DNA Sequencing

A Template

A single stranded piece of DNA, that will be used to to replicate. It also allows us to inferr the complimentary strand.

A primer

Short piece of DNA that bind to the template strand to start the DNA synthesis process.

Can a primer start on it’s own

No it can not it needs a pre - existing strand of 3’ OH end to add nucleotides.

dNTP’s

Deoxyribonucleotide triphosphates, the building blocks of DNA that provide the necessary nucleotides for DNA synthesis.

DNA polymerase

An enzyme that synthesizes new DNA strands by adding nucleotides to a pre-existing strand during DNA replication.

ddNTP’s

Dideoxyribonucleotide triphosphates, a modified form of nucleotide. The lack a 3’OH so no more nucleotides can be added.

Why is only 1 ddNTP used in each tube / reaction

If we use all 4 ddNTP it would be hard to tell which base cause the termination.

How did we use the dice in the lab and why did we use it in the way that we did.

the ddNTP are more rare so the dice represented the chance/ probability of rolling a ddNTP.

Why is sequencing DNA harder than sequencing proteins

They are 3 times longer.

In “classic” di-deoxy sequencing, how are the products visualized

Radioactive phosphate.

what are the advantages of using fluorescent dyes in DNA sequencing

No radiation involved and you only need to run reaction.

point mutation

one band is different at a specific position

Insertion

A extra band

Deletion

missing a band

what would happen if you add an extra ddNTP

their would be two bases showing up at the same position and on top of there single placements.

A ddNTP was omitted from the tube

it go all the way to the top there would be no stops.