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Roche 454 Sequencing System Principles

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Questions and Answers

What ensures that each small DNA fragment fixed on the beads can be the unique template for amplification in the sequencing process?

Use of single-stranded DNA binding protein

Presence of complementary oligos on the beads (correct)

Incorporation of PCR reagents in the incubation system

Combination with magnetic beads after PCR reaction

What is the purpose of using a PTP plate with special nanopores in the pyrosequencing process?

Fix the position of each bead for convenience in sequencing (correct)

Enhance fluorescence signal detection

Accelerate the degradation of ATP by diphosphatase

Accommodate multiple beads in each nanopore

What initiates the release of a pyrophosphate group in the pyrosequencing process?

Degradation of ATP by diphosphatase

Binding of single-stranded DNAs to DNA polymerase

Combination with magnetic beads after PCR reaction

Pairing of dNTP with template DNA (correct)

How does the pyrophosphate group produced during DNA synthesis lead to fluorescence in pyrosequencing?

Binds to ATP sulfuric acid chemical enzymes Signup and view all the answers

What is the significance of using a CCD camera in the pyrosequencing process?

To record the signal of fluorescence from fluorescein molecules Signup and view all the answers

Why does 454 sequencing technology conduct each reaction in an independent nanopore on the PTP board?

To reduce mutual interference and sequencing bias Signup and view all the answers

What is the main principle of sequencing in the Roche 454 system?

Using a spray method to break DNA samples into small fragments and adding adapters at both ends Signup and view all the answers

What distinguishes the DNA library construction process of Roche 454 from Illumina?

Using a spray method to break DNA samples into small fragments and adding adapters at both ends Signup and view all the answers

What is the key technology behind emulsion PCR in the Roche 454 system?

Forming a large number of independent reaction spaces for DNA amplification Signup and view all the answers

How does Roche 454 system ensure that each DNA template is amplified in an independent reaction space?

By fixing single stranded DNAs with 28um beads buried in emulsion Signup and view all the answers

What is the role of mineral oil in the emulsion PCR process of Roche 454 system?

To form numerous small water droplets wrapped by it Signup and view all the answers

Why is the use of emulsion PCR important in the Roche 454 system?

To amplify DNA templates in independent reaction spaces efficiently Signup and view all the answers