Copying DNA: The Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) POINT > Explain why copying DNA is useful POINT > Define PCR POINT > Describe the logic of PCR POINT > Describe the process of PCR POINT > Compare and contrast PCR vs. plasmid/bacterial cloning

DNA samples of interest often contain only a small amount of DNA: Crime scene evidence (hair, blood, skin) Testing blood (presence of disease) Identifying species (or how closely related they are) from fossils

PCR is a lab technique that allows scientists to make many copies of a specific DNA sequence in very little time Discovered by Kary Mullis in 1983 (Nobel Prize ‘93)

PCR produces millions (or even billions) of copies of a target DNA sequence in a few hours PCR is an artificial form of DNA replication How? Thermocycler

Give 2 examples of when it would be helpful to make copies of a DNA sequence. Who discovered PCR? What does the abbreviation PCR stand for?

PCR produces millions (or even billions) of copies of a target DNA sequence in a few hours PCR is an artificial form of DNA replication How?

N-terminus C-terminus POINT > Describe the logic of PCR Recall what you have learned about proteins (folding, denaturation)

POINT > Describe the logic of PCR Recall what you have learned about proteins (folding, denaturation)

POINT > Describe the logic of PCR

If we heat DNA we can break hydrogen bonds and separate the two strands, the first step in normal DNA replication But DNA Polymerase is an enzymatic protein, it will denature if heated We needed a heat-stable DNA Polymerase…..

Yellowstone National Park!

POINT > Describe the logic of PCR It occurred to scientists that the organisms living in hot springs must have heat-resistant proteins and enzymes We found and isolated them

Instead of DNA helicase to separate DNA, strands, PCR uses heat Raising the temperature breaks the hydrogen bonds that hold the two DNA strands together The covalent bonds that form the sugar-phosphate backbone can withstand this heat PCR utilizes heat-stable Taq DNA Polymerase (isolated from Thermus aquaticus bacteria)

Why doesn’t normal DNA Polymerase work when it gets heated up? Where did Kary Mullis find a heat resistant DNA Polymerase?

When heated, why does DNA separate into 2 strands, but each of the separated strands is okay? a)The covalent bonds that hold the strands to each other break before the hydrogen bonds in the sugar-phosphate backbone b)The covalent bonds that hold the strands to each other break before the covalent bonds in the sugar-phosphate backbone c)The hydrogen bonds that hold the strands to each other break before the covalent bonds in the sugar-phosphate backbone d)The hydrogen bonds that hold the strands to each other break before the hydrogen bonds in the sugar-phosphate backbone

PCR requires five components: 1. A DNA sample 2. Taq DNA Polymerase 3. Lots of the four nucleotides (A, T, C, G) 4. Two “primers” (short segments of DNA acting as starting points) 5. A thermocycler

1. Denaturation DNA is heated to high temps (94-98°C) This separates DNA molecules into single strands by breaking hydrogen bonds

2. Annealing Temp is lowered (50-65°C) This allows hydrogen bonding of primers to the ends of complementary DNA sequences

3. Extension/Elongation Temp raised slightly (72-80°C) Taq DNA Polymerase adds nucleotides to complementary strands until entire sequence is copied

4. Repeat over & over Each cycle doubles the number of DNA copies. Thirty cycles (~ 2 hrs) = over 1,000,000,000 copies!

Which is not required for PCR? a) DNA ligase b) Helicase c) A plasmid d) A thermocycler e) All of the above f) a-c g) a and b

Which is the correct order? a) DNA is heated to separate strands > Taq polymerase builds new strands > primers anneal to template DNA b) Taq polymerase builds new strand > primers anneal to template DNA > DNA is heated to separate strands c) DNA is heated to separate strands > primers anneal to template DNA > Taq polymerase builds new strands d) Taq polymerase builds new strands > DNA is heated to separate strands > primers anneal to template DNA

PCR products run by gel electrophoresis Expect a single band as product (all DNA is the same)

POINT > Compare and contrast PCR vs. plasmid/bacterial cloning Both can produce millions of copies of a DNA sequence Both produce DNA sequences that can be isolated using gel electrophoresis PCR is much faster, but primers must be built first Plasmid cloning allows for gene expression into protein

What is an advantage of using PCR compared to using bacterial plasmid cloning of DNA? What is a advantage that bacterial plasmid cloning has over PCR?

Homework: Finish Reading 15.2 pages Assess #1-3 page 427 Workbook pages 246,