PCR. Free nucleotide triphosphates are shown at
[textentry single_char=”true”]

[c*] 4

[f] Great! Free nucleotides are shown at “4.”

[c] Enter word

[f] No, that’s not correct.

[c] *

[f] No. Here’s a hint: free nucleotides are going to be incorporated into the newly synthesized strand, which is at “3.” What could be the monomers that get incorporated into that strand?

[q] During PCR, the highest heat is applied during the[hangman]phase

[c] denaturation

[q] During PCR, primers bind to DNA during the [hangman] phase.

[c] annealing

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