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BIO 201 > DNA Sequencing > Flashcards

DNA Sequencing Flashcards

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1
Q

First Generation Sequencing Technologies

A
  1. Maxam-Gilbert Sequencing
  2. Sanger chain termination method
  3. Automated Sanger sequencing
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2
Q

labeling the 5’ end of DNA fragments followed by chemical cleaving at random sites

A

Maxam-Gilbert Sequencing

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3
Q

enzymatic termination of DNA synthesis using dideoxynucleotides

A

Sanger method

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4
Q

Automated Sanger technology method

A
  1. reaction mixture (primers and DNA template, dNTPs, ddNTPs with fluorochromes, DNA pol)
  2. primer elongation and chain termination
  3. capillary gel electrophoresis
  4. laser detection
  5. computational sequence analysis
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5
Q

Second Generation Sequencing Technologies

A
  1. 454 (pyrosequencing)
  2. Illumina sequencing
  3. SOLiD (sequencing by oligonucleotide ligation and detection)
  4. Ion Torrent Technology
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6
Q

T or F: All NGS platforms require a library obtained either by amplification or ligation with custom linkers.

A

True

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7
Q

454 sequencing technology

A

it uses the sequencing-by-synthesis principle where a pyrophosphate is detected by chemiluminescence everytime a dNTP is incorporated

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8
Q

Illumina sequencing technology

A

it uses a sequencing-by-synthesis principle by measuring the fluorophores released by reversible terminators

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9
Q

Illumina sequencing technology method (video)

A
  1. sample prep
  2. cluster generation
  3. sequencing by synthesis
  4. data analysis
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10
Q

SOLiD

A

sequencing by oligonucleotide ligation and detection

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11
Q

DNA is sheared, and amplified, and 3’ end is modified to allow covalent binding to slide

A

SOLiD

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12
Q

ion torrent technology

A

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

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13
Q

3 advantages of NGS techs

A
  1. no electrophoresis
  2. no cloning
  3. massively parallel sequencing
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14
Q

applications of NGS techs (4)

A
  1. personal genomics
  2. gene expression
  3. SNP analysis
  4. metagenomics
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15
Q

Third Generation Sequencing Technologies

A
  1. PacBio Sequencing
  2. Oxford Nanopore Sequencing
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16
Q

Pacific Biosciences (PacBio) Sequencing

A

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

17
Q

nanopore method

A
  • the template-DNA enzyme attaches to a nanopore
  • the nanopore read base by base by measuring the electrical perturbations
18
Q

Application of genome sequencing (4)

A
  • identify the genes
  • understand the evolution
  • understand the function
  • develop products
19
Q

genome sequencing strategies

A
  1. ordered BAC clone sequencing
  2. shotgun sequencing
  3. targeted sequencing of gene-rich regions
20
Q

ordered BAC clone sequencing

A
  • 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
21
Q

shotgun sequencing

A
  • 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
22
Q

The Human Genome: Public effort

A
  • used Ordered BAC clone sequencing
  • headed by Francis Collins
  • published in Nature (2001)
23
Q

The Human Genome: Private effort

A
  • used shotgun sequencing
  • headed by Craig Venter
  • published in Science (2001)
24
Q

possible approaches for targeting gene-rich regions

A
  1. based on methylation
  2. based on Cot curve
  3. based on euchromatin regions

BIO 201 (10 decks)

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