1) PCR (Polymerase Chain Reaction) Tutorial - An Introduction

Summary

This video from Applied Biological Materials introduces PCR, a vital molecular biology technique used for DNA amplification in various applications like sequencing and diagnostics. It explains the PCR process, including denaturation, annealing, and elongation, and highlights the importance of factors like primer design and DNA polymerase choice. The video also discusses challenges and solutions, such as GC-rich templates and PCR inhibitors, and promotes abm's range of PCR products.

Takeaways

• 🔬 PCR, or Polymerase Chain Reaction, is a fundamental technique in genetic and molecular biology with applications in DNA sequencing, forensics, and disease diagnosis.
• 📈 PCR enables the rapid and cost-effective amplification of DNA fragments from a small quantity of nucleic acid, often referred to as 'molecular photocopying'.
• 🔄 The PCR process involves repeated cycles of DNA replication, doubling the number of DNA strands with each cycle, resulting in over a trillion copies from a single DNA molecule.
• 🔥 The PCR procedure consists of four main steps: initialization, denaturation, annealing, and elongation, each with specific temperature requirements.
• 🔬 Initialization involves heating to activate DNA polymerase and denature contaminants, facilitating cell lysis in the case of colony PCR screening.
• 🧬 Denaturation breaks the hydrogen bonds of double-stranded DNA by heating, separating the strands for primer binding and replication.
• 🔍 Annealing lowers the temperature to allow primers to bind to the DNA template, guiding the DNA polymerase for replication.
• 📝 Elongation involves the synthesis of a new DNA strand by DNA polymerase incorporating dNTPs, with conditions depending on the enzyme and target sequence.
• 🔥 Taq Polymerase is the most commonly used enzyme in PCR, being thermostable and allowing for multiple amplification cycles without the need for enzyme replenishment.
• 🛠️ Variations of Taq enzyme have been engineered for specific PCR applications, such as those with decreased error rates or the ability to amplify DNA directly from blood samples.
• 🧪 Gel electrophoresis is used to visualize and estimate the size of amplified DNA fragments, ensuring successful gene amplification.
• 🚫 Many factors can interfere with PCR, including DNA template composition, enzyme choice, buffer components, primer design, and the presence of inhibitors.

Q & A

What is Polymerase Chain Reaction (PCR)?

PCR is a molecular biology technique that allows for the fast and inexpensive amplification of DNA fragments, generating large quantities of DNA from a small amount of nucleic acid, and is often referred to as molecular photocopying.

What are some common applications of PCR?

PCR is widely used in DNA sequencing, DNA fingerprinting, forensics, detection of microorganisms, and diagnosis of hereditary diseases.

How does PCR work in terms of DNA replication cycles?

PCR depends on 20 to 40 repeated cycles of DNA replication by a DNA polymerase enzyme, doubling the number of DNA strands after each cycle, resulting in over a trillion copies from a single DNA molecule.

What are the four main steps of the PCR process?

The PCR process consists of initialization, denaturation, annealing, and elongation.

What happens during the initialization step of PCR?

In the initialization step, the reaction is heated to activate the DNA polymerase and to denature other contaminants, facilitating cell lysis to release DNA and denature cellular proteins in the case of colony PCR screening.

What is the purpose of the denaturation step in PCR?

The denaturation step involves breaking hydrogen bonds between the double-stranded DNA by heating, which separates the DNA strands to prepare for primer binding.

What role do primers play in the annealing step of PCR?

Primers bind to a complementary sequence in the DNA template during the annealing step, guiding the DNA polymerase enzyme for replication.

What is the function of DNA polymerase during the elongation step?

DNA polymerase incorporates dNTPs in a 5' to 3' direction to synthesize a new DNA strand during the elongation step.

Why is Taq polymerase commonly used in PCR?

Taq polymerase is a thermostable DNA polymerase that allows multiple cycles of amplification without the need for new enzyme addition after each denaturation step.

How can the size of amplified DNA fragments be determined after PCR?

Gel electrophoresis is used to visualize and estimate the size of amplified DNA fragments by comparing the gel bands to a molecular weight marker.

What are some factors that can interfere with a PCR reaction?

Factors that can interfere with PCR include nucleotide composition of the DNA template, choice of DNA polymerase enzyme, buffer components, primer design, additives, and inhibitors.

How can PCR be optimized for different types of DNA templates?

Optimizing PCR for different templates may involve adjusting the annealing temperature, using additives for GC-rich or AT-rich templates, and selecting the appropriate DNA polymerase and buffer.

What is the significance of depurination in long template amplification?

Depurination, where the β-N-glycosidic bond in purine nucleoside is cleaved, can lead to incomplete replication as Taq DNA polymerase will not extend through apurinic positions, making it a limiting factor for long template amplification.

How can PCR inhibitors be managed in a reaction?

PCR inhibitors can be managed by using sample-specific nucleic acid isolation protocols or by using additives that help reduce inhibition.

What resources does Applied Biological Materials (abm) offer for PCR?

Applied Biological Materials offers a range of DNA polymerases, formulated PCR MasterMixes for each polymerase, and kits for easy DNA amplification from various samples such as plants, tissue, and blood.

Outlines

00:00

🔬 Introduction to PCR Technology

This paragraph introduces the fundamental concept of Polymerase Chain Reaction (PCR), a critical technique in genetic and molecular biology. PCR is utilized for various applications such as DNA sequencing, fingerprinting, forensics, microorganism detection, and hereditary disease diagnosis. It enables the rapid and cost-effective amplification of DNA fragments from a minimal amount of nucleic acid, earning it the nickname 'molecular photocopying.' The process involves repeated cycles of DNA replication catalyzed by a DNA polymerase enzyme, resulting in an exponential increase in DNA strands. The PCR process is structured into four main steps: initialization, denaturation, annealing, and elongation, each with specific temperature requirements and functions. The video also mentions the use of a final elongation step and a holding step for short-term storage of PCR products.

05:01

🌡 PCR Process Details and Challenges

The second paragraph delves deeper into the PCR process, discussing the initialization step that activates DNA polymerase and denatures contaminants, as well as the denaturation step that separates DNA strands by breaking hydrogen bonds. Primers are highlighted as essential for guiding DNA replication during the annealing step, followed by the elongation step where DNA polymerase synthesizes new strands. The paragraph also addresses the challenges in PCR, such as dealing with GC-rich and AT-rich templates, which may require specific additives or adjustments in temperature to overcome issues like incomplete strand separation and secondary structures. Long templates present their own difficulties due to the risk of depurination, which can be mitigated with higher pH buffers or proofreading DNA polymerases. The paragraph emphasizes the importance of selecting the right DNA polymerase and buffer for the template, designing primers carefully to avoid dimer formation, and dealing with PCR inhibitors that can be present in natural substances. The video script concludes with a mention of the various PCR products offered by Applied Biological Materials, including DNA polymerases and PCR MasterMixes, and invites viewers to follow their next video and engage with the content.

Keywords

💡PCR

PCR, or Polymerase Chain Reaction, is a core technique in molecular biology used to amplify a specific DNA sequence. It is central to the video's theme, as it discusses the process and applications of PCR. The script mentions that PCR is indispensable for various applications such as DNA sequencing, forensics, and disease diagnosis.

💡DNA sequencing

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It is mentioned in the script as one of the applications of PCR, highlighting its importance in genetic research and molecular biology.

💡Denaturation

Denaturation in the context of PCR refers to the process of breaking the hydrogen bonds between the two strands of a DNA molecule, resulting in the separation of the strands. The script describes this as a step in the PCR process where the reaction is heated to break these bonds, allowing for primer binding and replication.

💡Annealing

Annealing is the process where primers bind to the complementary sequence on the DNA template. It is a critical step in PCR, as mentioned in the script, where the reaction temperature is lowered to allow primers to attach optimally before replication begins.

💡Elongation

Elongation in PCR is the step where DNA polymerase synthesizes a new DNA strand by adding nucleotides in a 5' to 3' direction. The script explains that the temperature and time for this step depend on the enzyme and target sequence, and it is where the actual replication of DNA occurs.

💡DNA polymerase

DNA polymerase is an enzyme that catalyzes the synthesis of a new DNA strand during PCR. The script emphasizes its importance, mentioning Taq polymerase as a commonly used enzyme that is thermostable and allows for multiple cycles of amplification.

💡Taq polymerase

Taq polymerase is a specific type of DNA polymerase that is heat-resistant and widely used in PCR. The script discusses its role and mentions that variations of Taq enzyme have been engineered for different PCR applications, such as those with decreased error rates or the ability to amplify DNA directly from blood samples.

💡Gel electrophoresis

Gel electrophoresis is a technique used to separate and visualize DNA fragments by size after PCR amplification. The script describes its use for determining the success of the PCR by comparing the size of the amplified products to a molecular weight marker.

💡Depurination

Depurination is a chemical reaction that can damage DNA by cleaving the β-N-glycosidic bond in purine nucleosides, releasing a nucleotide base. The script discusses its impact on long template amplification and how it can be mitigated by using a higher pH buffer or a proofreading DNA polymerase.

💡Primer

A primer in PCR is a short nucleotide sequence that provides a starting point for DNA polymerase to begin replication. The script explains that primers bind to the DNA template and are crucial for the specificity of the PCR reaction, with considerations such as avoiding self-dimer formation.

💡Inhibitors

Inhibitors are substances that can interfere with the PCR reaction, such as polysaccharides, tannic acid, and EDTA. The script mentions that these can be removed with specific protocols or mitigated with additives, emphasizing the need to manage them for successful PCR.

Highlights

PCR is an indispensable technology in genetic and molecular biology with a wide range of applications.

PCR allows fast and inexpensive amplification of DNA fragments from a small amount of nucleic acid.

The PCR process involves repeated cycles of DNA replication by a DNA polymerase enzyme.

After each PCR cycle, the number of DNA strands doubles, resulting in over 1 trillion copies from a single DNA molecule.

The PCR process consists of four steps: initialization, denaturation, annealing, and elongation.

Initialization involves activating DNA polymerase and denaturing contaminants at 94–96°C.

Denaturation breaks hydrogen bonds in double-stranded DNA by heating to 94–98°C.

Primers bind to the DNA template during annealing to guide DNA polymerase replication at 50-65°C.

Elongation involves synthesizing a new DNA strand with DNA polymerase at 72-78°C.

Taq Polymerase is a commonly used thermostable DNA polymerase in PCR.

Applied Biological Materials offers modified Taq enzymes with decreased error rates and robust amplification from blood samples.

Gel electrophoresis is used to visualize and estimate the size of amplified DNA fragments.

Factors such as nucleotide composition, enzyme choice, buffer components, and primer design are crucial for PCR success.

GC rich templates may require the addition of secondary structure destablizers like DMSO for successful PCR.

AT rich templates require attention to primer annealing temperatures and the use of additives to maintain specificity.

Long templates are challenging to amplify due to the risk of DNA template breakage or degradation.

Depurination is a limiting factor for long template amplification, which can be mitigated with higher pH buffers or proofreading enzymes.

The choice of DNA polymerase and buffer is critical for successful PCR with different DNA templates.

Primer design considerations include length, annealing temperature, and sequence to avoid dimer formation.

Natural substances like polysaccharides, tannic acid, and EDTA can inhibit PCR and may require specific isolation protocols or additives.

Applied Biological Materials provides a range of DNA polymerases and PCR MasterMixes tailored for various applications.

Transcripts

Welcome to Applied Biological Materials’ PCR video series! In this video,

we will introduce the basics of PCR. Polymerase chain reaction, or PCR is a technology

indispensible to genetic and molecular biology. Many of its applications are widely used in

the world around us, including DNA sequencing, DNA fingerprinting, forensics, detection of

microorganisms, and diagnosis of hereditary disease. PCR allows fast and inexpensive amplification

of DNA fragments. Because it is able to generate large quantities of DNA from a small amount

of nucleic acid, PCR is also referred to as molecular photocopying.

PCR depends on a series of 20 to 40 repeated cycles of DNA replication by a DNA polymerase

enzyme. After each cycle, the number of DNA strands is doubled, and at the end of a 40

cycle reaction, more than 1 trillion copies are generated from a single copy of the DNA

molecule. The PCR process is separated into four steps:

initialization, denaturation, annealing, and elongation. In the first initialization step,

the reaction is heated to 94–96°C for 2 to 10 minutes to activate the DNA polymerase

and to denature other contaminates in the mixture. In the case of colony PCR screening,

this step also facilitates cell lysis to release DNA and denature other cellular proteins.

In the denaturation step, hydrogen bonds between the double-stranded DNA are broken by heating

the reaction to 94–98°C for 20–30 seconds. After the DNA strands are separated, primers

bind to a complementary sequence in the DNA template to guide DNA polymerase replication.

In this step, the reaction temperature is lowered to 50-65°C for 20–40 seconds to

allow for optimal primer annealing. After the primers establish a starting point for

the enzyme, DNA polymerase starts to incorporate dNTPs in a 5’ to 3’ direction to synthesize

a new DNA strand. The temperature and extension time for this elongation step depend on the

type of DNA polymerase enzyme and the target amplicon. Commonly used Taq Polymerase polymerizes

at a speed of 1-1.5 kilobases per minute and works ideally at 72-78°C. The cycle then

repeats from denaturation to elongation 20 to 40 times. At the end of the last cycle,

there is a final elongation step that keeps the reaction mixture at 72-78°C for 5-15

minutes. This ensures that any remaining single-stranded DNA is fully extended after the last PCR cycle.

A final holding step keeps the PCR at 4-15°C for an indefinite time, keeping the products

for short-term storage. The most common DNA polymerase enzyme used

in PCR is Taq polymerase, a thermostable DNA polymerase that allows multiple cycles of

amplification without the addition of new enzyme after each denaturation step. Since

the initial discovery of Taq DNA polymerase, many variations of the Taq enzyme have been

engineered with different attributes that can be utilized for specific PCR applications.

For example, we at Applied Biological Materials, also known as abm, carry a modified Taq enzyme

that has decreased error rates. Also, abm has engineered a robust Taq enzyme that amplifies

DNA directly from blood samples. There are many more engineered Taq enzymes and other

DNA polymerases available. To visualize the DNA fragments after amplification

by PCR, gel electrophoresis is used. By comparing the gel bands to a molecular weight marker,

researchers can estimate the size of the amplified products to know if their desired genes were

successfully amplified. Many factors can interfere with a PCR reaction.

Some are easy to eliminate while others are trickier to manipulate and would require experience

to tackle. In general, factors that are important in PCR include the nucleotide composition

of the DNA template, DNA polymerase enzyme choice, buffer components, primer design,

additives and inhibitors. Depending on the sequence of the target DNA,

different strategies may be needed for a successful PCR. For example, GC rich templates have more

hydrogen bonds compared to AT rich templates. This results in incomplete strand separation

which may cause the PCR to fail. In addition, high GC content templates tend to lead to

more secondary structures, which can arrest the polymerase leading to pre-mature termination.

In these cases, secondary structure destablizers such as DMSO can be added.

At the same time, AT rich templates also require special attention because of their low primer annealing

temperatures. Non-specific primer annealing can occur under low annealing temperature,

and primer specificity can be retained using a combination of lowered extension temperature

and the use of additives such as TMAC. Long templates are difficult to amplify because

of their higher likelihood of DNA template being broken or degraded by depurination.

Depurination refers to a chemical reaction during which the β-N-glycosidic bond in the

purine nucleoside is cleaved to release a nucletide base due to hydrolysis. As Taq DNA

polymerase will not extend through apurinic positions during replication, depurination

is an important limiting factor of long template amplification. This can be avoided by using

a higher pH reaction buffer and decreasing the denaturation time to limit depurination,

or by using a proofreading DNA polymerase. As mentioned earlier, there are many variations

of DNA polymerases commercially available, each with different features. Depending on

the DNA template, choosing the correct DNA polymerase, and pairing it with an appropriate

buffer can be critical for a successful reaction. Apart from DNA polymerase choice, having a

good pair of primers can also dictate the outcome of a PCR. Some considerations to keep

in mind include the length of the primer, the annealing temperature, and the sequence

of the primer. For example, the pair of primers should not have complementary sequences; otherwise

they can easily lead to self-dimer or primer dimer formation and compete with template-primer

annealing. Many naturally occurring substances, such

as polysaccharides, tannic acid and EDTA, can also inhibit PCR. Some inhibitors may

degrade or modify the DNA template, while others can disturb the annealing of primers

to DNA or alter the DNA polymerase activity. Inhibitors can be removed with sample-specific

nucleic acid isolation protocols, or sometimes the use of additives can help in reducing

the inhibition. For a complete list of PCR additives and their mechanisms of action,

please refer to our knowledge base. A combination of favorable factors contributes

to a successful PCR. Taking all these factors into consideration, abm provides a wide range

of DNA polymerases, and has formulated optimal PCR MasterMixes for each polymerase. We also

offer kits that make DNA amplification from plants, tissue and blood samples easy! Check

out our comprehensive list of PCR products in the link provided below. Thank you for

watching and make sure you follow our next video on the variations of DNA polymerases.

Don’t forget to leave your comments and questions below.