Nucleic Acid Target Amplification + Focus on qPCR and RT-qPCR Techniques

What are the main 3 Nucleic Acid Amplification Techniques 

1. Target

2. Probe

3. Signal 

Who discovered Thermus aquaticus, and when?

In 1966, Thomas Brock discovered Thermus aquaticus, a thermostable bacterium found in the hot springs of Yellowstone National Park.

Why was Thermus aquaticus an important discovery?

It provided a source of thermostable enzymes, including Taq polymerase, which later became essential for PCR because it can withstand high temperatures during DNA denaturation.

Who postulated the concept of the Polymerase Chain Reaction (PCR), and when?

In 1983, Kary Mullis proposed the concept of PCR, a method to amplify specific DNA sequences exponentially.

When and by whom was the first practical application of PCR published?

In 1985, R.K. Saiki and colleagues published the first PCR application in the journal Science, demonstrating amplification of the beta-globin gene.

What company was involved in early PCR development, and what was their major contribution?

Cetus Corporation (a biotechnology company) played a major role in 1985 by isolating thermostable Taq polymerase from Thermus aquaticus.

Why was the isolation of Taq polymerase such a major breakthrough?

Before Taq polymerase, DNA polymerase had to be added after every PCR cycle because it was destroyed by heat. Taq polymerase, being heat-stable, allowed for automated and continuous cycling, revolutionizing PCR efficiency and automation.

How many years passed between the discovery of Thermus aquaticus and the isolation of Taq polymerase?

19 years (from 1966 to 1985).

Why did Kary Mullis receive the Nobel Prize in 1993?

For his invention of the Polymerase Chain Reaction (PCR)

Which DNA strand is designated as the "Sense strand," and what is its orientation?

Also known as the Coding strand is the top strand, and its orientation is 5' to 3' (left to right)

Which strand is referred to as the "Non-coding" or "Non-sense strand," and what is its role in the natural process of transcription?

Also known as the Template strand is the bottom strand (3’→5’). In transcription, it serves as the template upon which the complementary messenger RNA (mRNA) molecule is synthesized.

What is the fundamental role of the primers in this PCR setup?

Also known as the short, complementary DNA sequences provide the necessary free 3'-hydroxyl (3’-OH) group that DNA polymerase requires to initiate the synthesis of a new DNA strand.

What are key components required for a PCR reaction?

1. DNA template- contains the target sequence to be amplified 

2. Primers- two short synthetic DNA strands (FP and RP) 

3. DNA polymerase (Taq polymerase)- synthesize new DNA strands

4. dNTP mix- building blocks

5. Mg²⁺ ions- cofactor for DNA polymerase

6. Buffer- maintains optimal pH and ionic strength for the reaction

What is the basic principle of PCR amplification?

It amplifies a DNA sequence that lies between two regions of known sequence, using two sets of primers (forward and reverse primers, FP and RP).

What are dNTPs, and why are they needed?

Deoxynucleotide triphosphates: dATP, dTTP, dCTP, dGTP are the building blocks used by DNA polymerase to synthesize new DNA strands.

What is the role of Mg²⁺ ions in PCR?

Acts as a cofactor for DNA polymerase, stabilizing the reaction and helping the enzyme incorporate nucleotides efficiently.

What is the function of the PCR buffer?

Helps maintains the optimal pH and ionic environment for enzyme activity and DNA stability during PCR.

What is the typical composition of a PCR buffer?

• 100 mM Tris-HCl (pH 8.3) - maintains pH

• 500 mM KCl- provides ionic strength 

• 15 mM MgCl₂  - supplies cofactor

• 0.1% - gelatin stabilizes enzyme

What is the function of Taq polymerase?

Thermostable DNA polymerase (from Thermus aquaticus) that synthesizes new DNA strands during PCR cycles at high temperatures.

In which direction does DNA polymerase move along the template strand?

In the 3′ → 5′ direction along the template strand.

In which direction is the new (daughter) DNA strand synthesized?

In the 5′ → 3′ direction.

Why is the 3′-OH group of the primer essential in PCR?

DNA polymerase can only add new nucleotides to a free 3′-OH group, so primers provide the starting point for DNA synthesis.

How do primers determine the specificity of PCR?

They define the start and end points of amplification; only the DNA region between the two primers is copied.

What are primers in PCR, and what are their characteristics?

• Short, synthetically produced DNA sequences (usually 20–30 nucleotides long)

• Complementary to the 3′ ends of the target DNA strands

• Provide a free 3′-OH group required by DNA polymerase to begin synthesis

• Should not be complementary to each other to avoid primer-dimer formation

What is the purpose of using two primers in PCR?

• The forward primer (FP) binds to one DNA strand.

• The reverse primer (RP) binds to the complementary strand.
  Together, they define the region of DNA to be amplified.

Why must primers not be complementary to each other?

They may bind to each other instead of the template DNA, forming primer-dimers and reducing amplification efficiency.

What enzyme is most commonly used in PCR?

Taq polymerase, although other natural or recombinant thermostable DNA polymerases may also be used.

Why must PCR enzymes be thermostable?

Because PCR involves repeated heating steps (up to 95°C) to denature DNA; thermostable enzymes can withstand high temperatures without denaturing.

Up to what temperature is Taq polymerase stable?

It remains stable at temperatures up to 95°C.

What is meant by high processivity in PCR enzymes?

Means the enzyme can continuously add nucleotides to the growing DNA strand without frequently dissociating from the template, ensuring efficient DNA synthesis.

What does Taq polymerase’s 5′–3′ exonuclease activity mean?

It can remove nucleotides from the 5′ end of DNA during synthesis, which is useful in some detection systems (like TaqMan probes).

Does Taq polymerase have proofreading activity?

No. Taq polymerase lacks 3′–5′ exonuclease (proofreading) activity, so it cannot correct errors during DNA synthesis.

In which direction does DNA synthesis occur during PCR?

5′ → 3′ direction.

How many main steps are there in a PCR cycle?

• Denaturation

• Annealing (Primer Hybridization)

• Extension (DNA Synthesis)

What happens during the denaturation step, and at what temperature?

• The double-stranded DNA is heated to 95°C.

• This breaks the hydrogen bonds between strands, producing single-stranded templates for replication.

What occurs during the annealing step, and what is its temperature range?

• The reaction is cooled to 50–60°C, allowing primers to bind (hybridize) to their complementary sequences on the single-stranded DNA template.

• The exact temperature depends on the melting temperature (Tm) of the primers.

What is the melting temperature (Tm) of a primer?

The Tm is the temperature at which half of the primer molecules dissociate (fall off) from the DNA template.

• Below Tm: primers bind stably.

• At annealing temperature (below Tm): primers successfully attach, allowing DNA synthesis to begin.

What happens during the extension (primer extension) step, and at what temperature?

• Occurs at about 72°C (optimal temperature for Taq polymerase).

• Taq polymerase adds complementary nucleotides to the primer, using the template strand and the dNTP mix floating in the buffer.

• New DNA strands are synthesized in the 5′ → 3′ direction.

What is the outcome of one complete PCR cycle?

At the end of the first cycle, two copies of the target DNA sequence are produced — each containing one original and one newly synthesized strand.

How does PCR achieve exponential amplification?

Each new DNA strand from one cycle acts as a template in the next, doubling the number of DNA copies with each cycle (e.g., 2, 4, 8, 16…).

What is the primary function of TRIzol in RNA isolation?

It contains guanidinium thiocyanate and phenol and is used to cause cell or tissue lysis and to denature proteins to prevent RNA degradation 

How is the aqueous phase separated from the organic phase?

Chloroform is added, and the mixture is centrifuged to allow the formation of three separate phases

Which phase contains the purified RNA?

Aqueous phase (the top, clear layer).

Which components are found in the organic phase and interphase?

It contains the bulk of the cellular DNA and Protein.

What reagent is used to precipitate the RNA, and what is the process called?

Isopropanol is added to the aqueous phase to cause the RNA to precipitate (become solid), which is then collected as the RNA pellet after centrifugation.

Why is it important to prevent RNA degradation?

RNA is highly susceptible to degradation by RNases found in the cell. Rapid lysis with a strong denaturing agent like TRIzol is crucial to inactivate these enzymes.

How can PCR amplification products be detected?

• Gel electrophoresis

• Capillary electrophoresis

• Quantification assays

• Sequencing of the amplified fragment

• Southern blot hybridization

What is the principle of gel electrophoresis in detecting PCR products?

Gel electrophoresis separates DNA fragments based on size and charge using an electric field through an agarose gel matrix.

• DNA fragments move toward the positive electrode (anode) because DNA is negatively charged.

• Smaller fragments migrate faster and further than larger ones.

What percentage of agarose gel is commonly used for PCR product visualization?

A 2% agarose gel is typically used, as it gives good resolution for small PCR products.

What is the main purpose of RT-PCR?

It is used to amplify RNA sequences by first converting them into complementary DNA (cDNA) using reverse transcriptase.

What is the starting material in RT-PCR?

RNA, often mRNA, is the starting material.

What enzyme is used in RT-PCR for the first step?

Reverse transcriptase (RNA-directed DNA polymerase), which synthesizes cDNA from an RNA template.

What are the two steps of two-step RT-PCR?

1. Reverse Transcription (RT): Converts RNA into double-stranded cDNA (ds cDNA).

2. PCR Amplification: Uses Taq polymerase to amplify the cDNA.

What is produced at the end of the reverse transcription step?

Double-stranded complementary DNA (ds cDNA) derived from the RNA template.

Why must the RNA used in RT-PCR be free of RNase contamination?

RNases degrade RNA rapidly; contamination can destroy the RNA template, making it impossible to synthesize cDNA and leading to failed amplification.

What does the quality of rRNA indicate in an RNA sample?

The quality of rRNA reflects the overall integrity of the RNA sample and correlates with the quality of the mRNA population used for reverse transcription.

What is the purpose of the overall process of RT-PCR?

To generate a stable double-stranded DNA copy (cDNA) from a single-stranded mRNA template for use in cloning, PCR, or quantification (qRT-PCR)

What is the primer used in Step 1 of RT-PCR, and where does it bind?

The Oligo dT (deoxy-thymidine) primer is used, and it binds the poly-A tail at the 3′ end of the mRNA.

What is the enzyme used to synthesize the first cDNA strand in Step 1 of RT-PCR?

Reverse Transcriptase is used to create the complementary DNA strand, forming an mRNA-cDNA hybrid.

What is the key action of the enzyme RNase H in Step 2 of RT-PCR?

RNase H specifically degrades the RNA strand (the mRNA) from the RNA-DNA hybrid, leaving the single-stranded cDNA.

What enzyme synthesizes the second DNA strand in Step 3 of RT-PCR?

DNA Polymerase synthesizes the second DNA strand.

How does the single-stranded cDNA prime itself for the second strand synthesis in RT-PCR?

The single-stranded cDNA folds back on itself, forming a hairpin loop at its 3′ end, which acts as a self-primer for the DNA polymerase.

What enzyme is used in Step 4 in RT-PCR to prepare the cDNA for cloning?

Terminal transferase is used to add single-stranded tails (e.g., poly-C tails) at the 3′OH ends.

What is the final molecule created by RT-PCR?

Double-stranded cDNA ready for insertion into an appropriate cloning vector.

Name two downstream applications for the final double-stranded cDNA.

PCR (Polymerase Chain Reaction) and qRT-PCR (quantitative Real-Time PCR).

What enzyme (often implied, but not listed as an incubation step) is required to cut the hairpin loop after Step 3 in RT-PCR?

S1 nuclease (a single-strand-specific nuclease) is typically used to cut the loop to fully linearize the double-stranded cDNA.

What is Nested PCR?

A two-step PCR technique that uses two sets of primers (outer and internal/nested primers) to increase the specificity and sensitivity of DNA amplification.

Why is Nested PCR used?

It is used because primers in conventional PCR may sometimes bind to the wrong region of DNA, producing unexpected or nonspecific products.

How does Nested PCR solve nonspecific amplification problems?

By using two sets of primers — the outer primers for the first round of amplification and nested (internal) primers for the second — which ensures that only the correct target DNA is amplified in the second step.

How many sets of primers are used in Nested PCR?

• Outer primers – used in the first PCR reaction.

• Nested (internal) primers – used in the second PCR reaction to re-amplify the correct product.

What is the main purpose of Nested PCR?

To increase sensitivity and specificity of amplification.

What is Multiplex PCR?

A technique where several primer pairs are used in a single reaction tube to amplify different genes simultaneously.

What is the purpose of using multiple primer pairs in Multiplex PCR?

To amplify several target sequences in a single reaction, saving time and reagents compared with conventional (monoplex) PCR.

Why is Multiplex PCR considered faster than conventional PCR?

Because multiple targets are amplified at the same time in one tube, rather than running separate reactions for each gene (monoplex PCR).

What is an important consideration before performing Multiplex PCR?

Primer optimization is essential to ensure that all primer pairs work efficiently together and do not interfere with each other.

How is the initial template (gene of interest) prepared for universal priming in Multiplex PCR

Gene-specific primers are used in an initial reaction. These primers have an added universal sequence on their 5′ end, flanking the target gene with common sequences

What is the function of the "universal sequences" in multiplex PCR

They serve as the common binding sites for the single pair of labeled universal primers used in the main amplification, allowing multiple targets to be amplified at once.

What component is used to label the amplicons for detection in Mutltiplex PCR?

A labeled universal forward primer (fluorescently tagged) is used in the main amplification step, incorporating the label into all amplified fragments.

What is the relationship between fluorescence intensity and gene expression in Multiplex PCR

The Fluorescence Intensity of the final product is directly proportional to the starting amount (expression level) of the target gene.

What technique is used to analyze the final Multiplex PCR products?

Capillary Electrophoresis is used to separate the fluorescently labeled fragments based on their size.

How are the different genes distinguished during the analysis in Multiplex PCR?

Each gene yields an amplicon of a unique size. In capillary electrophoresis, the different sizes separate into distinct peaks, identifying the gene.

What does the height/area of the peak in the final graph represent in Multiplex PCR?

The peak height or area (Fluorescence Intensity) represents the abundance (expression level) of that specific target gene in the original sample.

What tool is recommended for designing primers?

Use of software is recommended

What should be avoided to prevent intermolecular bonding?

Inter-strand homologies between forward and reverse primers that are complementary to each other.

How should the Tm of primers be designed?

Tm of forward primer = Tm of reverse primer; ensures both primers bind efficiently.

What should be the G/C content of primers?

40–60% GC (average 50%) to ensure stable binding of primer and template.

What sequences should primers avoid to prevent dimerization?

Primers should not have palindromes or inverted repeats.

Why should primers avoid long stretches of Gs (e.g., GGGG)?

Long G stretches can result in primer-dimers and noise.

What is the recommended product size for PCR primers?

100–700 bp.

How should target specificity of primers be ensured?

Avoid primers with low annealing temperatures; ensure they bind specifically to the target sequence.

How should the annealing temperature relate to the primer Tm?

Annealing temperature should be less than the Tm of primers to allow proper binding.

What should be avoided to prevent intermolecular bonding?

1. Avoid complementary sequences between forward and reverse primers (INTER-strand homology), which can cause primers to bind to each other.

2. Avoid complementary sequences within the same primer (INTRA-strand homology of 3 or more bases), which can form hairpins or self-annealing structures.

How can mis-priming be avoided?

Use proper annealing temperature; avoid low annealing temperature because low temperature causes nonspecific binding.

What role does primer design play in preventing mis-priming?

Carefully designed primers (following previous rules) reduce nonspecific binding.

How can the buffer prevent mis-priming?

Adjust monovalent cation (Na⁺/K⁺) concentration to optimize primer binding and reduce nonspecific interactions.

What is the rationale for using hot-start PCR?

Low temperatures cause mis-priming. Hot-start PCR inactivates DNA polymerase at low temperature, preventing nonspecific amplification until the reaction is heated.

How is hot-start PCR performed?

• Prepare reaction mix on ice

• Place in a preheated thermal cycler

• Use sequestered enzyme: active at high temperature, inactive at low temperature

Give examples of hot-start enzymes.

• Platinum Taq

• AmpliTaq Gold

• HotStarTaq

Why is PCR product cleanup performed?

Cleanup is performed for further analysis, e.g., cloning or sequencing.

What is gel elution, and what does it remove?

Gel elution removes all reaction components, misprimed products, and primer dimers.