9. DNA SEQUENCING Flashcards
What is Dideoxy chain termination?
- Dideoxy chain termination is also known as Sanger sequencing
- It is a robust method with a low error rate so is considered to be the gold standard method for DNA analysis
- Developed by Fred Sanger in 1977
What are the similarities & differences between PCR & dideoxy chain termination?
- Both PCR & Dideoxy chain termination use DNA dependent DNA polymerase
- However, PCR uses TWO primers for exponential amplification
- But dideoxy chain termination only uses ONE primer. The amplification is limited & isn’t exponential, as the products produced don’t act as a template for the subsequent reaction
What are the 4 parts to the reaction for Dideoxy chain termination?
- DENATURATION/ Strand separation
- ANNEALING
- ELONGATION/ Extension
- TERMINATION
**Briefly describe the 5 steps of dideoxy chain termination?
- A template for the DNA sequence is made using PCR
- Dideoxy chain termination using DNA polymerase makes multiple copies of DNA
- Size separation of the molecules produced by capillary electrophoresis
- Sequential determination of the terminal nucleotide by using a fluorescence scanner
- Use the read out to reconstruct the sequence
Describe the process of strand separation & annealing in dideoxy chain termination?
- The strands are separated by denaturation
A single stranded primer anneals to the template - Annealing is favoured over renaturation in the competition due to the excess of primers
*What happens in the elongation phase of Dideoxy chain termination?
- Elongation is carried out by DNA dependent DNA polymerase which recognises primers, forms an initiation complex & adds nucleotides to the 3’ end.
- An ester bond is formed when the 3’OH of the base bonds with the phosphate of the deoxynucleotide triphosphate, presented by the polymerase
- The formation of the ester bond releases inorganic pyrophosphate & H+
*What determines whether the DNA dependent polymerase keeps elongating in Sanger sequencing?
- The DNA dependent DNA polymerase will keep going until:
1. It runs out of template
2. The reaction becomes rate inhibited due to acidification
3. Depletion of nucleotides
*What are the 4 requirements for dideoxy chain termination?
- A template
- All 4 deoxy nucleotide triphosphates
- All 4 dideoxy nucleotide triphosphates
- Free 3’OH
What’s the difference between deoxy nucleotide triphosphate & dideoxy nucleotide triphosphate?
- Deoxy nucleotide triphosphate (dNTP) = missing one OH
- Dideoxy nucleotide triphosphate (ddNTP) = missing two OH ( 2’ & 3’)
How does adding a ddNTP cause chain termination?
- ddNTP is missing two OHs, the most important being 3’OH, without a free 3’OH elongation cannot occur. So, if ddNTP is incorporated, elongation will be terminated
- DNA dependent DNA polymerase cannot distinguish between dNTP & ddNTP
What is the probability of ddNTP incorporation dependent on?
- As DNA dependent DNA [polymerase cannot distinguish between ddNTP & dNTP, the probability of incorporation id down to chance aswell as the proportion of ddNTp relative to dNTP
- Too much ddNTP = the sequence is terminated too frequently & no product will be produced
- Too little ddNTP = sequence is terminated too infrequently & the product will be incomplete
Why do the dideoxy chain termination reaction products vary in length?
- The molecules/products produced vary in size & length depending on where the ddNTP is incorporated
- If the ddNTP is incorporated closer to the 5’ end near the primer, it will be shorter
- If the ddNTP is incorporated closer to the 3’ end, the sequence will be longer
How can we use CE to order the reaction products of dideoxy chain termination by size?
- The products of dideoxy chain termination can be ordered by size with capillary electrophoresis
- A voltage can be applied across the gel layer causing DNA to migrate towards the positive electrode
- Smaller/shorter sequences will reach the end of the capillary first
- Larger sequnces will move more slowly through the matrix
- The molecules will reach the end of the capillary from shortest to longest (5’ to 3”),
- Sequential determination of the terminal nucleotide with a fluorescence scanner can allow us to determine all the positions of nucleotides in the sequence
- A fluorescence scanner will generate a trace known as a base call
How will size affect the migration of the products of dideoxy chain termination in CE?
- Smaller molecules will have a ddNTP incorporated closer to the 5’ end, so they will reach the end of the capillary first
- Larger molecules will have a ddNTp incorporated closer to 3’ end, so they will move the matrix slower & will take longer to reach the end of the capillary
What are the application of Sanger sequencing in health?
- Sanger sequencing is used to conform/detect all mutations including synonymous, non-synonymous & nonsense
- Identify HIV haplotypes which are resistant to antivirals to determine new therapy
