PCR and Dideoxy Sequencing

Concept: PCR Ingredients

6m

Concept: PCR Steps

6m

Concept: Dideoxy Sequencing

7m

PCR and Dideoxy Sequencing Additional Practice Problems

Why is an enzyme from a thermophilic bacterium used in PCR?

A. DNA is replicated at a high temperature that denatures most proteins.

B. The enzyme makes DNA that is more similar to human DNA.

C. It is cheaper to obtain from live microorganisms than producing the enzyme in a lab.

D. This thermophile's enzyme will synthesize DNA.

Watch Solution

Which of the following is not required for PCR?

A. DNA polymerase

B. dNTPS

C. Helicase

D. Primers

E. None of the above (all are required)

Watch Solution

You are interested in the sequence of a 10-base segment f a gene. Using the chain-terminator procedure, you obtain the results shown below after gel electrophoresis.

A. What is the sequence read from the gel (5'→3')?

B. What is the sequence of the gene segments (5'→3')?

Watch Solution

What is thesequence of the TEMPLATE strand synthesized using Sanger sequencing in the figure shown below? Report this sequence in the customary 5' to 3' direction.

Watch Solution

PCR is a molecular biology technique that allows us to replicate DNA in an in vitro system without the need for a 'replisome'. What conditions or elements used in PCR mimic the effects of the following parts in the replisome? Explain your answer in 1 or 2 sentences.

A. Helicase

B. Primase

C. DNA pol III

Watch Solution

In a PCR reaction, if you start with a single copy of a DNA template, how many copies will there be after:

A. 4 rounds of PCR amplification?

B. 25 rounds of PCR amplification?

Watch Solution

Complementary DNA (cDNA) is a double-stranded molecule. In the laboratory, how is cDNA generated from a eukaryotic messenger RNA (mRNA)?

A. Reverse transcriptase generates two single-stranded cDNAs from different mRNA molecules which hybridize to form double-stranded DNA.

B. Reverse transcriptase generates a single-stranded cDNA and then uses that cDNA as a template to synthesize a complementary RNA strand.

C. Reverse transcriptase generates a single-stranded cDNA and the DNA ligase synthesizes the complementary strand.

D. Reverse transcriptase generates a single-stranded cDNA and then DNA polymerase synthesizes the complementary strand.

Watch Solution

Which statement about PCR is INCORRECT?

A. is a way to duplicate DNA outside of an organism

B. involves increasing the temperature of the DNA to cause it to unwind

C. requires a restriction enzyme

D. requires DNA polymerase

E. does not use DNA helicase

Watch Solution

Why are you performing two PCR reactions on each DNA sample?

Watch Solution

Describe what DNA sequencing is

Watch Solution

A DNA fragment was sequenced using dideoxy DNA sequencing. In this procedure, ddATP was tagged with a green dye, ddCTP was tagged with a blue dye, ddGTP was tagged with a yellow dye and ddTTP was tagged with a red dye. The primer used had the sequence 5'-GACTC-3' . Given the gel electrophoresis representation shown below, what was the sequence of the DNA fragment (template strand)?

Watch Solution

In order to determine if a mutation has occured in Gene X, DNA is sequenced using the Sanger method. Briefly explain how this method works and then give the sequence of DNA (10 bases) that is represented by the following gel. Be sure to label the 5' and 3' end of the DNA.

Lane 1: dATP, dGTP, dCTP, dTTP, ddATP

Lane 2: dATP, dGTP, dCTP, dTTP, ddGTP

Lane 3: dATP, dGTP, dCTP, dTTP, ddTTP

Lane 4: dATP, dGTP, dCTP, dTTP, ddCTP

Watch Solution

A 13-nucleotide long DNA template was sequenced by the chain-terminator method (the Sanger method) using a 5'-TAG primer. The resulting fragments were analyzed by gel electrophoresis as shown below. In each of the four different gel lanes (or wells), a different ddNTP was used (i.e. ddATP for the 1st well, ddGTP for the 2nd, ddCTP for the 3rd, and ddTTP or the 4th).

Determine the sequence of the DNA template strand.

Watch Solution

List the three steps in a PCR, and briefly indicate what happens at each step. 

Watch Solution

Polymerase chain reaction is known for its power of amplifying a target DNA sequence at a high speed. Each cycle can double the number of DNA molecules (target sequence). Which of the following is CORRECT regarding PCR?

A. In order to make 10 copies of the DNA, you need at least 5 cycles of PCR.

B. Helicase is required in order to separate the two strands in PCR.

C. Dideoxynucleotides are used in PCR.

D. DNA primers are needed in PCR.

E. All of the above

Watch Solution

In the shotgun approach to whole-genome sequencing (shotgun sequencing), random DNA fragments of a chromosome are sequenced. The fragment sequences are then assembled into a continuous sequence that represents the DNA of the entire chromosome.

What are the steps in the shotgun approach to whole-genome sequencing?
Put the following in order from 1-5 (there will be one that is not used)

- multiple copies of the same chromosome are prepared

- the plasmids are sequenced

- 1-kb fragments are transcribed into RNA

- Chromosome copies are broken into 1-kb fragments

- A computer combines the fragment sequences

- 1 kb fragments are cloned into plasmids

Watch Solution

In Northern blotting, electrophoresis is used to resolve which biological molecules? What type of probe is used to identify the target molecule(s)?

Watch Solution

What is the important feature of DNA polymerases used for PCR that distinguishes them from all other DNA polymerases?

Watch Solution

How will larger pieces of DNA move compared to smaller pieces of DNA?

Watch Solution

During the PCR, the hydrogen bonds of the double-stranded DNA molecules are broken by the enzyme helicase.

a. True

b. False

Watch Solution

PCR can be used to amplify DNA from any source.

a. True

b. False

Watch Solution

The most likely source of the Taq polymerase used in PCR is a bacterium that lives in

a. soil.

b. arctic ice.

c. hot vents.

d. humans.

e. plants.

Watch Solution

If you start with one double-stranded DNA molecule and you perform SIX cycles of PCR, how many double-stranded copies of the DNA will you have?

a. 6

b. 8

c. 16

d. 32

e. 64

Watch Solution

PCR requires all of the following EXCEPT

a. primers.

b. DNA ligase.

c. DNA polymerase.

d. DNA of interest.

e. deoxyrobinucleotides.

Watch Solution

PCR is used to _____.

a. amplify a single gene or smaller piece of DNA

b. create DNA without introns

c. insert foreign DNA into a vector

d. All of these

Watch Solution

For the polymerase chain reaction (PCR) to work, the Taq _______ must be heat stable to avoid denaturation. 

a. DNA polymerase

b. RNA polymerase

c. Ribosome

d. Primase

e. DNA

Watch Solution

The polymerase chain reaction: 

a. Doubles a region of DNA each cycle

b. Makes a single copy of a region of DNA

c. Is used to determine the sequence of a region of DNA

d. All are correct

Watch Solution