Polymerase Chain Reaction

What is Polymerase Chain Reaction (PCR)?

A technique used to replicate large quantities of a specific sequence of DNA from an initial minute sample (e.g., a single drop of blood or nasal swab).

How much does DNA double in each PCR reaction?

Each reaction doubles the amount of DNA

What does PCR target?

A specific segment of DNA.

How do scientists ensure PCR copies only the desired DNA region?

They use primers specific to that sequence of DNA.

What is a DNA primer?

A short, single-stranded sequence of DNA that serves as a starting point for DNA synthesis.

How is PCR used in COVID-19 testing?

Primers specific to genes within the COVID-19 virus are amplified billions of times and then analysed.

What are the three main stages of a PCR cycle?

1. Heat

2. Cool

3. Copy

1. Heat

DNA sample is heated (94-98ºC) to denature and break the hydrogen bonds between bases

• This separates the two strands

2. Cool

The sample is cooled (50-68º) and add RNA primers to anneal (stick)

• primers lock onto a short target sequence to mark the starting point for copying

3. Copy

The sample is reheated to (75º) for the enzyme Tag Polymerase to copy a complete complementary strand

• This is the optimal temperature for Taq polymerase to function

What happens to polymerase enzymes at high temperatures needed to run each PCR cycle?

Denature

What is thermus aquaticus?

A bacteria - it is tough and it thrives at 75ºC

Why is Taq polymerase used in PCR?

It is heat-stable and functions at high temperatures without denaturing.

What is Thermus aquaticus?

A bacterium from which Taq polymerase is isolated

What is the purpose of PCR in DNA profiling?

To amplify small DNA samples to provide clear and accurate profiles for analysis via gel electrophoresis.

What is gel electrophoresis?

A lab technique used to separate DNA (or protein) fragments based on mass, size, and electric charge.

How are DNA samples prepared for gel electrophoresis?

DNA is cut at specific points using restriction enzymes and labelled with a dye.

How does gel electrophoresis separate fragments?

An electric current pulls negatively charged DNA through the gel

Why will all proteins and nucleic acids be slowly attracted across the gel?

They have a negative charge

Why do smaller samples move faster through gel?

They are less impeded by the gel matrix

What does this cause samples of different sizes to do?

Separate as they travel at different speeds

Why does gel electrophoresis produce a unique DNA fingerprint?

Each individual has unique DNA sequences and fragment lengths.

What is DNA profiling commonly used for?

Criminal investigations (forensic) and settling paternity disputes.

What are other applications of DNA profiling?

Medical diagnostics, genealogy and ancestry, wildlife conservation, agriculture (identifying/selecting genetically desirable traits in crops and livestock).

What is DNA profiling based on?

The presence of short tandem repeats (STRs) in DNA sequences.

What are short tandem repeats (STRs)?

Blocks of DNA that repeat themselves a random number of times.

Why does each individual have a unique DNA profile?

Each person can have a different number of STRs at a given DNA location.

How are DNA fragments separated for profiling?

Using gel electrophoresis.

How are DNA profiles compared to find a match?

By comparing the lengths of DNA fragments separated via gel electrophoresis.

Why does gel electrophoresis generate a unique fingerprint?

Because individuals have slightly different sequences of DNA.

What are the steps in the DNA profiling process?

1. What happens to a DNA sample?

It is collected and then amplified using PCR

2. What is sued to cut DNA at specific points?

A special ‘cutting enzyme’ called a restriction enzyme

3. What is gel electrophoresis used to separate>

The cut fragments according to their length

How accurate is DNA profiling?

Comparing fragment lengths allows identification with 99.9% accuracy.

What must happen for DNA evidence to convict a suspect?

There should be a complete match between the suspect's DNA profile and the crime scene sample.

What is the probability of two unrelated individuals having the same DNA profile?

On average, less than 1 in 1 billion.

Applications of PCR in medical diagnostics?

Detecting pathogens and genetic mutations associated with diseases.

Applications of PCR in forensic science?

Amplifying DNA from crime scene evidence for identification purposes.

Applications of PCR in genetic research?

Cloning and analysing genes to understand their function.

Applications of PCR in paternity testing?

Amplifying DNA samples to establish biological relationships.

Applications of PCR in cancer research?

Detecting and studying genetic mutations involved in cancer.

Applications of PCR in agriculture?

Genotyping plants and animals to improve breeding programs.

Advantages of PCR for viral infections?

High sensitivity, high specificity, and rapid results.

High Sensitivity

Very small amounts of viral DNA/RNA can be used making it possible to identify infections early

High Specificity

Targeting specific viral sequences (reduces the likelihood of false positives from other viruses)

Disadvantages of PCR for viral infections?

False negatives, over-sensitivity (detects non-viable virus), and cost.

False negatives

Improper sample collection, degradation of viral RNA/DNA, or inhibitors in the sample can lead to false-negative results

Over-sensitivity

PCR can sometimes detect viral genetic material from non-viable virus particles