Unit 2: Sequencing DNA

Question 1

Why do we sequence DNA (2) ?

Answer 1

• To determine whether a cloned DNA fragment or PCR product is correct
• Detect mutations

Question 2

What are the 2 methods of DNA sequencing ?

Answer 2

• ‘Sanger’ or ‘chain termination’ method
• Next generation sequencing (NGS)

Question 3

Explain the process of Sanger DNA sequencing (3)

Answer 3

1) Mix a small amount of dideoxy with deoxy nucleotides
2) Cycle through multiple rounds of degeneration, primer annealing and elongation
3) Gives multiple DNA fragments different in length by single nucleotide- all ending in ddNTP

Question 4

What is chain termination ?

Answer 4

When adding ddNTPs to DNA sequence this leads to premature stopping of DNA strand elongation due to no OH on 3’

Question 5

What is a sequencing trace ?

Answer 5

A graphical representation of raw data from Sanger sequencing

Question 6

In a sequencing trace what are the colours of the 4 bases ?

Answer 6

Thymine- Green
Adenine- Red
Guanine- Black
Cytosine- Blue

Question 7

Why would there be a double peak in a sequencing trace graph ?

Answer 7

Due to heterozygosity of mutant and wild type alleles

Question 8

What are the 2 limitations of the Sanger sequencing method for DNA diagnostics ?

Answer 8

1) Can only read 500-1000bp accurately
2) Some regions are difficult to sequence

Question 9

What are the 4 steps of NGS ?

Answer 9

1) Library preparation
2) Cluster amplification
3) Sequencing
4) Alignment and data analysis

Question 10

What happens during library preparation ?

Answer 10

• DNA is sheared or RNA is converted to cDNA
• Fragments of 200-400bp are isolated and adaptors are ligated to ends

Question 11

What happens during cluster amplification (6) ?

Answer 11

1) Single stranded fragments are hybridised to the flowcell via adaptors
2) Second strand is copied, DNA is denatured and template (first) strand is washed away
3) Strands bend over and bind to the other oligo via the second adaptor forming a bridge
4) Second strand synthesis happens
5) Process is repeated hundreds of times to create a ‘cluster’ of identical molecules
6) Each cluster arises from a single DNA molecule

Question 12

What is a flow cell ?

Answer 12

A flat slide that allows for sliding of lots of DNA

Question 13

What happens during sequencing (4) ?

Answer 13

1) Primer is added aswell as florescent dNTPs that are blocked at the 3’ end
2) Polymerase adds 1st corresponding fluorescent dNTPs
3) All clusters are read with a laser and 3’end is de blocked so dNTP can be added
4) Process repeated with fluorescent dye read at end of each cycle

Question 14

What happens during analysis (2) ?

Answer 14

1) Fluorescent colour data for each cluster is converted to a DNA sequence
2) Bioinformatics matches up overlapping sequence reads to generate an overall sequence

Question 15

What are the 3 types of NGS approaches ?

Answer 15

1) Exome sequencing
2) Transcriptome sequencing
3) Whole genome sequencing

Question 16

What is exome sequencing ?

Answer 16

All exons are sequenced to catch all the protein coding sequences

Question 17

What is transcriptome sequencing ?

Answer 17

All RNA in a cell is sequenced (mRNA, tRNA and non coding RNA)

Question 18

What is whole genome sequencing ?

Answer 18

All DNA of an individual is sequenced

Question 19

What are 5 limitations of NGS ?

Answer 19

1) NGS generates huge amounts of data
2) Short reads
3) Requires special software to interpret data correctly
4) Big variation between individuals
5) Exome sequencing only identifies variants in protein coding sequences