Jordans Notes 16

Question 1

How does solid phase PCR work? (huge card)

Answer 1

1. Single strand DNA attach to flowcell surface (adapter ligates to
   oligonucleotides)
2. free end curves down and attaches to flowcell forming a bridge
3. Polymerase create double stranded bridge
4. Denaturation separates strands to repeat cycle

extra reading:
Single stranded DNA is fragmented into shorter fragments
Ends are repaired and A tails are added Different adapters with T tails are ligated to the complimentary A tails of the fragments (one end gets a barcode adapter for identification)
Fragments are flushed over the flow cell which has attached oligonucleotides that are complimentary to the adapters.
This explanation is now in regards to a single nucleotide on a single point on the flowcell. In reality this occurs in mass over the entire cell with many different fragments Adapters anneal to oligonucleotides, attaching them to the flowcell Thermostable DNA polymerase forms a double strand onto the end of the attached oligonucleotide.
Denaturation releases the original fragment which is flushed away. Now there is a complimentary strand attached to the flowcell.
The free end of the fragment (also with an adapter) curves over and anneals to an oligonucleotide complimentary to this second adapter forming a “bridge”. Again, thermostable DNA polymerase generates a double strand.
This double strand is then denatured, releasing a single end from each fragment that is not attached to the permanently attached oligonucleotide.
This process can then be repeated with the single stranded fragments to form a cluster of many fragments of the same sequence.
Because there are many copies of the same fragment clustered in parallel, it makes subsequent sequencing more accurate due to a high depth of coverage

Question 2

What are 4 components of natural selection?

Answer 2

1. trait must be heritable
2. must be variation present in the population
3. differential reproduction (not all individuals get to reproduce)
4. therefore, advantageous traits that increase survivability will be passed on more frequently

A response to environmental pressures (Not random!)

Question 3

what kind of nucleotides are used during illumina
sequencing?

Answer 3

fluorescent dNTPs with reversible terminators (inactive 3’ hydroxyl groups)

Question 4

What is the PCR product also called?

Answer 4

Amplicon

Question 5

a primer is _____ to the conserved region it binds
with

Answer 5

Complimentary

Question 6

What can scan the quality of DNA extracted before
conducting a PCR?

Answer 6

Spectrophotometer

estimates the ratio of DNA to other molecules such as proteins and other contaminants

Question 7

What does non-coding strands of DNA do?

Answer 7

1. Functional RNA molecules
2. Transcription Regulation
   a. Promoter regions
   b. regulatory sites

Question 8

How is nucleic acid released from the silica
membrane?

Answer 8

Elution buffer AE or water hydrates the DNA
If there is any ethanol present the DNA will not fully hydrate

Question 9

Ingredients for putting DNA in gel

Answer 9

3µL DNA and 3μL of loading dye

Question 10

What allows you to take into account differences in
substitution rates between LINEAGES?

Answer 10

Relaxed molecular clock assigning a different rate to different lineages

Question 11

What is used to determine SNPs in association
with binary traits?

Answer 11

Genome-Wide Association Studies