Nucleic Acid Amplification

What are the two main types of amplification techniques in molecular diagnostics?

Target amplification and signal amplification.

What is the polymerase chain reaction (PCR)?

A primer-directed enzymatic reaction for producing specific DNA fragments.

What are the steps involved in the PCR process?

Denaturation, annealing, and elongation.

What is the purpose of controls in PCR?

To ensure specificity, sensitivity, and to control for contamination.

What is the role of primers in PCR?

Primers are short DNA fragments that determine the specificity of the PCR reaction.

What is the significance of Taq polymerase in PCR?

Taq polymerase is a heat-stable enzyme used for DNA synthesis in PCR.

What is the function of dNTPs in a PCR reaction?

dNTPs are the building blocks for DNA synthesis during PCR.

What is the difference between target amplification and signal amplification?

Target amplification increases the copy number of the target sequence, while signal amplification increases detection sensitivity without changing the copy number.

What are some examples of transcription-based amplification systems?

Nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), and self-sustaining sequence replication (3SR).

What is the purpose of a blank reaction in PCR?

To control for contamination by containing all reagents except the DNA template.

What is a positive control reaction in PCR?

A reaction that contains all reagents and a known target-containing DNA template to ensure sensitivity.

What is the amplification program in PCR?

A series of 20-50 cycles of denaturation, annealing, and extension.

What is the typical temperature for denaturation in PCR?

90-96°C.

What is the annealing temperature range in PCR?

40-68°C.

What is the extension temperature in PCR?

70-75°C.

What is the expected number of amplicons after N cycles of PCR?

The number of amplicons is 2^N, where N is the number of cycles.

What are some common issues that can lead to contamination in PCR?

Carelessness, bad technique, and exposure of pre-PCR reagents to post-PCR contaminants.

What is the role of synthetic probes in amplification techniques?

Synthetic probes bind to target sequences and are amplified to increase detection sensitivity.

What is the purpose of a negative control reaction in PCR?

To control for specificity by containing all reagents and a DNA template lacking the target sequence.

What is the significance of the GC content in primers?

GC content of 45-60% is optimal for primer stability and specificity.

What does the term 'amplicon' refer to in PCR?

The amplified product of the PCR reaction.

What are some modifications of PCR?

Multiplex PCR, reverse-transcriptase PCR, nested PCR, real-time PCR, and arbitrarily primed PCR.

What is the purpose of using dedicated pipettors in PCR?

To prevent contamination between pre- and post-PCR processes.

What is the role of magnesium ions (Mg++) in PCR?

Mg++ is a cofactor necessary for the activity of DNA polymerase during amplification.

What is the purpose of air-locks and positive airflow in PCR?

To ensure physical separation and prevent contamination.

What role does dUTP and uracil-N-glycosylase play in PCR?

They help prevent contamination by degrading any uracil-containing DNA.

What is the function of Psoralen in PCR?

It is used in conjunction with UV light, depending on the wavelength and distance to the surface.

What is the most effective solution for surface decontamination in PCR?

10% bleach.

What are some common contaminants that can affect PCR?

Detergents, phenol, heparin, heme, dyes, and body fluids like CSF, urine, and sputum.

What should be checked if there is a lack of PCR product?

Verify all components were added, check pipettors and reagents, and review the detection method.

What can cause too many bands in a PCR result?

Issues with primer specificity, low annealing temperature, or excessive Mg++ or cycles.

What are primer dimers in PCR?

They occur when primers anneal to each other instead of the target DNA, resulting in a band size equal to the sum of the two primer lengths.

What is the typical duration for a PCR process?

Usually 2 to 5 hours.

What is the amplification range of DNA or RNA in PCR?

From minute amounts directly from clinical samples, achieving high sensitivity and specificity.

What is the maximum DNA sequence length that can be amplified using PCR?

Up to 30 kb.

What factors affect the specificity of PCR amplification?

Temperature and Mg++ concentration.

What is the purpose of a thermal cycler in PCR?

To change temperatures in a block or chamber holding the samples for repeated cycles.

What type of polymerases are used in PCR?

Thermostable polymerases that can withstand high denaturation temperatures.

What is the significance of the expected size of the PCR product?

The product should match the expected size, and no product should be present in the reagent blank.

What is the role of gel elution in PCR?

It removes all reaction components, including misprimes and primer dimers.

What is nested PCR?

A technique that uses two pairs of primers to amplify a single target in two separate PCR reactions for increased sensitivity and specificity.

What is reverse transcription in PCR?

The process of generating complementary DNA (cDNA) from an RNA template using reverse transcriptase.

What is quantitative real-time PCR (qPCR)?

A method that detects PCR products by fluorescence during the reaction, allowing for quantification of the target DNA.

What are the two common methods of quantification in qPCR?

Fluorescent dyes that intercalate with double-stranded DNA and modified DNA oligonucleotide probes that fluoresce when hybridized with complementary DNA.

What is the significance of the exponential growth of PCR products?

PCR products grow exponentially, doubling at each cycle, which is observed as an exponential curve during detection.

What does a threshold level of fluorescence indicate in qPCR?

It is determined based on the signal and background, indicating the presence and amount of the target DNA.

What is the relationship between input and threshold cycle in qPCR?

Input is inversely proportional to the threshold cycle, which is the cycle at which fluorescence crosses the threshold fluorescence level.

What are DNA-specific dyes used in qPCR?

Ethidium bromide and SyBr® green.

What are the two types of hybridization probes used in qPCR?

Cleavage-based probes (TaqMan®) and displaceable probes (Molecular Beacons®, FRET®).

How do DNA-specific dyes function in qPCR?

They bind and fluoresce double-stranded DNA nonspecifically.

What is the role of hybridization probes in qPCR?

They only bind and fluoresce the intended PCR product.

What is the function of primer-incorporated probes in qPCR?

They label the PCR product.

How does SyBr® Green work in qPCR?

It binds to the minor groove of double-stranded DNA and can be tested in a post-amplification melt curve.

What is the significance of Taq polymerase's 5' to 3' exonuclease activity in qPCR?

It allows for the measurement of fluorescent signals generated by the separation of dye and quencher.

What is a TaqMan® probe?

A probe that measures the accumulation of product at the annealing step in PCR and is modified to avoid degradation during extension.

How does the hairpin structure of Molecular Beacons® function?

When not bound to the template, the hairpin brings the fluorophore close to the quencher, preventing fluorescence.

What happens to the fluorescence signal when the probe binds to the template in qPCR?

Fluorescence occurs upon binding, and the signal is restored when the probe is displaced by Taq polymerase.

What is the principle behind FRET® in qPCR?

Fluorescent resonance energy transfer occurs when donor and acceptor fluorophores are brought within 1-5 nucleotides of each other.

What are the components of a FRET® probe?

A 3' fluorophore (acceptor) and a 5' catalyst for fluorescence (donor).

What is the relationship between template availability and fluorescence generation in qPCR?

The more template available for binding, the more fluorescence is generated.

What type of equipment is used for real-time PCR detection?

Thermal cyclers with fluorescent detection and specialized software.

What is the purpose of using optically clear plates in qPCR?

To allow for fluorescent detection during the PCR reaction.

What is the significance of probe design in qPCR?

Probe design is essential for successful qPCR as it affects the accuracy and efficiency of the assay.

What is the role of excess probe in qPCR?

It ensures that with every doubling of the target, more fluorescence is generated.

What is the purpose of a negative control in PCR?

To check reagents for contamination by not including any DNA.

What does a no reverse transcriptase control detect?

It detects if the signal is coming from contaminating DNA.

What is the function of a positive control in PCR?

To check that reagents and primers work, especially important for showing absence of gene expression.

What is a major problem in PCR that can lead to false results?

Contamination.

Why is amplification of dsDNA more sensitive than that of cDNA?

Because dsDNA can be amplified from lower quantities and is less prone to degradation.

What is the significance of using a medium copy number in PCR?

It allows for more accurate corrections in quantification.

What are some commonly used standards in PCR?

Glyceraldehyde-3-phosphate dehydrogenase mRNA, Beta-actin mRNA, MHC I mRNA, Cyclophilin mRNA, and ribosomal protein mRNAs.

What is the role of reverse transcriptase in PCR?

To copy RNA into complementary DNA (cDNA).

What does the term 'D Ct' represent in PCR analysis?

The difference in threshold cycle between the target and reference genes.

What is the purpose of purification methods in PCR?

To remove PCR inhibitors and ensure the sample is free of contaminants.

What is the Transcription-Mediated Amplification (TMA) process?

An isothermal reaction that amplifies RNA or DNA targets using reverse transcriptase and RNA polymerase.

What is NASBA in molecular biology?

Nucleic Acid Sequence-Based Amplification, a method for amplifying RNA targets.

What is the Ligase Chain Reaction (LCR)?

A method that uses ligation of adjacent probes to amplify specific sequences.

What does the term 'primer-dimer' refer to in PCR?

Non-specific amplification products formed when primers anneal to each other instead of the target DNA.

What is the significance of using oligonucleotide-T7P primers in NASBA?

They hybridize to target sequences for amplification and detection.

How does the Hybridization Protection Assay (HPA) work?

It allows for simultaneous detection of multiple targets through hybridization of probes.

What is the main advantage of using isothermal reactions in amplification?

They eliminate the need for a thermocycler, reducing the total time for the procedure.

What are the key components of a successful PCR reaction?

Specific primers, high efficiency, and absence of primer-dimers.

What does 'ssDNA or RNA' refer to in the context of PCR?

Single-stranded DNA or RNA that serves as the target for amplification.

What is the role of RNase H in the NASBA process?

It digests RNA in RNA:DNA hybrids to facilitate further amplification steps.

What is the importance of ensuring samples are free of protein in PCR?

To avoid interference with the PCR reaction and ensure accurate results.

What is the primary purpose of Ligase Chain Reaction (LCR)?

To amplify genomic DNA by detecting target sequences.

What are the two main stages of the Ligase Chain Reaction?

Target generation and exponential amplification.

At what temperature is the target DNA denatured in LCR?

95°C.

What role do primers play in Ligase Chain Reaction?

Primers bind close to the target sequence and are extended by DNA polymerase.

What is the function of the probes in Ligase Chain Reaction?

Probes have a recognition sequence for a restriction endonuclease and are displaced during amplification.

What type of DNA polymerase is used in LCR?

Exonuclease-deficient DNA polymerase.

What is the significance of the modified nucleotide used in LCR?

It is incorporated during primer extension to facilitate the amplification process.

What happens to the probes after they are displaced in LCR?

A second set of complementary primers binds to the displaced probes, leading to the production of double-stranded versions of the probes.

What is the temperature at which the modification of the probe occurs after adding the restriction enzyme?

52°C.

What is the outcome of the restriction enzyme action on the ds probe DNA?

It cuts only one strand of the probe to form a nick, which is then extended by DNA polymerase.

What is the result of the Strand Displacement Amplification (SDA) process?

Millions of copies of the initial probe are produced.

What is the role of the branched DNA (bDNA) in detection sensitivity?

It amplifies the signal rather than the product itself, enhancing detection sensitivity.

What is the purpose of the capture probes in branched DNA detection?

To hybridize with target nucleic acid sequences for signal amplification.

How does quantitative PCR differ from traditional PCR?

Quantitative PCR quantifies the target in addition to amplifying it.

What is the significance of contamination control in PCR laboratories?

It is crucial for ensuring the accuracy and reliability of results.