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Questions and Answers
What is the read length for Sanger dideoxy chain terminator sequencing?
Which DNA sequencing method is known for its reversible terminator chemistry?
In nanopore DNA sequencing, what is one of the main disadvantages that can be compensated for by sequencing the same DNA sequence multiple times?
Which DNA sequencing technique is associated with the Human Genome Project?
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Which of the following methods is NOT considered a form of next-generation sequencing?
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What feature distinguishes massively parallel sequencing from single molecule sequencing approaches?
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What is one of the main advantages of Sanger dideoxy sequencing?
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Which factor contributed significantly to the high cost of the Human Genome Project?
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What is a key disadvantage of Sanger dideoxy sequencing that led to the development of 'next-generation' methods?
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Which modern sequencing method offers a reversible chain terminator mechanism?
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In comparison to other methods, what makes Nanopore sequencing stand out?
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Which method has the highest accuracy among the three compared sequencing methods?
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What is the purpose of attaching different fluorescent colors to each type of ddNTP in DNA sequencing?
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What type of gel is usually used in automated fluorescent dideoxy sequencing to separate single-stranded DNA fragments by size?
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What allows researchers to distinguish DNA fragments that differ by only 1 base in size in gel electrophoresis during DNA sequencing?
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Why is automated fluorescent sequencing faster compared to the original method of Sanger dideoxy sequencing?
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What is a significant advantage of using automated fluorescent sequencing in comparison to Sanger dideoxy sequencing?
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Which technology was a significant improvement over the original Sanger dideoxy sequencing method, allowing for a higher throughput of sequencing samples?
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Study Notes
Sanger Dideoxy Sequencing
- Very accurate, with an accuracy of 99.99%
- Relatively long sequencing reads, up to nearly 1,000b, although 650b is more common
- Easy to do and can be automated
- Low cost for small numbers of samples
- Continues to be used due to its advantages
Limitations of Sanger Dideoxy Sequencing
- Too slow for many applications, such as genome sequencing
- Costly when scaled up to acquire large amounts of data
- Requires purification and preparation of each individual DNA sequence being studied
Human Genome Project
- Cost around $3 billion (U.S.)
- Sequencing the first human genome was incredibly expensive due to the limitations of Sanger dideoxy sequencing
- A human genome sequence now costs around $100,000
Comparison of Sequencing Methods
- Sanger: non-massively parallel, sequencing by synthesis, no single molecule, chain terminator, 99.99% accuracy, 650-1,000b read length
- Illumina: massively parallel, sequencing by synthesis, no single molecule, reversible chain terminator, up to 99.9% accuracy, 75-600b read length
- Nanopore: massively parallel, no sequencing by synthesis, single molecule, no chain terminator, up to 98-99% accuracy, up to 100 kb read length
How Sanger Dideoxy Sequencing Works
- Uses dideoxy chain terminator method to sequence DNA
- Attaches fluorescent colors to each type of ddNTP to keep track of which bases are terminating which fragments
- Uses gel electrophoresis to sort the fragments by size
- Gel electrophoresis uses denaturing polyacrylamide gel to separate single-stranded DNA fragments by size
- Automated fluorescent sequencing is usually used
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Description
Learn about the advantages and disadvantages of Sanger dideoxy sequencing. Explore why it is considered accurate, allows for relatively long sequencing reads, is easy to perform, and cost-effective for small sample sizes. Discover the drawbacks such as being slow for certain applications, costly when scaled up, and requiring individual DNA sequence preparation.
