Lecture 5

Question 1

What are the steps of dideoxy or chain termination (Sanger method)

Answer 1

• Deoxyribonucleotide primer
• ssDNA template
• DNA polymerase
• Dideoxynucleotides= ddNTPS
  These can be incorporated into a growing DNA chain by DNA polymerase. The chain does not contain a 3’ OH group so no nucleotide can be added

Question 2

What is cycle sequencing

Answer 2

Adaptation of PCR using heat stable DNA polymerase, a single primer and differentially fluorescently labelled ddNTPs

Question 3

What are the general principles of NGS

Answer 3

• No cloning step required
• Short synthetic DNA adaptors are ligated
• Adaptors are specific to each platform/machine and allow individual fragments to be captured on a solid surface by hydrogen bonding.
• Unique clusters created.
• Amplification required

Question 4

What are the steps of Illumina sequencing

Answer 4

1) Fragment genomic DNA
2) Trim fragment ends to make them blunt
3) Phosphorylate 5’ nucleotide
4) Add adenine 3’ overhang
5) Ligation to adaptors

When termination occurs a flash of fluorescence shows, showing the base sequence

Question 5

List the sequencing methods relative to bp size

Answer 5

Biggest
1) Sanger (300-1200bp)
2) 454 (400bp)
3) Ion Torrent (100bp)
4) Illumina and SOLiD (75bp)
5) Helicos (30-35bp)
Smallest

Question 6

What are applications of NGS

Answer 6

• Re-sequencing for mutation
• Metagenomic sequencing (entire genomes from mixed populations)
• Transcriptome sequencing

Question 7

What are the steps of Nanopore sequencing

Answer 7

1) Protein and accessory protein attached to membrane
2) Top protein has the duty to unwind DNA and second protein creates a pore in the membrane to hold adapter molecules.
3) Flow pore creates a current. Each base blocks the flow to a different gene which alters the current