C785 Genetic Testing

8. Assuming there is one copy of the target DNA sequence before PCR, how many copies of DNA are there after 5 PCR cycles?

32 Each cycles double the numbers of DNA copy. Round 1: 1->2. Round 2: 2 ->4. Round 3: 4 -> 8. Round 4: 8 ->16. Round 5: 16 ->32.

14. DNA polymerase can synthesize new DNA strands in which direction?

5' to 3' DNA needs a free 3' end to bind to and initiate synthesis of a DNA. It synthesizes in a 5' to 3' direction.

9. DNA polymerase can synthesize new DNA strands in which direction?

5' to 3' DNA needs a free 3' end to bind to and initiate synthesis of a DNA. It synthesizes in a 5' to 3' direction.

16. You are designing a piece of DNA for a project and must select the appropriate nitrogenous bases. Which combination of nitrogenous bases would you find most suitable for your work?

Adenine, Guanine, Thymine, Cytosine DNA is composed of A, C, T, and G. It does not include U (uracil).

2. Which ingredients/molecules (at least four) are required to set up a polymerase chain reaction?

DNA polymerase DNA nucleotides Template DNA Primers In PCR, a DNA sample is separated into single strands and incubated with DNA polymerase, deoxynucleotides (dNTPs), and two short DNA primers whose sequences flank the DNA segment of interest and thus define the region to be amplified. Recall from the section on DNA replication that DNA polymerase needs a primer to begin DNA synthesis. This requirement means the primers will direct the DNA polymerase to only synthesize complementary strands of the target DNA.

11. Which of the following steps in PCR occurs first?

Denaturation separated the DNA strands. The DNA strands must be separated to expose the bases to which the primers will bind.

10. The following are steps involved in a polymerase chain reaction. Which is the correct order:

Denaturation, annealing, and elongation PCR uses repeated cycles of temperature to amplify particular DNA segments. In the first step, the reaction mixture is heated to separate the DNA strands (denaturation). The reaction is then cooled to allow the DNA primers, which define the sequence to be amplified, to anneal (base pair) with the template DNA. In the third step, DNA polymerase extends the DNA primers to create a copy of the target DNA sequence. Heating the reaction to stop polymerization and separate the DNA strands starts the cycle over again

6. Which of the following changes can NOT be detected using PCR?

Epigenetic changes Epigenetic changes do not affect the sequence of the DNA. PCR is used to look at the DNA sequence.

9. DNA ligase is a protein that connects primers to the template strand during PCR.

False In DNA replication, RNA primers are used at several places along the two strands of DNA, which means the new DNA is created in fragments (known as Okazaki fragments for the person who discovered them). However, RNA is much less stable than DNA, and needs to removed to prevent degradation. As each Okazaki fragment is finished, the RNA primer is removed by RNase H and replaced with DNA by DNA polymerase. This process leaves nicks in the sugar-phosphate backbone of the DNA. DNA ligase seals the nicks to create a continuous strand of new DNA.

19. Unlike DNA replication, in PCR, Okazaki fragments are used as primers.

False Okazaki fragments are involved in DNA replication, not PCR.

1. The polymerase chain reaction is a tool used to study protein structure.

False Recall from the section on DNA replication that DNA polymerase needs a primer to begin DNA synthesis. This requirement means the primers will direct the DNA polymerase to only synthesize complementary strands of the target DNA. (Note: In DNA replication, the primers are RNA primers, while PCR generally uses DNA primers because they are more stable.)

8. Template DNA is broken down into fragments prior to being copied.

False The template DNA is never broken in in the process of PCR or DNA replication.

10. How would you design primers to amplify a mutation (indicated by the figure below) on one of your parental DNA strands? Starting with one of the primers, fill in the 3' and 5' ends, then complete the remaining DNA strands. Keep in mind the direction that DNA polymerase synthesizes new DNA strands.

From left to right, top to bottom: 3', 5', 5', 3', 3', 5', 5', 3' DNA polymerase synthesizes 5' to 3'. The 3' ends of the primers need to flank the mutation (starred).

7. Which of the following components is NOT used in PCR?

RNA polymerase PCR makes a DNA copy, so DNA polymerase is used.

2. What color is the primer in the following diagram?

Red Recall from the section on DNA replication that DNA polymerase needs a primer to begin DNA synthesis. This requirement means the primers will direct the DNA polymerase to only synthesize complementary strands of the target DNA. (Note: In DNA replication, the primers are RNA primers, while PCR generally uses DNA primers because they are more stable.)

3. What color is the primer in the following diagram?

Red Recall from the section on DNA replication that DNA polymerase needs a primer to begin DNA synthesis. This requirement means the primers will direct the DNA polymerase to only synthesize complementary strands of the target DNA. (Note: In DNA replication, the primers are RNA primers, while PCR generally uses DNA primers because they are more stable.)

Learning Objective

The student identifies the use of PCR in identifying diseases. (2 questions on the assessment)

5. DNA polymerase is used in DNA replication and in PCR.

True Both PCR and DNA replication require DNA polymerase to make new copies of DNA.

7. DNA polymerase is used in DNA replication and in PCR.

True Both PCR and DNA replication require DNA polymerase to make new copies of DNA.

4. A thermocycler is a machine used for PCR that varies the temperature of a sample.

True For PCR, in each cycle, the two strands of the duplex DNA are separated by heating, then the reaction mixture is cooled to allow the primers to anneal (or pair) to their complementary segments on the DNA. Next, the DNA polymerase directs the synthesis of the complementary strands. The use of a heat-stable DNA polymerase eliminates the need to add fresh enzyme after each round of heating (heat inactivates most enzymes). Hence, in the presence of sufficient quantities of primers and dNTPs, PCR is carried out simply by cycling through the different temperatures for strand separation, primer annealing, and DNA synthesis. The thermocycler is the machine that is used to vary the temperature of the samples.

18. A primer creates a "handle" for DNA polymerase to grab onto in order to synthesize a copy of the DNA.

True Primers provide a free 3' end ('handle") for DNA polymerase to grab on to, allowing for DNA polymerase to begin synthesis in the 5' to 3' direction.

3. dNTPs are DNA nucleotides used in PCR.

True dNTPs stands for deoxynucleotide triphosphates, which are the nucleotides used in DNA synthesis.