ANSC20003 Chap.9 PCR and Nucleic Acid Based Diagnostics
PCR and Nucleic Acid Based Diagnostics
A direct test with a sampling problem
The polymerase chain reaction amplifies DNA sequences from a very small input, from as little as one copy to millions, and it is a direct test: it finds the pathogen's own genome, or a particular gene carried by the animal.
Because it is direct, the organism has to be present in the material taken, which makes sample choice a pathogenesis question rather than a laboratory one and is the most common reason a well-run reaction returns a negative on an infected animal.
Extraction method also varies with the setting, the purity required and whether the target is DNA or RNA.
Five reagents, and the primers are the assay
A thermostable polymerase extends the new strand and survives repeated heating. Deoxynucleotides supply the building blocks. Forward and reverse primers of around 18 to 25 bases bind the complementary template and define both ends of the product.
Buffer with magnesium sets the conditions. Template supplies the target.
What an assay can and cannot report is decided by which sequence the primers bind: a conserved region detects a whole family without separating its members, while a variable region can be species specific but may miss a divergent strain.
Cycling, and the cost of more cycles
Denaturing at around 95 degrees separates the strands, annealing between about 45 and 60 degrees lets primers bind, and extension near 72 degrees builds the new strand, repeated for 30 to 45 cycles.
More cycles raise the signal and also raise the chance of amplifying something that is not the target, so a band appearing only at a raised cycle number is a question about specificity before it is a finding about a low-level infection.
Reading and confirming a product
Product is resolved in an agarose gel containing a DNA-binding dye.
DNA carries a negative charge and migrates towards the anode, with smaller products travelling faster, and bands are visualised under ultraviolet light and sized against a molecular weight ladder. A positive control shows the reaction worked; a no-template control shows nothing was contaminated. Confirmation of identity takes a further step, either sequencing of the purified product or a melt analysis for shorter products.
Quantitative and isothermal variants and sequencing at scale answer different questions again.
What this chapter covers
- 01
Amplification from a very small input, and why sample choice decides the result
- 02
The five reagents and the specific job each one does
- 03
Primer design as the thing that sets an assay's specificity
- 04
Thermocycling temperatures, cycle number, and the cost of adding cycles
- 05
Gel electrophoresis, migration by size, and sizing against a ladder
- 06
The positive and no-template controls, and what each one rules out
- 07
Sequencing and melt analysis for confirmation, and the quantitative, isothermal and large-scale variants
Deciding what to do about a band in the negative control
- 1State the conclusion about the run as a whole.
- 1Explain why the valid positive control does not rescue it.
- 1Give the action and say why partial salvage is not an option.
Key terms
- Thermostable polymerase
- The enzyme used in an amplification reaction, chosen because it survives repeated heating to strand-separation temperature.
- Primer
- A short oligonucleotide that binds a complementary template sequence and defines one end of the amplified product.
- Annealing temperature
- The stage temperature at which primers bind template, and the setting that determines how tolerant an assay is of an imperfect match.
- Molecular weight ladder
- A mixture of DNA fragments of known size run alongside samples so that product size can be judged by comparison.
- High resolution melt
- A method that raises temperature in small increments while recording fluorescence, distinguishing short products by their melting behaviour.
PCR and Nucleic Acid Based Diagnostics FAQ
Why can a well-run reaction be negative in an infected animal?
Because amplification is a direct method and the organism has to be in the material that was taken. If the sample came from a site the agent has not reached, or was taken when the animal was not shedding, the reaction has nothing to amplify. Sample choice follows from the pathogenesis of the agent.
Does a band of the expected size identify the organism?
Not by itself. It shows that something of about the right length was amplified by those primers, which for a family-level assay is compatible with several organisms. Sequencing the purified product, or a melt analysis if the product is short enough, is what converts the band into an identity.
What is the risk of simply increasing the cycle number?
Extra cycles increase the signal and also increase the chance of amplifying something that is not the target. A faint band that appears only after the cycle number is raised should prompt a question about non-specific amplification before it prompts a conclusion about a low-level infection.
Why does the run include water as a control?
The no-template control detects contamination. Because the method can amplify a single copy, a single copy carried over from a previous reaction produces the same band as a genuine positive, and the water lane is the only thing that reveals it.
Exam move
Learn the three cycle temperatures and the cycle range as exact figures, and be able to say what each stage achieves in one clause. Practise the controls answer until it is automatic, because it appears in both the practical and the written papers and is scored on order as much as content.
For primer specificity, rehearse the two-way argument: given an assay description, predict what a positive can and cannot claim, and given a required claim, say what the primers would have to target.
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