18 Nucleic Acid Sequencing

Question 1

What is the type of sequencing called where dideoxy nucleotides are used?

Answer 1

Chain-terminator sequencing

Aka Sanger sequencing
Aka dideoxy sequencing

Question 2

What type of gel are synthesized fragments of chain-terminator sequencing resolved on?

Answer 2

Polyacrylamide gel

Question 3

What are the three steps of chain-terminator sequencing?

Answer 3

• Prepare ssDNA template
• Anneal oligonucleotide primer to template
• Perform synthesis reactions (four tubes) with a DNA polymerase enzyme in the presence of dNTPs and small amount of chain terminator (ddNTP analog)

Question 4

Each reaction tube of chain-terminator sequencing contains? (5)

Answer 4

• ssDNA template
• Primer (sometimes labeled)
• Polymerase
• All four dNTPs
• One of four ddNTPs at low concentration

Question 5

What is thermal cycling sequencing?

Answer 5

A DNA sequencing method that uses PCR to generate chain-terminated polynucleotides.

Question 6

How is thermal cycling sequencing carried out?

Answer 6

• Similar to PCR but with one primer only
• Uses dsDNA template to generate set of fluorescently labeled chain termination products (terminated by ddNTPs) to be analyzed using electrophoresis
• Very small amount of template needed (no cloning needed)

Question 7

What type of gel electrophoresis is used to separate DNA fragments from automated DNA sequencing?

Answer 7

Capillary gel electrophoresis, allows samples to be quickly run in a single lane on the gel, runs past a laser beam which causes fluorescence.

Question 8

In automated DNA sequencing, what type of chain terminated reaction is used? And how many reaction mixtures are made?

Answer 8

Thermal cycle sequencing reaction used to incorporate ddNTPs and produce labeled chain terminated products. This is all done in one mixture.

Question 9

What two factors determine the type of primer used for DNA sequencing?

Answer 9

• The type of vector

- The size of the insert

Question 10

What type of primers exist for DNA sequencing by chain termination?

Answer 10

• Universal primers (one primer for an entire DNA insert)

- Internal primers (many primers start along different areas of the length of insert)

Question 11

What are the steps of shotgun sequencing?

Answer 11

• DNA is extracted from cells
• It is sonicated to break it up into random sized fragments
• Agarose gel electrophoresed
• Purified DNA bands are isolated and chain-terminated sequenced
• The end sequences are compared by a computer to construct a sequence contig and reproduce the genome

Question 12

What is ‘sequencing by synthesis’ called? What is this method of sequencing?

Answer 12

Pyrosequencing

The order in which dNTPs are incorporated is detected and the sequence is read as the reaction takes place.

Question 13

How is pyrosequencing carried out?

Answer 13

• Primers anneal to purified template in very small reaction volume
• dNTPs are added individually in a specific order and either incorporated or degraded
• Complex multi-enzyme system detects light released when a dNTP is successfully incorporated and degrades dNTPs if incorporation is not successful

Question 14

Data from pyrosequencing is recorded on a ___

Answer 14

pyrogram