Lecture 11 - DNA Sequencing

Question 1

What is pyrosequencing

Answer 1

based on sequencing by synthesis principle, stepwise synthesis of DNA by the addition of dNTPs, light signal when dNTP is added

Question 2

What enzymes are present in pyrosequencing

Answer 2

DNA polymerase
ATP sulfurylase
Luciferase
Apyrase

dNTPs are added one at a time

Question 3

What primers are required for pyrosequencing

Answer 3

1. forward primer
2. sequencing primer downstream of the forward primer but upstream of the target
3. reverse primer that is biotinylated

Question 4

What are the general steps of pyrosequencing

Answer 4

1. run PCR with one of the primers biotinylated
2. immobilized biotinylated PCR products onto streptavidin coated beads
3. separate strands by denaturation in NaOH
4. Wash/neutralize the immobilized strand
5. anneal sequencing primer

Question 5

What is the pyrosequencing workflow

Answer 5

extraction
PCR
sample preparation
pyrosequencing
analysis

Question 6

What is pyrosequencing

Answer 6

a method to determine a DNA sequence without having to make a sequencing ladder, relies on the generation of light when nucleotides are added to the growing strand

Question 7

What are the steps of pyrosequencing

Answer 7

1. based on the generation of light signal through the release of pyrophosphate upon nucleotide addition
2. pyrophosphate is used to generate ATP from adenosine phosphosulfate, ATP and luciferase generate light by a conversion of luciferin to oxyluciferin which is detected by a luminometer
3. the system is regenerated with apyrase that degrades residual free dNTP and dATP, as nucleotides are added to the system one at a time the sequence is determined by which of the four nucleotides generates a light signal

Question 8

What is pyrosequencing used for

Answer 8

short-to moderate sequence analysis
commonly used for applications in infectious disease typing and HLA typing
Used for bisulfite sequencing

Question 9

What is bisulfate sequencing

Answer 9

a modification of chain termination sequencing designed to detect methylated nucleotides

Question 10

What is methylated DNA involved in

Answer 10

gene expression regulation
chromatin structure regulation
cell differentiation
implicated in a number of diseases

Question 11

What is the procedure for bisulfite sequencing

Answer 11

1. cut genomic DNA with restriction enzymes
2. run an agarose gel and fragments of interest are purified from the gel
3. DNA is denatured by heat and exposed to bisulfite solution
4. bisulfite incubation results in the deamination of cytosines converting them to uracils
5. treated template is used for PCR amplification
6. PCR amplicons are sequenced

Question 12

How is methylation detected in bisulfite sequencing

Answer 12

by comparing the treated sequence with an untreated sequence noting where the treated sequence C base pairs are not changed to T