WHY IS DTT USED IN PCR?

Deoxyribonucleic acid (DNA) is the blueprint for life. It holds the genetic instructions that determine the traits and characteristics of an organism. DNA can be extracted from cells and amplified using a technique called polymerase chain reaction (PCR). PCR is a powerful tool that allows scientists to make millions of copies of a specific DNA sequence in a short amount of time.

Principle of PCR

PCR is a three-step process that is repeated for 30-40 cycles. The steps are:

• Denaturation: The DNA is heated to a high temperature (95°C) to separate the two strands of the DNA.

• Annealing: The temperature is lowered (55-70°C) to allow short DNA sequences called primers to bind to the single-stranded DNA. Primers are complementary to the ends of the DNA sequence that is being amplified.

• Extension: The temperature is raised again (72°C) to allow a DNA polymerase enzyme to extend the primers, synthesizing new DNA strands.

This cycle of denaturation, annealing, and extension is repeated until millions of copies of the DNA sequence are produced.

Why is DTT Used in PCR?

Dithiothreitol (DTT) is an antioxidant that is commonly used in PCR. It helps to prevent the formation of disulfide bonds between cysteine residues in the DNA polymerase enzyme. Disulfide bonds can inactivate the enzyme and prevent it from synthesizing new DNA strands.

DTT acts in PCR in two ways:

• It reduces disulfide bonds between cysteine residues in the DNA polymerase enzyme, keeping it active.

• It scavenges free radicals, which can damage DNA and interfere with PCR.

Benefits of Using DTT in PCR

Using DTT in PCR has several benefits, including:

• Increased PCR efficiency: DTT helps to ensure that the DNA polymerase enzyme is fully active, which leads to more efficient amplification of the DNA sequence.

• Reduced background noise: DTT helps to reduce the formation of non-specific PCR products, which can interfere with the interpretation of the results.

• Improved DNA yield: DTT helps to improve the yield of DNA that is produced by PCR. This makes it easier to obtain enough DNA for downstream applications, such as DNA sequencing.

When to Use DTT in PCR

DTT is not always necessary for PCR. It is typically used when the DNA sample is known to contain high levels of cysteine residues or when the DNA polymerase enzyme is prone to inactivation by disulfide bonds.

Conclusion

DTT is a useful additive for PCR that can help to improve the efficiency, specificity, and yield of the reaction. It is commonly used in PCR when the DNA sample is known to contain high levels of cysteine residues or when the DNA polymerase enzyme is prone to inactivation by disulfide bonds.

Frequently Asked Questions (FAQs)

1. What is the role of DTT in PCR?
DTT acts as an antioxidant, preventing the formation of disulfide bonds between cysteine residues in the DNA polymerase enzyme. This helps keep the enzyme active and improves PCR efficiency.

2. When is DTT used in PCR?
DTT is used when the DNA sample contains high levels of cysteine residues or when the DNA polymerase enzyme is prone to inactivation by disulfide bonds.

3. What are the benefits of using DTT in PCR?
Using DTT in PCR can increase PCR efficiency, reduce background noise, and improve DNA yield.

4. Are there any alternatives to DTT that can be used in PCR?
Yes, there are other antioxidants that can be used in PCR instead of DTT, such as beta-mercaptoethanol (BME).

5. What is the optimal concentration of DTT for PCR?
The optimal concentration of DTT for PCR typically ranges from 1 to 10 mM.