DNA Sequencing

Question 1

Chain Termination Sequencing

Answer 1

Method of sequencing DNA by using dideoxynucleotides to terminate synthesis of DNA chains.

Question 2

What does chain termination sequencing require?

Answer 2

1) a primer
2) DNA polymerase
3) a single-stranded DNA template
4) Deoxynucleotides
5) Dideoxynucleotides

Question 3

Automatic DNA Sequencers

Answer 3

Machine that separates a dye-labeled dideoxy sequencing reaction into increasing larger fragments, analyzes the final nucleotide of each fragment by recording the color, and prints the results in a graphical format to determine the sequence

Question 4

What is the first important thing that needs to happen with chain termination sequencing?

Answer 4

Stop the synthesis of the newly synthesized DNA chains at each base pair
(each fragment of DNA will differ by one base pair)

Question 5

What are the advantages of cycle sequencing?

Answer 5

1) destruction of secondary structures/double-stranded regions because of 95 degree temp.
2) control of primer hybridization
3) requires very little template DNA

Question 6

How are these fragments then separated?

Answer 6

Using polyacrylamide gel electrophoresis

Question 7

What are the steps of the thermocycling process?

Answer 7

1) template DNA is denatured at a high temp
2) temp is lowered to anneal primer
3) temperature is raised to 72 C
4) polymerization

Question 8

How do you read the sequence once the fragments have been separated?

Answer 8

From the bottom of the gel to the top
(the shortest fragment traveled the farthest)

Question 9

Polymerization step of thermocycling

Answer 9

dideoxynucleotides are incorporated to cause chain termination. fragments are made that stop at each base pair, and four different colors will be able to be seen based off of the fluorescent labeling.

Question 10

cycle sequencing

Answer 10

Technique that combines PCR and chain termination sequencing to determine the sequence of a template DNA

Question 11

Dideoxynucleotides

Answer 11

Nucleotides with no 3’- hydroxyl group
(therefore no additional nucleotide can be added and sequencing is terminated)

Question 12

what are the components of cycle sequencing?

Answer 12

1) all four deoxynucleotides
2) all four dideoxynucleotides
3) a single primer
4) template DNA
5) Taq polymerase

Question 13

how is the identity of a dideoxynucleotide determined?

Answer 13

each dideoxynucleotide is linked to a unique fluorophore for each of the four nucleotides