PCR and Dideoxy Sequencing

Concept: PCR Ingredients


Concept: PCR Steps


Concept: Dideoxy Sequencing


PCR and Dideoxy Sequencing Additional Practice Problems

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

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Describe what DNA sequencing is

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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)?

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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

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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.

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

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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

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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

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

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

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

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

a. True

b. False

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PCR can be used to amplify DNA from any source.

a. True

b. False

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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.

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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

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PCR requires all of the following EXCEPT

a. primers.

b. DNA ligase.

c. DNA polymerase.

d. DNA of interest.

e. deoxyrobinucleotides.

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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

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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

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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

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