9.3 Sequencing DNA

Question 1

What enzyme does Sanger sequencing use to make new DNA?

Answer 1

DNA polymerase

Question 2

What are the three requirements of DNA polymerase in order for Sanger sequencing?

Answer 2

1. Template strand (a single strand of DNA to copy)
2. Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
3. RNA Primer (short single stranded DNA molecule that is complementary to the template)

Question 3

What is a good template for Sanger sequencing?

Answer 3

Recombinant plasmid

Question 4

What are the steps of Sanger sequencing?

Answer 4

WORD DOCUMENT!

Question 5

_________ is designed to be complementary to the plasmid sequence adjacent to the unknown insert sequence

Answer 5

Plasmid

Question 6

the template strand and primer interact through _____________

Answer 6

hybridization

Question 7

Sanger sequencing generates a series of single-stranded ________________

Answer 7

DNA fragments

Question 8

DNA fragments include:

Answer 8

the primer and nucleotides complementary to the unknown DNA

Question 9

What is meant by “the DNA fragments are a nested array”?

Answer 9

They each differ in length from the proceeding and succeeding fragment by one nucleotide

Question 10

Why is it that the incorporation of dideoxynucleotide (ddNTP) terminates DNA synthesis?

Answer 10

this is because they lack a 3’-OH

Question 11

Nested fragments are separated by size using _____________

Answer 11

electrophoresis

Question 12

Explain this “Nested fragments are separated by size using electrophoresis”

Answer 12

A special gel can separate DNA fragments that differ in size by only one nucleotide.
Smaller DNA fragments migrate quickly and appear at the bottom of the gel.

Question 13

Explain “Each ddNTP is labeled with a different fluorescent dye for detection of the sequence.”

Answer 13

Each lane displays the sequence obtained from a separate DNA sample and primer.
Each fragment has terminated with a specific ddNTP labelled with a specific fluorescent dye.

Question 14

define Hybridization

Answer 14

The natural tendency of complementary single-stranded molecules of DNA or RNA to base pair and form double helixes.

Question 15

“Automated DNA sequencing” What occurs after electrophoresis?

Answer 15

DNA fragments flow through a detector and the color of the fragment is digitally recorded

Question 16

Explain “DNA sequence trace and inferred DNA Computer reads of sequence from automated Sanger sequencing”

Answer 16

The computer reads of sequence complementary to the template strand
The sequence is read from the left to the right (5’-3’ synthesis from primer)
Ambiguity in the sequence is recorded as “N”.