Electrophorhesis

Question 1

inc/dec [agarose] = _er pores

Answer 1

inc. [agarose] = smaller pores

Question 2

How are NA visulaised in gel electrophorhesis?

Answer 2

• use intercalating dye (ethidium bromide or SYBR Green/gold/safe) intercalate into major groove of NA
• stain can be added during gel casting or post-stain (run gel sits in dye & de-stain)
• visible under UV light

Question 3

What is a disadv of post staining & staining during?

Answer 3

during: stain binds to DNA = slow migration
post: longer process

Question 4

What you need to know for interpreting a gel

Answer 4

Know
• expected band size
• size of each band from marker

Question 5

when running RNA through gel electrophorhesis what must be done (&why)

Answer 5

formamide is added to loading buffer & formaldehyde to buffer > denature RNA & maintain denatured state

• prevent formation of 2º structures = affect migration
• must be done in a fumehood bc formaldehyde

Question 6

Characteristics of polyacrylamide gel electro

Answer 6

• inc [acrylamide] = dec pore size
• smaller pore size than agarose
• better resolution of small NA fragments (not really for large fragments)
• Must be post stain

Question 7

Describe capillary electrophorhesis

Answer 7

• samples injected into silicon tubing & electrophorhesed under denaturing cond
detector present near anode > detect DNA passing
through & time is recorded
• Adv.: fast & high resolution & Hi voltage applied to sample (100V+)

Question 8

method of sequencing w/ electrophoresis & describe

Answer 8

• sanger sequencing OR chain termination sequ.

* uses ddNTP - missing -OH on 3’ ≠ make PO bond ≠ elongate

Question 9

How Sanger sequencing works

Answer 9

• 4x tube rxns: contain dNTP + ddNTP (1 type per tube)
• ssDNA template
• primer
• polymerase
• rxn buffer
• use fluorescently labelled oligonucleotide primer
• each tube put in wells of gel electrophoresis=> read bottom-up to get sequence

Question 10

to analyse DNA for mutations you need:

Answer 10

• reference gene sequence
• mutation details, if known (e.g. dNTP change, the position)
• “test” DNA sequence
• a program to compare reference & test sequence

Question 11

Describe what pyrosequencing is

Answer 11

• DNA amplification
• pyrophosphate is released when dNTP is inserted in DNA = light
• intensity light produced = amount of ATP produced = # dNTP incorporated (in each step)

Question 12

Describe process of pyrosequencing

Answer 12

1. DNA polymerase incorporates dNTP present in the rxn mixture
2. ATP sulfurylase (if there’s Adenosine 5’ phosphosulfate) converts pyrophosphate => ATP
3. ATP used by Luciferase to convert Luciferin => LIGHT
4. Apyrase degrades remaining dNTP present
5. next dNTP is added & rxn repeats (*work w/ 1 dNTO @ a time)

Question 13

Describe pH-mediated sequencing

Answer 13

• dNTP added to DNA strand by DNA pol => H+ ion released
• pH is measured
• cycle repeated w/ remaining dNTPs