DNA Sequencing Flashcards
First Generation Sequencing Technologies
- Maxam-Gilbert Sequencing
- Sanger chain termination method
- Automated Sanger sequencing
labeling the 5’ end of DNA fragments followed by chemical cleaving at random sites
Maxam-Gilbert Sequencing
enzymatic termination of DNA synthesis using dideoxynucleotides
Sanger method
Automated Sanger technology method
- reaction mixture (primers and DNA template, dNTPs, ddNTPs with fluorochromes, DNA pol)
- primer elongation and chain termination
- capillary gel electrophoresis
- laser detection
- computational sequence analysis
Second Generation Sequencing Technologies
- 454 (pyrosequencing)
- Illumina sequencing
- SOLiD (sequencing by oligonucleotide ligation and detection)
- Ion Torrent Technology
T or F: All NGS platforms require a library obtained either by amplification or ligation with custom linkers.
True
454 sequencing technology
it uses the sequencing-by-synthesis principle where a pyrophosphate is detected by chemiluminescence everytime a dNTP is incorporated
Illumina sequencing technology
it uses a sequencing-by-synthesis principle by measuring the fluorophores released by reversible terminators
Illumina sequencing technology method (video)
- sample prep
- cluster generation
- sequencing by synthesis
- data analysis
SOLiD
sequencing by oligonucleotide ligation and detection
DNA is sheared, and amplified, and 3’ end is modified to allow covalent binding to slide
SOLiD
ion torrent technology
everytime an ion is released upon incorporation of a nucleotide, the change in pH from the charge of the ion is detected through an ion sensor
3 advantages of NGS techs
- no electrophoresis
- no cloning
- massively parallel sequencing
applications of NGS techs (4)
- personal genomics
- gene expression
- SNP analysis
- metagenomics
Third Generation Sequencing Technologies
- PacBio Sequencing
- Oxford Nanopore Sequencing
Pacific Biosciences (PacBio) Sequencing
uses SMRT (single molecule real-time) sequencing
- thousands of zero-mode waveguides (ZMWs) in a SMRT cell
- the DNA template-DNA pol complex is immobilized at the bottom
- fluorophore-labeled nucleotide is introduced
- light emission is detected
nanopore method
- the template-DNA enzyme attaches to a nanopore
- the nanopore read base by base by measuring the electrical perturbations
Application of genome sequencing (4)
- identify the genes
- understand the evolution
- understand the function
- develop products
genome sequencing strategies
- ordered BAC clone sequencing
- shotgun sequencing
- targeted sequencing of gene-rich regions
ordered BAC clone sequencing
- the BAC clones are aligned against a genetic map to construct the physical map
- the BAC clones are sequenced individually and aligned to identify the minimum tiling path
- each BAC clone in the minimum tiling path is sequenced by shotgun method and aligned
- genomic sequence
shotgun sequencing
- the genomic DNA is divided into many fragment DNA molecules using the shotgun method
- the fragment DNA molecules are incorporated into universal cloning vectors to form the small insert libraries
- the insert libraries are sequenced and aligned to generate the genomic sequence
The Human Genome: Public effort
- used Ordered BAC clone sequencing
- headed by Francis Collins
- published in Nature (2001)
The Human Genome: Private effort
- used shotgun sequencing
- headed by Craig Venter
- published in Science (2001)
possible approaches for targeting gene-rich regions
- based on methylation
- based on Cot curve
- based on euchromatin regions
