W14 L2 - DNA Analysis II

Flashcards about DNA analysis and sequencing techniques discussed in the lecture.

What is the purpose of using agarose gels?

A method used to estimate the size of unknown DNA fragments using size markers and agarose gels.

How is a standard curve linearized for DNA fragment size estimation?

By plotting the logarithm of the size in base pairs against the migration distance.

What causes DNA to absorb light at a particular wavelength?

Aromatic bases (purines and pyrimidines) in the DNA.

At what wavelength does pure DNA have a peak absorbance?

Approximately 260 nanometers.

What does an absorbance of one at 260 nm indicate about DNA concentration?

An absorbance of one at 260 nanometers indicates a concentration of roughly 50 micrograms per milliliter of double-stranded DNA.

Why is absorbance also measured at 280 nanometers?

To check the purity of the DNA sample; a ratio around 1.8 (or between 1.7 and 1.9) indicates a fairly pure sample.

What is Sanger sequencing?

A technique developed by Frederick Sanger to determine the base sequence of DNA.

What are the initial steps in Sanger sequencing?

Heating the DNA to separate strands, adding a primer, and then adding nucleotides with a DNA polymerase.

What is the function of dideoxynucleotides (ddNTPs) in Sanger sequencing?

ddATP, ddTTP, ddCTP, and ddGTP cause strand termination because they lack a 3' OH group necessary for further extension.

How is the DNA sequence read from the colored bands (or peaks) in Sanger sequencing?

The colors indicate the sequence of the DNA fragment, with each color representing a specific base (A, T, G, or C).