Nucleic acid sequencing technologies Flashcards
What is required for in vitro DNA replication?
- DNA polymerase
- dNTP
- Buffer
- Template
- Primer
Describe
The evolution of sequencing technologies
- 1st gen (Sanger)
- 2nd gen (parallel)
- 3rd gen (single molecule, real time)
- 4th gen (in situ)
What are the similarities between Sanger seq. and MG seq?
- Radioactive tracer: end-labelled 32P primer (Sanger) or template (MG)
- Polyacrylamide gel electrophoresis
- Autoradiography
Both methods were reported in 1977
What are the marks of a high-quality sequencing?
- Number of contigs = number of chromosomes
- T2T contigs
Summarize
Chemical DNA sequencing
Maxam-Gilbert sequencing
- End label template with radioactive nucleotide
- Separate the 2 strands on polyacrylamide gel
- Perform 4 sets of base-specific chemical reactions to randomly break DNA
- Separate fragments by size on acrylamide gel
- Expose to X-ray film and read sequence
Chain termination DNA sequencing
Sanger sequencing
- End label primer with radioactive nucleotide
- Perform 4 synthesis reactions, each with a different chain terminator (ddNTPs)
Describe
Fluorescence-based DNA sequencing
- A fluorophore is covalently linked to the oligonucleotide primer, a different color for each reaction
- The four reactions are pooled and electrophoresed on a single lane
- The separated bands are detected near the bottom of the gel
- Computer records sequence information
This is a half-automated version of Sanger sequencing
What are some methods to increase the throughput of Sanger sequencing?
- M13 phage cloning (= ss DNA templates)
- Modified polymerase that can incoporate fluo ddNTPs (one reaction)
- Cycle sequencing with a thermocycler (less template needed)
- Use of non-cross-linked polyacrylamide for separation by capillary electrophoresis
What are some of the objectives of genomic libraries?
- Maximizing the proportion of the entire genome present in the library
- At least 1 clone/locus
What are the defining features of second-gen sequencing?
- Massively parallel
- PCR-based
- DNA cloning not required
Describe
454 Pyrosequencing
- Ligate 5 and 3 primers to fragmented DNA
- Denature to single strands
- Immobilized onto beads
- Emulsion PCR
- Pyrosequencing and detection of chemiluminescence
Issue: error in homopolymer stretches
Describe
ABI-SOLiD technology
- Ligation of primers to fragmented DNA
- Immobilize DNA on primer coated beads
- Emulsion PCR to amplify templates
- Modify 3 end of DNA to promote binding on glass slide
- A series of ligation of fluorescently tagged dinucleotides, detection, and cleavage generates sequence information.
Signal detected post ligation, shows identity of second base of probe
Describe
Illumina technology
- Attach adapters to ends of fragmented DNA
- Attach strands to surface of flow cell
- Bridge amplification
- Add all four labelled reversible terminators, primers and DNA polymerase
- Capture image and record identity of first base of each cluster, added post bridge amplification
- Remove blocked 3 terminus and fluorophore
What are the advantages of Illumina?
- The fluorophores are cleavable
- Polymerases are engineered
- Bridge-amplification chemistry on each DNA strand to create clonal, spatially separated cluster
Describe
Ion torrent
- For each added nucleotide, a flow of H+ ions lowers the pH and allows detection
- Issue with homopolymer stretches
