Molecular Techniques

Question 1

What does gel electrophoresis do?

Answer 1

Separates genes and proteins according to SIZE.

Question 2

How does gel electrophoresis work?

Answer 2

All proteins and placed on an electrical field covered in gel.
There is a positively charged side (cathode) and negatively charged (anode).
As all proteins are negatively charged (due to phosphate backbone), they will move towards to anode end.
However, the smaller the protein fragment the FURTHER it will travel.

Question 3

How can you tell the size of a protein fragment in gel electrophoresis?

Answer 3

You used a ‘DNA ladder’, a protein where you know exactly how big the fragments / base pairs are and use this as a comparison to the protein you are testing.

Question 4

What can gel electrophoresis be used for?

Answer 4

To detect small deletions/ insertions in base pairs.

Question 5

What is an aganose gel used for?

Answer 5

Used for big fragments (up to 50 bp).

Question 6

What is SDS page gel used for?

Answer 6

Smaller fragments or proteins.

S = small

Question 7

What is 2D-PAGE gel electrophoresis used for?

Answer 7

Looking at whole cells / tissues - separating organelles.

Question 8

What is gene cloning?

Answer 8

Making identical copies of a piece of DNA.

Question 9

How does gene cloning work?

Answer 9

Restriction enzymes are used to cut out a gene from a piece of double stranded DNA.
This is then placed in a circular plasmid that has the right sequence at its ends to bind, and DNA ligase attaches the added strand.
The plasmid is then added to a bacterial cell and multiplied in a Petri dish.

Question 10

What are restriction enzymes?

Answer 10

Enzymes that cut DNA at a particular place.

Question 11

What can gene cloning be used for?

Answer 11

To make useful proteins e.g. Proinsulin.

Question 12

What does PCR stand for?

Answer 12

Polymerase Chain Reaction

Question 13

What does PCR do?

Answer 13

Make lots of copies of a DNA fragment

Question 14

What temperature is used to denature and separate the double stranded DNA in PCR?

Answer 14

96 degrees Celsius.

Question 15

What temp is a PCR solutions warmed up to after denaturation?

Answer 15

56 degrees Celsius.

Question 16

What are primers and their role in PCR?

Answer 16

Primers serve as a starting point for DNA synthesis.
In PCR, specific primers are added to the solution and attach to the separated single strands of DNA before primer extension occurs.

Question 17

What enzyme is used to extend the strands and why?

Answer 17

DNA TAQ Polymerase - heat resistant (72 degrees).

Question 18

How many times is PCR repeated and why?

Answer 18

35 times producing about a billion DNA fragments. This is so you can amplify a particular section of DNA.

Question 19

What is PCR used for?

Answer 19

To amplify DNA fragments, investigate single bases mutations, insertions, deletions.

Question 20

What is Isoelectric Focusing (IEF)?

Answer 20

Separates proteins according to CHARGE.

Question 21

How does IEF work?

Answer 21

Proteins are placed on an electrical field separate by ph (e.g. Ph 3 one end and ph 9 the other).
Proteins migrate until their ph = their pl.

Question 22

What is a proteins pl?

Answer 22

The isoelectric point - the ph in which a molecule carries no electric charge.

Question 23

What are proteomics?

Answer 23

The analysis of ALL proteins expressed from the genome.

Question 24

What is molecular diagnosis?

Answer 24

The analysis of a single purified protein.

Question 25

What happens to protein identification proteomics?

Answer 25

In a solution, protein is digested with TRYPSIN.
A mass spectrometry is performed (separating according to mass), and a database of known proteins is used to compare peptide sizes and identify the protein.

Question 26

What is Western Blotting?

Answer 26

Where you form an antibody that attaches to a specific protein that we want to locate.
A MONOCLONAL antibody - specific to one protein.

Question 27

Explain the three stages of Western Blotting.

Answer 27

1. Proteins are separated by size using SDS electrophoresis
2. A primary antibody is added to the gel, which binds to its specific protein.
3. A secondary antibody is added which binds to the primary antibody, isolating the protein you want to locate.

Question 28

What does ELISA stand for?

Answer 28

Enzyme-linked immune-absorbent assay

Question 29

What does ELISA do?

Answer 29

Is used to detect the presence of an antibody or antigen in a sample.

Question 30

Explain the four stages of ELISA?

Answer 30

1. In a solution, antigens are coated to a well.
2. Specific antibodies are added which binds to its specific antigens. The well is washed of excess antibodies.
3. Enzyme linked antibodies are added to the solution, which bind to antibodies. The well is washed of excess enzymes.
4. Specific substrates are then added, changing the colour of the solution according to how much antibody is present.

Question 31

What an ELISA be used for? Give a clinical example.

Answer 31

Used to measure the concentration of proteins e.g. ELISA is the gold standard diagnosis for detecting troponin in heart failure patients.

Question 32

What is an enzyme assay?

Answer 32

Identifying a protein through its enzyme activity.

Question 33

What two things are enzyme assay used to detect?

Answer 33

1. Metabolic disorders - in tissues

2. Diagnosis of disease - serum enzymes e.g. Lipase for pancreatitis.