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14 Cards in this Set
- Front
- Back
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What is DNA sequencing? |
process of determining the precise order of nucleotides within a DNA mol. |
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What does DNA sequencing include? |
Method/technology that determine the order of 4 bases |
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What is the principle of sanger sequencing? |
Chain termination method Terminating DNA synthesis one nucleotide at a time |
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What are the components of sanger sequencing? |
DNA primers DNA pol. dNTPs ddNTPs |
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What is ddNTPs? |
Chain terminating nucleotides Lack 3'-OH group Required for the formation of phosphodiester bond during DNA strand elongation Can be radioactively labeled |
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What are the challenges of sanger sequencing? |
Detection of DNA differing in length by one nucleotide Distinguishing nucleotides |
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What is dye terminator sequencing |
Sequencing that is sanger-based, non-radioactive and more automated |
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What does dye terminator sequencing based on? |
ddNTPs is labeled with different dye which permits sequencing in a single reaction DNA detection via optical system |
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What is the major advantage of automated sequencing? |
greater efficiency and speed |
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What are the issues of dye terminator sequencing? |
Poor quality in the first 15-40 bases of sequence Deteriorating quality seq. traces after 700-900 bases resulting seq. may contain parts of cloning vector if DNA fragments are cloned before seq. Current methods can directly seq. only relatively short DNA fragments in a single rxn Main obstacle to sequencing DNA fragments above size limit is the insufficient power of separation |
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What is pyrosequencing? |
Sequencing that relies on the detection of pyrophosphate release on nucleotide incorporation |
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What does pyrosequencing based on? |
Pyrophosphate which is released during addition of nucleotide to growing chain of DNA |
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What happens in pyrosequencing? |
Pyrophosphate released can be used by ATP sulfurylase to generate ATP ATP can be used by enzyme luciferase to produce light |
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How does sanger and pyrosequencing compare to each other? |
Sanger Accurate and reproducible Expensive, time-consuming, and labor-intensive Requires capillaries/sequencing gels Method capable of producing long sequencing runs (200-800 bp) Pyrosequencing High-throughput Accurate and reproducible Almost fully automated Less expensive, time-consuming and labor- intensive Easier to perform Only short sequence runs (~100 bp) Capable of sequencing roughly 100 megabases of raw DNA per 7 hour run |
