Molecular Biology Techniques

Question 1

What is the central dogma of gene expression?

Answer 1

DNA –> RNA –> protein

Question 2

What are 3 ways to analyse gene expression?

Answer 2

• RNA expression
• Protein expression & localisation
• Analysis of molecular interaction

Question 3

What is does a hybridisation method include?

Answer 3

One strand of DNA & one strand of RNA

Question 4

What is a probe?

Answer 4

DNA sequence that is complimentary to the RNA sequence you want to analyse - labelled in some way that we can analyse.

Question 5

What are 2 ways of analysing RNA expression?

Answer 5

• Northern blot
• Microarray

Question 6

Explain the steps of Northern blotting

Answer 6

1. RNA separated by electrophoresis - will see major bands of rRNA
2. RNAs are transferred to a membrane
3. Probe - complementary to your chosen sequence - labelled.

Question 7

Explain the steps involved of microarrays

Answer 7

1. Oligonucleotides (short DNA molecules) attached to a spot in a chip.
2. Each spot has a different oligonucleotide corresponding to a specific gene.
3. Fluorescent cDNA is made from RNA
4. Fluorescent cDNA is applied to the chip and allowed to hybridise.

Question 8

How does microarrays measure relative mRNA levels?

Answer 8

Lots of fluorescence would signify that lots of cDNA has bound to the oligonucleotides. As a result, this would mean that there was lots of mRNA

Question 9

How do we compare levels of transcription using microarrays?

Answer 9

Analysis of the colours produced via fluorescence will signify how much cDNA has been produced by each source

Question 10

What is the technique used to analyse RNA localisation?

Answer 10

Fluorescent in situ hybridisation

Question 11

Describe the steps of FISH (fluorescence in situ hybridisation)

Answer 11

Probe is labelled with a fluorescent marker & visualised using microscopy. Different colours will fluoresce in different locations, depending on where they have bound to.

Question 12

How can reporter genes be useful?

Answer 12

If reporter genes are cloned next to the promoter region of a gene of interest, expression of the reporter gene (which may fluoresce) can signify the expression of the gene of interest.
- as a result, high level of fluorescence = high level of gene expression.

Question 13

What are 2 uses of reporter genes

Answer 13

• protein localisation
• to measure expression

Question 14

What are 2 ways of analysing protein expression & localisation?

Answer 14

• using protein-specific antibodies
• fusion proteins & reporter genes

Question 15

How do protein-specific antibodies work?

Answer 15

• The primary protein-specific antibody attached to the protein. - - - The secondary antibody attaches to the primary antibody.
• The secondary antibody is usually conjugated to a molecule that allows detection

Question 16

Describe the steps of Western blotting

Answer 16

1. Proteins separated by electrophoresis
2. It is then transferred to a membrane
3. Detection is using a protein-specific antibody and a labelled secondary antibody

Question 17

What type of detection molecule is usually conjugated to a secondary antibody?

Answer 17

A fluorescent molecule

Question 18

What is an example of fluorescent protein?

Answer 18

GFP (green fluorescent protein)

Question 19

How can fusion of the fluorescent protein (GFP) bound with a gene of a protein of interest be useful?

Answer 19

Expression of the protein will lead to fluorescence and therefore detection

Question 20

What is the use of a pull-down assay?

Answer 20

A way of analysing protein interaction in vitro

Question 21

What is an organism that can be used to make a lot of recombinant DNA?

Answer 21

E. coli

Question 22

What is the process of a pull-down assay?

Answer 22

1. Make a cell lysate
2. Bind GST (Glutathione S-Transferase) to the affinity ligand.
3. Wash away any unwanted stuff (purified recombinant protein)
4. Investigate what can bind to protein

Question 23

Explain the process of immunoprecipitation

Answer 23

1. Use antibiotics to directly bind the preferred protein.
2. See what interacts with it (co-immunoprecipitation)

Question 24

What are the disadvantages of immunoprecipitation?

Answer 24

• Antibodies aren’t easy to make (expensive, not readily available, often in limited supply)

Question 25

What is a yeast two-hybrid?

Answer 25

The fusion of proteins. This occurs when transcription is activated. This is facilitated as ‘bait’ binds to the DNA binding domain attached to the promoter, as well as the prey being bound to the transcription activator.

Question 26

What is the use of chromatin immunoprecipitation (ChIP)?

Answer 26

Used to study interactions of proteins with DNA. Antibodies are then used to purify the protein.

Question 27

What is the first step of chromatin immunoprecipitation?

Answer 27

Formation of a cross-link DNA-protein, in order for the interaction to survive harsh purification conditions.