MOLECULAR DIAGNOSTICS

Question 1

Describe the steps that are involved in PCR reaction and their temperatures:

Answer 1

• denaturation- 95 degrees
• annealing- 50 to 60 degrees
• extension - 72 degrees

Question 2

What are the ingredients that are required in PCR:

Answer 2

• target DNA, primer, DNA polymerase, dNTP’s, machine to cycle temperatures- thermocycler, typically analyse products using gel electrophoresis

Question 3

What is an oligonucleotide?

Answer 3

• serves as primer for RNA synthesis

Question 4

What are the advantages of PCR?

Answer 4

• very specific assay determined by 2 primers
• start with very small amounts of DNA
• not dependant on DNA being intact
• can be used in combination with other techniques

Question 5

Describe quantitative PCR:

Answer 5

• based on detection and quantitation of a genetic reporter

- signal increases in direct proportion to the amount of product that is present

Question 6

Describe digital PCR:

Answer 6

• works by separating the mixture into an array of different partitions
• individual molecules are targeted and amplified

Question 7

What are the three applications of PCR:

Answer 7

• detection of infectious/genetic diseases
• Sex ID and screening of males versus females
• Genotyping

Question 8

Outline 3 applications of next generation sequencing:

Answer 8

• mixed breed identification
• understanding resistance to infection
• understanding disease transmission and vaccination requirements
• microbiome analysis