Sanger Sequencing Flashcards
Applications of DNA sequencing (4)
- Detecting mutations
- Typing microorganisms
- ID human haplotypes
- Designating polymorphisms
Describe the most common method of DNA sequencing
Dideoxynucloeotide (Sanger) sequencing:
- uses 2’,3’-ddNTPs; has 3’ hydrogen groups instead of OH
- ddNTPs are labeled with a fluorescent dye (in 4 different colours)
- reaction involves template DNA + primers + DNA polymerase + regular dNTPs
Describe Overall Sanger Sequencing Workflow (7)
- Extract DNA
- Amplify DNA (PCR)
- Sample prep; removal of PCR reagent, quantitiation of DNA
- Sanger sequencing
- Clean-up of sequencing reaction; remove primers, dNTPs, ddNTPs
- Capillary electrophoresis
- Data analysis
Sanger sequencing denatuartion
Apply heat = complementary DNA separate
Sanger sequencing annealing
Primer binds beside region of interest = free 3’ OH
Sanger sequencing extension
DNA polymerase binds and adds dNTPs, complementary of template
Sanger sequencing termination
DNA polymerase adds a fluorescently labeled ddNTP = 3’ H terminates elongation
How are terminated sequences detected ?
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 are detected
In electropherograms analysis, each coloured peak represents what ?
In electropherograms analysis, each coloured peak represents each letter of the sequence
Cause of no electropherogram outputo
- insufficient template
- thermal cycle malfunction
- loss of product during cleanup
- reagent not added/ deteriorated
Cause of messy electropherogram outputo
- insufficient sample volume
- failed injection
- old buffer
- broken/ blocked capillary
Cause of broad peaks in electropherogram output
- occurs within first 120 bases
- poor clean-up of sequencing reaction
Cause of shoulders on all peaks in electropherogram output
- capillary array needs to be replaced
- overloaded sample
- homopolymeric region
Cause of double peaks in electrophoregram output
- occurs at the beginning of sequence
- seen when PCR products are present in the reaction