PCR as a Diagnostic Tool (Week 1, Mod 9)

What is the Polymerase Chain Reaction? (PCR)

The amplification of a specific DNA sequence (target) within a sample

• Is used to detect / isolate specific nucleic acid sequences related to particular conditions or diseases (viral, bacterial, autoimmune, etc.)

What are the 5 key components that are needed to complete a PCR test?

• Template DNA – e.g. blood, tissue, swabs

• Primers – short synthetic oligonucleotides; starting points for DNA synthesis, defining the region of the template DNA to be copied

• Taq Polymerase – DNA polymerase

• Deoxynucleotide triphosphates (dNTPs) – DNA building blocks

• Buffer (Mg²⁺)

What are the 3 major steps of PCR?

1) DENATURING - Target DNA is denatured into separate strands (95 degrees C)

2) ANNEALING - Reaction is COOLED to allow primers to anneal to the target DNA (55-65 degrees C)

3) EXTENDING - Primers are then extended using Taq polymerase (72 degrees C)

Cycle is then repeated 25-30 times, resulting in billions of copies (amplicons)

What is the major difference between PCR and Reverse Transcriptase PCR (RT-PCR)?

Starting out with an RNA segment instead of DNA… need to first convert it to a complementary DNA (cDNA) using the reverse transcriptase enzyme

• After that is done, the cDNA is then used in the classic PCR reaction

What is the difference between Conventional PCR and qPCR (aka Real TIme PCR)?

Conventional PCR → gives you QUALITATIVE RESULTS, not quantitative like qPCR

• Product goes through electrophoresis gel; if something is highlighted, that means that your target DNA WAS in the sample

• BUT doesn’t say how much of your target was in the sample either

  • Usually have to recover the product for further investigation via gel / capillary electrophoresis

SEE IMAGE: this would be the last step in Conventional PCR; shows the different size of your products as it travels down the gel in comparison to the marker

What is Real-time PCR (qPCR)?

Is QUANTITATIVE

• Based on PCR principles

.. product is measured as the reaction progresses, in real time, with product quantification after each cycle.

.. To enable detection, the amplified product is labelled with a fluorescent dye

What are the 2 different fluorescent dyes used in qPCR? Which one is better to use?

• SYBR Green

  • non-sequence-specific fluorescent dyes that intercalates with all dsDNA product

  • Fluorescence proportional to all amplified product (specific and non-specific) 

• TaqMan

  • sequence-specific DNA probes attached to fluorochrome are added which bind through sequence complementarity to dsDNA product

  • fluorescence proportional to specific product

TaqMan would be best to use… more specific

What are the 2 different phases in qPCR data as its being recorded?

1) Exponential phase

• Reagents are abundant and non-limiting

• At 100% efficiency doubling of product each cycle

2) Non-exponential phase

• Reagents running out until they are exhausted

• No further amplification can occur

What is the threshold line in this graph? What does the Ct value represent?

Threshold line – machine calculated level of fluorescence which is (statistically) 

significantly higher than baseline

Ct (threshold cycle) – cycle number at which sample fluorescence crosses threshold; tells you how much target was in your sample

• Low Ct = high amount of target in samples, high Ct = low amount of target in samples

What are 4 things that could affect the PCR results?

1. Poor sample collection technique

2. Sample contamination

3. Sample degradation: RNA viruses (RNA degrades faster than DNA) 

4. PCR inhibitory substances - Substances contained within the clinical sample that interfere with the PCR reaction: FALSE NEGATIVE 

E.g. (Lithium) heparin, fluorescein, cat litter, charcoal, Heme, bilirubin, bile salts (faeces).

What are 2 things that could cause a false positive PCR?

• Laboratory contamination

• Non-specific primers: then some products may not be of desired sequence

What are 3 things that could cause a false negative PCR?

• Non-specific primers: target sequence may not be amplified efficiently

• Primers may not bind to mutant strains 

• Reaction failed

What are 7 advantages to using PCR testing?

• Rapid (24-48hr report), sensitive, specific diagnosis

• Identify pathogens non-cultivable/slow to culture/dangerous to culture/overgrown by other agents

• Can be performed on formalin-fixed, paraffin-embedded tissues

• Identification of carriers + shedders

• Strain-specific identification

• Vaccine vs field infections (sequence differences)

• Quantitative: pathogen load

What are 3 limitations in PCR?

• No information on viability/infectivity

• No data on antimicrobial susceptibility (bacterial pathogens)

• Commensal or not clinically significant