Dna Sequencing Flashcards
What is Dideoxy chain termination also known as?
Sanger Sequencing
What is the history about this technique?
- Developed to sequence DNA in 1977
- Technology has since been improved, modified and semi automated
- Very robust with low error rate therefore highly reliable
What is the automated DNA sequencing by ABI 3730?
- Samples are prepared by dideoxy chain termination on large scale by robotics
- Has a read length of upto 900 bp & 99.95% accuracy
- Handles 48 or 96 samples simultaneously
- > 1000 samples per day
- But only performs separation of labelled DNA & determines the sequence
Briefly describe the Automated DNA sequencing by ABI 3730
- Technique was used to sequence the human genome
- Produced 23 thousand million bases of sequencing
- But took 13 years & $2.7 billion to complete
How does Dideoxy chain termination work in steps?
Template -> Enzymatic sequencing reaction -> Size separation of products by capillary electrophoresis-> Detection of reaction products -> readout of sequence
What happens in the enzymatic sequencing reaction phase?
DNA polymerase makes multiple copies of the DNA
How are the products sorted in the “Size separation of products by capillary electrophoresis” phase?
Sorted by size
What happens in the “Detection of reaction products phase”?
Sequential detection of the terminating nucleotide to identify the base
What happens in the “Readout of sequence” phase?
Re-Constructing the sequence
Name one way in which Sanger sequencing is similar to PCR
- Some protocols cycle through repeated temperatures BUT only uses a single forward primer
- Application is limited and Not exponential
Name another way in which Sanger Sequencing is similar to PCR
Also uses DNA polymerase
- If cycling is performed, a thermostable polymerase would be necessary and is usually used
PCR: What are the 4 stages also known as?
- Sequencing reaction
- Strand Separation
- Annealing primer
- Extension
- Chain termination
What happens in the first 3 stages?
Sequencing using DNA polymerase
- DNA is mixed with the reaction components including both Dideoxy and deoxy nucleotides
1. A single stranded oligonucleotide (primer) is bound to the template
2. The polymerase recognises the DNA structure, then forms an initiation complex
What happens in the “Extension” stage?
DNA dependent DNA polymerase
Which requires:
1. A template that extends beyond a primer
2. Free 3’ OH group on the primer
3. All 4 deoxy nucleotide triphosphates
4. MG2+ ions
What is required in the “Chain termination” stage?
All 4 Dideoxynucleotide triphosphates are required
- In this phase DNA elongation is terminated by the addition of Dideoxynucleotide as Dideoxynucleotide prevents elongation.
What happens in the “Chain termination” phase?
Sequencing using DNA polymerase
- DNA is mixed with the reaction components including both Dideoxy and deoxy-nucleotides
1. The polymerase commences elongation from the 3’ terminus
2. As the enzyme encounters a particular nucleotide in the sequence it picks out and incorporates a complementary nucleotide into the elongating strand
- If a Dideoxy nucleotide is incorporated into the strand, then elongation is terminated otherwise elongation continues
What does the reaction products represent after the reaction?
- Products where a ddCTP is incorporated therefore represent all positions within the sequence where a “Cytosine” occurs
- Since all four labelled Dideoxynucleotides are present in the reaction, the population of molecules represent all the possible positions in the sequence from the same point to the end
What does ordering the molecules by size allow us to determine?
Determine the sequence of the new strand
How is this size separation carried out?
By Gel Electrophoresis
How does this process work?
- The nucleic acid passes through a gel matrix by applying a voltage across two electrodes
- Negatively charged nucleic acid migrates towards the positive electrode
- The matrix retards the molecule according to their size
- Those that are larger are retarded to a greater extent and as a consequence move through the matrix more a slowly
How are the sequences determined?
By the direct comparison of the lengths of products terminated by each of the four Dideoxy nucleotides
What are the uses of DNA sequencing by Dideoxynucleotide termination in today’s society?
Used in
- Health
- Silent, Misense and mis splicing
- Used to confirm all types of mutations
- Identifying HIV haplotypes resistant to anti retrovirals to determine therapy HAART
- Mammalian and pathogen gene sequencing
- Cloning
