Int 7: Molecular Techniques

Question 1

Nucleic Acid Hybridisation (Northern Blotting)

This method is based on the fact that complementary ssDNAs or ssRNAs form a duplex, the stability of which depends on the a)____________ and b)__________- of the nucleic acid.

Short pieces of DNA/RNA can be labelled during synthesis and can be separated via c)_____________ on the basis of size.

The separated blots are then transferred to a nylon membrane via d)____________. This immobilizes the RNA.

Complementary sequences e)__________ and can be detected by f)___________

• capillary flow
• hybridize
• base sequence
• autoradiography
• length
• hybridization

Answer 1

a) base sequence
b) length
c) hybridization
d) capillary flow
e) hybridize
f) autoradiography

Question 2

What can Northern Blotting indicate and what can it not?

Answer 2

• Can detect if more than on mRNA is transcribed from a gene (could indicate alternative intron/exon processing / multiple transcription initiation points)
• Cannot know where within a tissue a gene is being transcribed (in situ hybridization can!)

Question 3

In contrast to Northern blotting, In situ hybridization involves the labelling of DNA/RNA with enzyme-linked colour metric assay. In situ hybridisation can also:

Answer 3

• provide info on cell type specific gene expression in tissue
• give cellular resolution

Downsides are:
- only analyse one or two gene products at a time
- not particularly sensitive

Question 4

How do micro-arrays work?

Answer 4

-Label all the transcripts instead of sequence specific probes
-Then simultaneously analyse a large number of immobilised test sequences
- Expensive + much data analysis needed

Question 5

What is a common downside of northern blots, in itu hybridisation and PCR?

Answer 5

They all only follow the transcription of a small number of genes

Question 6

NOTES:

Micro arrays follow the transcription of
10,000s of genes. Patterns of transcription
rather than the activity of individual genes.

RNAseq offers the potential to sequence a
whole mRNA population.

In situ methods provide good cell and tissue
specificity, but are not particularly sensitive, quite
difficult to quantify, only analyse one or two gene
products at a time and are technically difficult.

Quantitative Real Time Polymerase Chain Reaction,
“qRT-PCR” , is exquisitely sensitive, can analyse
more than one gene product at a time.

Microarray hybridization and RNAseq methods can
analyse the expression from a much greater number
of genes. Expensive, and extensive data analysis
required.

Question 7

a) What does SDS-PAGE do?
b) what does 2-D PAGE do?

Answer 7

a) Separates 10-100s of proteins on the basis of size!
b) separates on basis of both size and charge

Question 8

How does SDS-PAGE work?

Answer 8

• Proteins treaded with SDS, causes unfolding
• SDS molecules bind to amino acid residues
• SDS is -ive so goal is to make all the amino acid residues the same charge so any difference is solely due to molecular weight

Question 9

What are the following probes used to find in 2-D PAGE?
a) Stains or Dyes
b) Antibodies

Answer 9

a) To detect proteins in general
b) To detect specific proteins

Question 10

What is the difference between polyclonal and monoclonal?

Answer 10

Polyclonal - several antibodies to different epitopes
Monoclonal - single antibody to singe epitope

Question 11

What is the kid of blotting that is sued to detect the presence of proteins in a complex mixture (also called 1-D PAGE)

Answer 11

Western / Immuno blotting

Question 12

How does co-immunoprecipitation work? (pretty self explanatory)

Answer 12

• use an antibody that binds to the target protein
• incubate in solution with this and another coupled antibody that binds the primary antibody
• wash away unbound proteins and analyse

Question 13

Why is PCR so sensitive?

Answer 13

Because through reiterated cycles of polymerisation it can exponentially amplify very few or a single molecule. The exponential amplification is achieved because the DNA strand of one polymerization is the template for the other second polymerization.

Question 14

Which of the methods of in situ hybridization, northern blotting, qRT-PCR and RNAseq analyses most RNAs in a single reaction?

a) Northern blotting
b) qRT-PCR
c) RNAseq

Answer 14

C

Question 15

How can mass spectrometry be used to identify proteins?

Answer 15

• Proteins broken up into fragments depending on primary structure
• Unique patterns