DNA Sequencing

Question 1

Describe in detail the process of Dideoxy sequencing, also known as Sanger Sequencing

Answer 1

Process:
Enzymatic Sequencing reaction: DNA polymerase makes multiple copies of the DNA
Size Separation of products by capillary electrophoresis
Sequential Detection of the terminating nucleotide to identify the base

Question 2

State some similarities and differences between Sanger sequencing and PCR

Answer 2

Similarities:
- protocols cycle through repeated
temperatures
- cycling is performed a thermostable
polymerase so would be necessary and is
usually used
Differences:
- only uses a single forward primer
- amplification is limited and NOT exponential

Question 3

Describe the detail of the four stages of the reaction itself

Answer 3

Sequencing reaction
1* Strand separation
2* Annealing primer
3* Extension
4* Chain Termination

Question 4

Explain the process of size separation of the reaction products by gel electrophoresis

Answer 4

Gel electrophoresis:
* 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 molecules according to their size
* Those that are larger are retarded to a greater extent
and as a consequence move through the matrix more slowly

Question 5

Explain how base-calling by software works

Answer 5

base calling software
measures fluorescence absorbance of each base which generates trace

Question 6

What are the applications of Sanger sequencing in healthcare and research?

Answer 6

Healthcare:
- confirmatory test for specific genetic mutations in patients with suspected genetic diseases
- Identifying HIV haplotypes resistant to anti-retrovirals to determine therapy HAART
- EXCEPTION: low freq. mosaicism
Research:
- Mammalian and Pathogen Gene sequencing
- “Walking” a gene to identify a causative mutation in candidate gene studies