W3L5 - Gel and Capillary Electrophoresis

Question 1

What is the pore size of agarose gel dictated by?

Answer 1

Agarose concentration

Higher [agarose] = smaller pores

Question 2

Visualising the Nucleic Acids

Answer 2

Use Ethidium Bromide or SYBR Green/Gold/Safe
These intercalate into major groove of NA
Stain can be added to gel during casting or gel can be post-stained
Visualise under UV light

Question 3

Polyacrylamide Gel Electrophoresis (PAGE

Answer 3

Higher [acrylamide] = lower pore size
Polyacrylamide has a smaller pore size than agarose
Polyacrylamide gels provide better resolution of small NA fragments
Not useful for large fragments
Gels must be post stained

Question 4

Capillary Electrophoresis

Answer 4

Uses thin silicon tubes containing a linear acrylamide polymer
Samples injected into tubing and electrophoresed under denaturing conditions
Smaller fragments travel faster than larger fragments
Detector near anode to detect DNA as it passes through tubing and the time taken to travel the tubing is recorded

Question 5

Sanger DNA Sequencing

Answer 5

Instead of only deoxynucleotides (dNTPs), it uses a combination of dNTPs and labelled dideoxynucleotides (ddNTPs)
DNA Polymerase cannot produce a phosphodiester bond between adjacent nucleotides without the 3’ OH group
Amplification of the specific DNA chain terminates with addition of a ddNTP
Once cycling is complete, the reaction products put through capillary electrophoresis
Different sized fragments elute at different times
Fluorescent label read by detector therefore sequence can be determined

Question 6

What does a Sanger Sequencing Reaction contain?

Answer 6

DNA to be sequenced
Polymerase
Reaction buffer
dNTPs and ddNTPs
- each ddNTP is fluorescently labelled with a different colour
Primer

Question 7

Homozygous vs Heterozygous Mutations

Answer 7

For example Wild type alleles is TT
Homozygous mutation could change it to CC (for example)
Heterozygous mutation could change it to TG

Question 8

What do you need to analyse DNA for mutations?

Answer 8

The reference gene sequence (NCBI)
Mutation details, if known
The ‘test’ DNA sequence
Program to compare the reference for test sequences

Question 9

Pyrosequencing Steps

Answer 9

1. DNA polymerase incorporates the nucleotide that is present in the reaction mixture
2. ATP Sulfurylase, in the presence of Adenosine 5’ phosphosulfate, converts pyrophosphate to ATP
3. ATP is used by Luciferase that ultimately converts Luciferin to light
   - amount of light produced is proportional to the amount of ATP produced which is proportional to no. of dNTPs incorporated
4. Apyrase degrades the remaining dNTP present
5. The next dNTP is added to the reaction and the process begins again

Question 10

What does the initial Pyrosequencing reaction mixture contain?

Answer 10

DNA to be sequenced
DNA polymerase
Sequencing primer
One of the four dNTPs
ATP Sulfurylase
Adenosine 5' Phosphosulfate
Luciferase
Luciferin
Apyrase

Question 11

pH-Mediated Sequencing

Answer 11

Based on process of DNA amplification
When a dNTP is added to a DNA strand by DNA polymerase a H+ ion is released
Reaction mixture contains:
- DNA to be sequenced
- DNA polymerase
- sequencing primer
- one of the four dNTPs
If the dNTP is incorporated, H+ is released and this is measured as a change in pH
Cycle is repeated with the remaining dNTPs