PCR

Question 1

What is the purpose of PCR?

Answer 1

Creating millions of copies of DNA for diagnostics, research, forensic medicine

Question 2

What is the target?

Answer 2

DNA in the sample

Question 3

What are primers?

Answer 3

Short sequences of DNA (oligonucleotides), designed to complement the sequence on the target DNA. 3’ and 5’.

Question 4

What are dNTP?

Answer 4

Nucleotides used to create copies of DNA

Question 5

What is taq polymerase?

Answer 5

Enyme which amplifies DNA at a high temperature

Question 6

What is the purpose of a buffer?

Answer 6

To maintain an appropriate reaction environment

Question 7

What are the 2 types of PCR?

Answer 7

Reverse transcriptase PCR (RT-PCR)

Real time PCR (qPCR)

Question 8

What are the steps in RT-PCR?

Answer 8

1. Mixture heated to 45-55 degrees for 15-30 mins to allow reverse transcriptase to act (unzip DNA helix)
2. Heated to 95 degrees, for 15s, to denature the DNA
3. Cooled to 60 degrees, for 30s, to allow primers to anneal to target DNA
4. Heated to 72 degrees to extend the primers using taq polymerase

Cycle repeated 25-30 times

Question 9

How are the strands of DNA seperated?

Answer 9

DNA is placed onto agarose gel. An electrical current is then passed through it causing DNA of different lengths to travel different distances through the gel.

Question 10

How is the product of PCR verified?

Answer 10

Sequencing of product

Expected size of product

Question 11

How can the product be used?

Answer 11

1. Indicates presence of taret in sample - diagnosis in clinical case
2. Analysis of product
3. Cloning of product for use in research based/therapeutic based context

Question 12

What are the precautions that need to be taken when carrying out RT-PCR?

Answer 12

1. ‘Clean’ rooms for setting up of master mixes
2. Addition of target rooms where product is not amplified
3. Wearing of protective gear
4. Use of UV hoods which irradiate contaminating DNA

Question 13

What are issues with RT-PCR?

Answer 13

1. Contamination of sample
2. Specificity - primers can bind elsewhere in the sequence, target sequence may not be amplified efficiently
3. Sequence variation across primer binding site - any mismatches across primer binding site means efficiency of reaction reduced (possible false negatives)

Question 14

What is the difference between RT-PCR and qPCR?

Answer 14

qPCR - Amplified product is labelled with a fluorescent dye for detection. Product is detected in real time and is quantified.

Question 15

What kinds of dye are used in qPCR?

Answer 15

1. dsDNA intercalating dye - non-sequence specific, intercalate with DNA product
2. Sequence specific probe - probes are attached to fluorochrome and bind to complementary DNA product

Question 16

What are veterinary uses of PCR?

Answer 16

1. Disease diagnosis
2. Monitoring disease/infection progress
3. Screening to prevent intrduction of infection into a negative area
4. Research - gene expression and disease

Question 17

What are the advantages to qPCR?

Answer 17

More sensitive
More quantitative
Faster
Safer
More specific
Lowered risk of contamination

Question 18

What are the disadvantages of qPCR?

Answer 18

Need more specialised equipment
More expensive
Requires known sequence across the primer and probe regions

Question 19

What are the diagnostic advantages of PCR?

Answer 19

1. Allows fast diagnosis of disease

2. Can identify the strain associated with the outbreak by using strain specific primers

Question 20

What are the disadvantages to PCR?

Answer 20

1. Requires a specialist lab
2. Steps need to be taken to avoid contamination
3. Existing primers may not recognise new strains of pathogen

Question 21

What do vets need to be aware of?

Answer 21

1. Make sure the test being used has been used in refereed publications
2. Be clear about the purpose of your test
3. Check to make sure the best sample is submitted
4. Be aware of limitations of PCR
5. The presence of an infectious organism doesn’t always mean that it is the cause of the disease