Amplification techniques Flashcards
Who invented the PCR machine, and when?
Kary Mullis invented the PCR machine in 1983.
What is the role of thermostable polymerases in PCR?
Thermostable polymerases (e.g., Taq polymerase) allow DNA replication at high temperatures, improving PCR efficiency.
What are the three major stages of PCR?
- Denaturation
- Primer Annealing
- Elongation
What are the essential components required for PCR?
- Thermostable DNA polymerase
- Deoxynucleotides (dNTPs)
- Target DNA sequence
- A pair of oligonucleotide primers
What is the purpose of primers in PCR?
Primers bind to complementary sequences on the target DNA, providing a starting point for DNA synthesis.
What happens during the denaturation step of PCR?
The DNA is heated to 94-98°C to separate the double-stranded DNA into single strands.
What is the annealing temperature range in PCR?
50-65°C, where primers bind to the target DNA.
What occurs during the elongation step of PCR?
The temperature is raised to 72°C, allowing DNA polymerase to extend the primers and synthesize new DNA strands.
What factors affect PCR efficiency?
- Concentration of primers and DNA polymerase
- Temperature cycling protocol
- Presence of polymerase inhibitors
How does the size of the PCR product affect the reaction time?
Larger PCR products require longer elongation times, increasing the overall reaction time.
What is digital PCR?
A method that partitions a sample into thousands of individual reactions, allowing absolute quantification of nucleic acids.
What is the purpose of ligase chain reaction (LCR)?
LCR amplifies DNA by ligating adjacent probes that are complementary to the target sequence.
What is strand displacement amplification (SDA)?
An isothermal amplification method that uses a DNA polymerase with strand-displacing activity to amplify DNA.
What are the two main methods of nucleic acid detection?
- Generic measurement or visualization
- Quantification of specific nucleic acid sequences
How is UV spectroscopy used in nucleic acid detection?
Nucleic acids absorb UV light at 260 nm, allowing measurement of their concentration and purity.
What is the significance of the 260:280 ratio in nucleic acid purity?
A ratio of 1.7 to 2.0 indicates pure nucleic acid, while lower ratios suggest protein contamination.
What are some common fluorescent dyes used in nucleic acid detection?
Ethidium bromide and SYBR Green I.
Why are fluorescent dyes more sensitive than UV absorbance?
Fluorescent dyes are 1,000 to 10,000 times more sensitive because they only fluoresce when bound to nucleic acids.
What is the purpose of labeled probes in nucleic acid detection?
Labeled probes bind to complementary sequences, allowing specific detection of target nucleic acids.
What are the limitations of radioactively labeled probes?
They have short half-lives, are unstable, and pose safety and disposal concerns.
What are some non-radioactive labels used in probes?
Biotin and digoxigenin, which require enzyme-linked detection systems.
What is the main advantage of real-time PCR?
It allows simultaneous amplification and detection of nucleic acids, providing real-time quantification.
What are some common dyes and probes used in real-time PCR?
SYBR Green I, TaqMan probes, and molecular beacon probes.
How is fluorescence used to quantify nucleic acids in real-time PCR?
Fluorescence increases as the dye binds to double-stranded DNA, and the signal is proportional to the amount of target DNA.
What is the GeneXpert system used for?
It automates sample purification, nucleic acid amplification, and detection using real-time PCR for pathogens like HIV, TB, and SARS-CoV-2.
What are some limitations of the GeneXpert system?
- It cannot differentiate between live and dead organisms.
- Mutations in primer or probe binding regions may affect results.
What are the internal quality controls in the GeneXpert system?
- Sample Volume Adequacy (SVA)
- Probe Check Controls (PCC)
- Sample Processing Controls (SPC)
Why is Taq polymerase used in PCR?
Taq polymerase is thermostable, allowing it to withstand the high temperatures used in PCR cycles.
What is the difference between SYBR Green I and TaqMan probes?
SYBR Green I binds to any double-stranded DNA, while TaqMan probes are sequence-specific and provide higher specificity.
How does real-time PCR differ from traditional PCR?
Real-time PCR monitors amplification in real-time using fluorescence, while traditional PCR requires post-amplification analysis (e.g., gel electrophoresis).
