Lecture 24 - Molecular Genetics in Research 1

Question 1

What is PCR

Answer 1

Polymerase Chain Reaction

Question 2

What is Real-time PCR used for

Answer 2

This method can be used for a semi-quantitative determination of the amount of a particular DNA/RNA species of interest present within a heterogeneous sample

The method relies on specific enzymatic properties of DNA polymerase and real-time fluorescence detection

Some DNA polymerases, such as Taq DNA polymerase, harbour 5’ to 3’ exonuclease activity

Question 3

What is Digital PCR

Answer 3

‘Digital PCR’ enables an absolute quantification of DNA/RNA

As it is relatively expensive and time-consuming compared to Real-Time PCR, it is not as widely used

It is instead applied in more special circumstances, for example in determining viral loads from patient samples

Question 4

What is Nucleic Acid hybridisation

Answer 4

Nucleic acid hybridization-based techniques generally involve the use of synthesized oligonucleotide probes to detect the presence of a DNA/RNA species of interest

The probe or the DNA/RNA sample is usually fixed to some type of solid support which allows binding/detection to be visualised spatially

The basic principle can be used in a variety of different ways depending on the application

Question 5

What is in situ hybridisation

Answer 5

Can be used to detect the presence/localisation of DNA/RNA in cells/tissues following fixation to a solid support

Probes are usually fluorescently labelled (i.e. FISH)

Widely used to visualise the patterns of mRNA transcript localisation in developing embryos, tissue sections, and cell cultures

Question 6

What are Microarrays

Answer 6

Used in large-scale applications i.e. genome/transcriptome wide analyses

Very commonly used for large-scale gene expression studies

probes are complementary to all 20,000 protein coding genes

Also used to differentiate between diseased and normal samples