5-6: SDS-PAGE and PCR

Question 1

What are the two (mentioned) options for gel material in gel electrophoresis, and when would you use each one?

Answer 1

Agarose for large molecules e.g., DNA
Polyacrylamide for smaller nucleic acids and proteins

Question 2

Describe the three-step process to ensure that proteins migrate according to Molecular Weight ONLY

Answer 2

1. Heat to disrupt hydrophobic and dipole-dipole interactions
2. Add a Thiol Reducing Agent (DTT / dithiolthreitol) to disrupt S-S bonds
3. Add SDS to solubilise, denature and coat the proteins with negative charges

Question 3

Name the dye that is added during SDS-PAGE and its function

Answer 3

Bromophenol Blue
It is impossible to see the proteins while the gel is running
Bromophenol Blue is smaller and migrates faster than the proteins, so when it reaches the bottom of the gel, the power is turned off and the proteins will not have migrated too far

Question 4

What is the significance of the lipophilic tail of SDS in this experiment?

Answer 4

It can dissolve hydrophobic molecules - this ensures that when a cell is incubated with SDS, the membranes are dissolved and the proteins solubilised

Question 5

What is the significance of the anionic head of SDS in this experiment?

Answer 5

By binding non-covalently to proteins (about 1 SDS molecule per 2 AAs) SDS covers all proteins (regardless of their intrinsic net charge) with a similar negative charge

Question 6

Why are (some) protein samples incubated at 95C for 10 minutes prior to loading?

Answer 6

This helps denature them further

Question 7

Name all the components added to a PCR tube

Answer 7

1. DNA Template (containing original DNA sequence to be amplified)
2. Two single-stranded primers (which target the sequence to be amplified)
3. Taq polymerase
4. Free nucleotides
5. Some reaction buffers

Question 8

Name and describe the FIRST step in the PCR cycle

Answer 8

DENATURATION (90-95C for 30 seconds) - double stranded DNA denatures into single-stranded DNA molecules

Question 9

Name and describe the SECOND step in the PCR cycle

Answer 9

ANNEALING (around 50-60C for 30 seconds) - drop in temperature allows primers to anneal to their complementary sequences on the single stranded DNA

(NOTE: template strands can also REanneal, so primers are added in excess)

Question 10

Name and describe the THIRD step in the PCR cycle

Answer 10

EXTENSION (72C for 60 seconds) - the primers provide a short sequence of dsDNA for the Taq polymerase to extend from (in the 5’->3’ direction)

Question 11

When will Taq polymerase STOP adding new dNTPs in the extension stage of PCR?

Answer 11

When there is no template left to read, or when extension time is over (i.e. when temperature is increased to 95C again)

Question 12

What determines the actual DNA fragment to be amplified?

Answer 12

Where the primers bind - they determine the start and end of the amplified fragment

Question 13

Why is it important to design primers carefully using software?

Answer 13

Each primer must bind to the genome only ONCE, and they must have similar annealing temperatures to each other

Question 14

Name the 3 (or 4) types of polymorphisms

Answer 14

1. Single Nucleotide Polymorphisms (SNPs)
2. Copy Number Variants (CNVs) -> can be subdivided into Short Tandem Repeats (STRs) and Variable Number Tandem Repeats (VNTRs)
3. Transposable Elements (TEs) - also known as jumping genes

Question 15

How do the three main types of polymorphisms arise?

Answer 15

SNPs - due to errors in DNA replication
CNVs - due to duplication or deletion
TEs - viral origin