PCR

Question 1

What does PCR stand for?

Answer 1

Polymerase Chain Reaction.

Question 2

What is the fundamental purpose of PCR?

Answer 2

To amplify specific DNA sequences, creating millions to billions of copies from a small initial amount.

Question 3

Why is PCR considered powerful?

Answer 3

Because it allows exponential amplification from minimal starting DNA, even a single molecule.

Question 4

What fields or applications use PCR?

Answer 4

Genetic research, environmental microbiology, medicine, phylogenetics, forensics, agriculture, food security, and consumer genomics.

Question 5

What are the 5 main components required for PCR?

Answer 5

DNA template
DNA primers (forward and reverse)
DNA polymerase (e.g., Taq polymerase)
Nucleotides (dNTPs)
Buffer solution (including Mg²⁺)

Question 6

What is the role of the DNA template in PCR?

Answer 6

It is the original DNA that contains the sequence to be amplified.

Question 7

What are PCR primers?

Answer 7

Short single-stranded DNA oligonucleotides that are complementary to the target sequence, initiating DNA synthesis.

Question 8

What does Taq polymerase do?

Answer 8

It synthesizes new DNA strands from the primers using the template DNA at high temperatures.

Question 9

Why is Taq polymerase commonly used?

Answer 9

It is heat-stable and can withstand the high temperatures used during the denaturation step.

Question 10

What is the role of the buffer in PCR?

Answer 10

Maintains the pH and ionic strength needed for optimal polymerase activity.

Question 11

What are the three main steps of a PCR cycle?

Answer 11

Denaturation (94–95°C): DNA strands separate.
Annealing (50–65°C): Primers bind to complementary sequences.
Extension (72°C): Taq polymerase synthesizes new DNA.

Question 12

How many cycles are typically run in PCR?

Answer 12

Usually 25–35 cycles.

Question 13

What happens to the DNA amount in each PCR cycle?

Answer 13

It doubles—leading to exponential amplification.

Question 14

What is a typical PCR reaction volume?

Answer 14

round 20 µL (0.02 mL), in a 200 µL PCR tube.

Question 15

What is the practical minimum PCR product size?

Answer 15

~60 base pairs (bp), though ~100 bp is preferred.

Question 16

What is considered a long PCR product?

Answer 16

Typically 8–9 kb (kilobases); some protocols report up to 20–50 kb.

Question 17

What types of DNA can be used as PCR templates?

Answer 17

Genomic DNA, plasmids, vectors, or even products of previous PCR reactions.

Question 18

Name at least 5 real-world applications of PCR.

Answer 18

Genetic disease testing
Pathogen detection
Forensics (e.g., hair/DNA at crime scenes)
Ancestry and consumer genomics
GMO detection in crops

Question 19

How is PCR used in evolutionary studies?

Answer 19

By amplifying introns or non-genic regions for phylogenetic analysis.

Question 20

How does PCR contribute to food and agriculture?

Answer 20

By validating plant varieties, checking for GMOs, and improving breeding programs.

Question 21

What are the three main steps in a PCR cycle and their typical temperatures?

Answer 21

Denaturation (~94°C)
Annealing (~50–60°C)
Extension (~72°C)

Question 22

What is the purpose of PCR?

Answer 22

To amplify a specific DNA sequence using repeated thermal cycling.

Question 23

What four key ingredients are needed for PCR?

Answer 23

Template DNA, primers, DNA polymerase (e.g. Taq), and dNTPs.

Question 24

What is Taq polymerase and why is it used in PCR?

Answer 24

A heat-stable DNA polymerase from Thermus aquaticus that can withstand high temperatures.

Question 25

Why is magnesium (Mg²⁺) included in the PCR buffer?

Answer 25

It’s a cofactor required for DNA polymerase activity.

Question 26

What are primers and what do they do in PCR?

Answer 26

Short DNA sequences that bind to the target DNA and provide a starting point for DNA synthesis.

Question 27

What happens during the denaturation step of PCR?

Answer 27

The double-stranded DNA is separated into single strands by heat.

Question 28

What happens during the annealing step of PCR?

Answer 28

Primers bind (anneal) to complementary sequences on the single-stranded DNA template.

Question 29

What happens during the extension step of PCR?

Answer 29

DNA polymerase synthesizes a new DNA strand by adding nucleotides to the primer.

Question 30

How does PCR achieve exponential DNA amplification?

Answer 30

Each cycle doubles the amount of DNA, so repeated cycles rapidly multiply the target sequence.

Question 31

Why is 72 degrees used for the extension step in PCR ?

Answer 31

Its the optimal temperature for Taq DNA polymerase to function efficiently without dislodging primers

Question 32

What is the result of one complete PCR cycle?

Answer 32

One double-stranded DNA becomes two double-stranded DNA molecules — a doubling of the template.

Question 33

How long does each PCR step typically last?

Answer 33

Denaturation: 5–30 seconds at ~94°C Annealing: 5–30 seconds at 50–60°C Extension: Varies by target length; short targets (~60 bp) may only take 5 sec

Question 34

How fast does Taq polymerase synthesize DNA?

Answer 34

Around 1,000 base pairs every 40 seconds.

Question 35

Why might short PCR products be fully extended before reaching 72°C?

Answer 35

Polymerase can begin working during the temperature ramp-up, completing synthesis for short targets early.

Question 36

What is exponential amplification in PCR?

Answer 36

The product of each cycle becomes the template for the next, doubling DNA with each cycle.

Question 37

How many cycles are typically used in PCR, and what’s the theoretical yield?

Answer 37

Usually 35–40 cycles; theoretically up to 2⁴⁰ (~1 trillion) copies if 100% efficient.

Question 38

Why does PCR amplification eventually plateau?

Answer 38

Due to depletion of reagents like primers and dNTPs, and enzyme degradation over time.

Question 39

What happens if extension time is longer than needed?

Answer 39

Taq polymerase may continue beyond the primer-binding site, producing longer, non-specific products.

Question 40

What is the difference between linear and exponential amplification in PCR?

Answer 40

Linear: longer, off-target products accumulate slowly; Exponential: desired products double each cycle.

Question 41

What are 'amplicons' in PCR?

Answer 41

The DNA fragments (products) generated through PCR amplification.

Question 42

What is a genetic marker in the context of PCR?

Answer 42

Often refers to a specific DNA region amplified by a primer pair, used to identify genetic traits.

Question 43

Why are there long-tailed PCR products early in the reaction?

Answer 43

Because Taq polymerase doesn't know to stop at the intended primer site and keeps extending if given time.

Question 44

What is PCR and why is it important?

Answer 44

PCR (Polymerase Chain Reaction) is a method to amplify specific DNA sequences, allowing detection and analysis from very small or degraded samples.

Question 45

How is PCR performed in the lab?

Answer 45

PCR is set up on ice to prevent premature enzyme activity, using precise pipetting and sterile techniques. A thermal cycler is used for rapid heating/cooling to cycle through denaturation (~95°C), annealing (~50–65°C), and extension (~72°C).

Question 46

How do you confirm PCR worked?

Answer 46

Run the product on gel electrophoresis; compare band sizes to a DNA ladder. Correct-sized bands suggest successful amplification.

Question 47

What precautions prevent PCR contamination?

Answer 47

Use DNA-free reagents, change gloves, physically separate PCR setup from post-PCR areas, and include negative controls (extraction and PCR blanks).

Question 48

What are some PCR types and their uses?

Answer 48

qPCR (Quantitative/Real-Time): Measures DNA in real time via fluorescence RT-PCR (Reverse Transcription): Converts RNA → cDNA before amplification Multiplex PCR: Amplifies multiple targets in one reaction

Question 49

Who invented PCR and when ?

Answer 49

Kary Mullis, 1983 — while working at Cetus Corporation near San Francisco.

Question 50

What was Kary Mullis known for besides PCR?

Answer 50

He was a Nobel Prize winner, but also controversial — he denied the HIV-AIDS link and was a climate change sceptic.

Question 51

How was PCR done before thermal cyclers?

Answer 51

Early PCR used three water baths for cycling temperatures; samples were manually moved between them or later by robotic arms.

Question 52

What is the purpose of the “hot lid” in modern thermal cyclers?

Answer 52

It prevents evaporation of the PCR reaction mix and eliminates the need for oil overlays.