Nucleic Acid Sequencing

Question 1

Chain Terminator Sequencing (dideoxy/Sanger sequencing)

Answer 1

1. Preparation of ss template
2. Anneal oligonucleotide primer to template
3. perform synthesis rxn w/ DNA pol enzyme in the presence of dNTPs and small amnt of chain terminator

Question 2

Chain Terminator sequencing

Reaction done in 4 separate tubes, each with (5):

Answer 2

• ss template
• primer (sometimes labeled)
• polymerase
• all 4 dntps
• one of 4 ddNTPs (low conc, not labeled)

Question 3

Automated DNA sequencing

Answer 3

• uses fluorescently labeled ddNTPs
• a PCR like rxn w/ only 1 primer is used to incorporate the dNTPs and ddNTPs and produce labeled chain terminated products
• all 4 rxns done in the same tube on a thermal cycler

Question 4

Automated DNA sequencing- Capillary gel electrophoresis

Answer 4

• used to separate DNA fragments
• sample run in a single lane on the gel, runs past a laser which causes fluorescence
• nucleotide specific fluorescence received by a detector converted into a chromatogram
• highly automated, longer read lengths

Question 5

Thermal Cycle Sequencing

Answer 5

• uses PCR to generate chain-terminated polynucleotides
• similar to PCR, except uses only 1 primer and no exponential growth
• use dsDNA as template, generates fluorescently labeled chain termination products to be analyzed using electrophoresis
• only small amnt of template needed
• use thermal stable polymerases

Question 6

Primers for sequencing cloned products

Answer 6

2 types used depends on type of vector and size of insert

1. A universal primer
2. internal primers

Question 7

“Shotgun” sequencing a genome

Answer 7

• randomly sheared DNA to make genomic library
• clones picked at random from the library and sequenced using universal primers
• individual sequence reads assembled in computer to produce contigs
• contigs put together to produce genome assembly-a computer generated representation of what the genome is actually like in the cell (errors depend on how large the genome is)

Question 8

Next-Gen sequencing-pyrosequencing (454)

Answer 8

• the order in which dNTPs are incorporated is detected, sequence read as rxn takes place
• primer annealed to purified template in small rxn volume
• dNTPs added individually in specific order and either incorporated or degraded
• complex multi-enzyme system detects light when dNTP is incorporated, degrades dNTP if no incorporation

Question 9

Pyrogram

Answer 9

-signal strength is proportional to the number of nucleotide incorporations

Question 10

Pyrosequencing and shotgun sequencing (3)

Answer 10

• using pyrosequencing a genome can be shotgun sequenced faster and cheaper than with sanger sequencing
• read lengths are 500 bp
• accuracy of base-calling lower than for automated sanger sequencing

Question 11

Next-Gen sequencing-Reversible Terminator Sequencing

Answer 11

1. A flow cell containing clusters of immobilized, randomly sheared DNA fragments is created (fragments all have the same adaptor added to their ends)
2. A primer (specific to the adaptor) is added, along w/ DNA pol, fluorescently labeled, 3’OH blocked dNTPs
3. nucleotide incorporation occurs, fluorescence detected and recorded by instrument, elongation can’t continue due to blocking groups
4. blocking groups and fluorescent labels are removed, flow cell washed, fresh pol and blocked dNTPs added
5. repeat 50-300x