Sanger Sequencing

Question 1

Applications of DNA sequencing (4)

Answer 1

1. Detecting mutations
2. Typing microorganisms
3. ID human haplotypes*
4. Designating polymorphisms

*NOTE: A set of closely linked genetic markers or DNA variations on a chromosome that tend to be inherited together

Question 2

Describe the most common method of DNA sequencing

Answer 2

Dideoxynucloeotide (Sanger) sequencing:
- uses ddNTPs; have 3’ hydrogen groups instead of OH
- 2’3’-ddNTPs are labeled with a fluorescent dye (in 4 different colours)
- reaction involves template DNA + primers + DNA polymerase + regular dNTPs

Question 3

Describe Overall Sanger Sequencing Workflow (7)

Answer 3

1. Extract DNA
2. Amplify DNA (PCR)
3. Sample prep; removal of PCR reagent, quantitiation of DNA
4. Sanger sequencing
5. Clean-up of sequencing reaction; remove primers, dNTPs, ddNTPs
6. Capillary electrophoresis
7. Data analysis

Question 4

Sanger sequencing denatuartion

Answer 4

Apply heat = complementary DNA separate

Question 5

Sanger sequencing annealing

Answer 5

DNA primer binds beside region of interest = provides free 3’ OH

Question 6

Sanger sequencing extension

Answer 6

DNA polymerase binds and adds dNTPs, complementary of template

Question 7

Sanger sequencing termination

Answer 7

DNA polymerase randomly adds a fluorescently labeled 2’3’-ddNTP = 3’-H terminates elongation

Question 8

How are terminated sequences detected in Sanger Sequencing ?

Answer 8

Capillary electrophoresis (introduced via electrokinetic injection)
- terminated sequences flow from cathode (-) to anode (+)
- flowable polymer minimizes electro-endosmosis
- migrate by size (smaller sequences flow faster)
- fluorescent labels (2’3’-ddNTPs) are detected

Question 9

In electropherograms analysis, each coloured peak represents what ?

Answer 9

In electropherograms analysis, each coloured peak represents each letter of the sequence (a nucleotide: G/C/A/T)

Question 10

Cause of no electropherogram output

Answer 10

• insufficient template
• thermal cycle malfunction
• loss of product during cleanup
• reagent not added/ deteriorated

Question 11

Cause of messy/ poor electropherogram output

Answer 11

• failed injection = insufficient sample volume
• old buffer
• broken/ blocked capillary

Question 12

Cause of broad peaks in electropherogram output

Answer 12

• poor clean-up of sequencing reaction (primers, dNTPs, ddNTPs)

NOTE: occurs within first 120 bases

Question 13

Cause of “shoulders” on all peaks in electropherogram output

Answer 13

• capillary array needs to be replaced
• overloaded sample
• homopolymeric region (stutter)

Homopolymeric region - stretches of the same nucleotide (i.e. AAAAA or TTTTTT)

Question 14

Cause of double peaks in electrophoregram output

Answer 14

• more than one PCR product is present in the reaction
• residual PCR primers and dNTPs

NOTE: occurs at the beginning of sequence