Questions and Answers
What is the difference between dNTP and ddNTP?
dNTP has an OH on 3'C of sugar; ddNTP has an H on 3'C of sugar.
What are the two ways that 'cycle sequencing' is set up differently than a regular PCR reaction?
- Only 1 primer instead of 2. 2) ddNTPs are added in addition to the dNTPs.
Why have the use of capillary electrophoresis and different color labeling of ddNTPs improved the efficiency of sequencing?
- Capillary column setup is easier than polyacrylamide gel. 2) Four fluorescent labels allow all reactions to run on one lane.
How is pyrosequencing different from chain termination sequencing?
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What was the difference between NIH's and Celera's approaches to sequencing the human genome?
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How does the HapMap project build on the Human Genome Project?
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How does the 1000 Genomes project build on the Human Genome Project?
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Study Notes
DNA Sequencing Fundamentals
- dNTP contains a hydroxyl group (OH) at the 3' carbon of the sugar, while ddNTP has a hydrogen atom (H) at the same position.
- Incorporation of ddNTP leads to chain termination because Taq DNA polymerase cannot form a phosphodiester bond without the OH group.
Cycle Sequencing Setup
- Only one primer is used in dideoxy chain termination reactions, unlike conventional PCR which requires two primers.
- ddNTPs are included in the reaction alongside dNTPs, but are added in significantly lower concentrations.
Advancements in Automated Sequencing
- Capillary electrophoresis simplifies the sequencing process compared to traditional polyacrylamide gels.
- Each ddNTP is tagged with a distinct fluorescent label, enabling all four sequences to be analyzed simultaneously in one lane, enhancing sample throughput.
Pyrosequencing vs. Chain Termination Sequencing
- Pyrosequencing represents a "next generation sequencing" method, offering up to 10,000 times more data output at a reduced cost compared to Sanger sequencing.
- Current limitations in sequencing efficiency arise from the need for advanced bioinformatics rather than technical capabilities.
Sequencing Approaches for the Human Genome
- NIH used a hierarchical shotgun sequencing method, employing systematically designed primers for organized progression through the genome.
- In contrast, Celera utilized a whole shotgun sequencing approach, randomly shearing chromosomal DNA and relying on computational analysis to reconstruct the sequence.
Contributions of the HapMap Project
- The HapMap project established a public SNP database that connects genetic variations in the human genome to specific disease associations.
Expansion of the 1000 Genomes Project
- The 1000 Genomes Project involves aligning over 1000 complete human genomes to explore genetic variability (SNPs) among individuals, providing deeper insights into human genetics.
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Description
This quiz explores key concepts in DNA sequencing, focusing on the differences between dNTPs and ddNTPs. It covers the structural implications of these nucleotides and their functions in chain termination during DNA synthesis. Perfect for students looking to solidify their understanding of molecular biology.
