RR3

Question 1

What are some uses of PCR?

Answer 1

Sequencing // DNA cloning // Pathogen detection // Gene editing

Question 2

What is required for PCR?

Answer 2

DNA template // Taq (DNA polymerase) // primers // dNTPs

Question 3

What does PCR stand for?

Answer 3

Polymerase Chain Reaction

Question 4

Describe PCR steps

Answer 4

Denature protein (heat shock) // Annealing: Temperature lowered a bit for (“artificial”) primers to latch onto ssDNA // Extension: form nascent DNA strand // Repeat over and over again // Gel electrophoresis (analysis)

Question 5

From what point on are the DNA fragments the right size in PCR?

Answer 5

Starting from the 3rd cycle, they get more and more abundant.

Question 6

What are some uses of DNA sequencing?

Answer 6

Ancestry and phylogenetic relations // Catching criminals // Diagnosis

Question 7

What is required for DNA sequencing?

Answer 7

DNA polymerase // Primer // DNA template // dNTPs // ddNTPs

Question 8

What’s the point of using ddNTPs in DNA sequencing?

Answer 8

They’re chain terminators. They lack a second OH group which prevents them from attacking a phosphate group: stops chain growth.

Question 9

What happens after all “ingredients” have been incubated together in DNA sequencing?

Answer 9

You end up with several daughter cells of varying lengths. You can denature and separate them through SDS-PAGE: allows to read base sequence (gives you what base is added to go from one length to the next)

Question 10

True or False? “Technological advances have sped up how long it takes to sequence an entire genome”

Answer 10

True. Automated DNA sequencer (also capillary gel electrophoresis).

Question 11

Describe Illumina sequencing

Answer 11

(The idea is to use PCR on a solid support where DNA copies cluster up in the same space.) // A known sequence is ligated to linkers // Only one strand is covalently attached to the plate // A complementary strand is synthesized (the template is washed) // The open end of the strand attaches to another linker (forms upside down “U”) // Primer is added as well as reversible terminators (fluoro-labelled dNTPs) // Imaging tells which base is bound (remove label after) // New strand is synthesized and double strand is cut // Repeat from the annealing step

Question 12

Describe single molecule sequencing (Nanopore sequencing)

Answer 12

The order of bases is recognized as DNA passes through a channel (different voltage change means different base).

Question 13

What are some advantages to nanopore sequencing?

Answer 13

We don’t have to assemble the pieces of DNA as much (longer chunks are read) // Portable (laptop) // Offers possible solutions to new questions