Sanger Sequencing Notes

These flashcards cover key concepts and methods of DNA sequencing, including techniques, components, and their significance.

What is the purpose of DNA sequencing?

To determine the order of nucleotides in a piece of DNA.

What is Sanger sequencing?

A method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

What are the key components required for chain termination sequencing?

1. ssDNA template, 2. DNA polymerase, 3. Primer, 4. dNTPs and ddNTPs (around 100:1 ratio), 5. Label for detection, 6. High resolution separation of reaction products.

What is the significance of cycle sequencing?

It utilizes a thermostable DNA polymerase and ddNTPs in a cycling format to generate chain-terminated sequences with less required template DNA.

What does automated DNA sequencing involve?

Labeling each set of ddNTPs with distinct fluorescent dyes, allowing all four sequencing reactions to be performed in one tube.

What are some advantages of capillary electrophoresis for DNA sequencing?

1. Short run time, 2. Nanogram quantities of DNA required, 3. Easy loading, 4. Lower cost and higher reproducibility.

What is the typical output of an automated sequencing experiment?

About 500 to 700 bases of sequence.

What is the accuracy of Sanger sequencing?

99.99% accuracy, making it the gold standard for biomedical research sequencing.

What is primer walking in DNA sequencing?

A method of designing a primer when the initial sequence is unknown, relying on plasmid-encoded primer binding sites.

Why is it important to sequence PCR clones?

To confirm the presence and integrity of plasmid inserts and to reveal any mutations that may have been introduced.