PCR_and_Sequencing_Flashcards
What factors influence the choice of DNA extraction technique?
Target nucleic acid, starting material, expected results, and downstream applications.
What are examples of target nucleic acids?
RNA, circulating DNA, or other nucleic acids.
How does the starting material affect DNA extraction?
Tissue, cell culture, or biopsy dictates the approach to release nucleic acids.
Why is the extraction method important for downstream applications?
It must be compatible with the intended molecular biology test.
What is PCR used for?
Amplifying a specific DNA region in vitro to obtain sufficient quantities for study.
What is the outcome of PCR amplification?
Exponential replication of a DNA template.
What are the main steps of PCR?
Denaturation, hybridization, and polymerization.
What happens during denaturation in PCR?
Double-stranded DNA is separated into single strands by heating.
What occurs during hybridization?
Primers bind to the single-stranded DNA, defining the region to amplify.
What is polymerization in PCR?
DNA polymerase extends primers, creating complementary strands.
What are primers?
Short DNA sequences (forward and reverse) that bind to the target DNA region.
What is a thermocycler?
A machine that controls temperature cycles for denaturation, annealing, and extension.
What are PCR tubes made of?
High-quality polypropylene for efficient heat transfer.
What is the purpose of a PCR buffer?
To provide optimal conditions for the reaction.
How is electrophoresis used in PCR?
To separate DNA fragments by size using an electric charge.
What dye is used in electrophoresis to visualize DNA?
Ethidium bromide.
What is conventional PCR?
A basic method where amplified DNA fragments are visualized at the end using gel electrophoresis.
What is capillary electrophoresis?
A high-resolution technique that separates DNA fragments in a capillary tube.
What is real-time PCR used for?
Monitoring DNA amplification in real time, useful for quantitative analysis.
What is multiplex PCR?
A technique that detects multiple targets in a single reaction using different primer pairs.
What is a common challenge in PCR?
Contamination, which can cause false-positive results.
How can PCR contamination be prevented?
Using blank control samples and strict contamination controls.
Who developed Sanger sequencing?
Frederick Sanger, a two-time Nobel Prize winner.
What does Sanger sequencing use to terminate DNA synthesis?
Dideoxyribonucleotides (ddNTPs).
How does Sanger sequencing determine DNA sequences?
By analyzing the lengths of terminated DNA fragments.
What is Single Molecule Real Time (SMRT) sequencing?
A technique that sequences DNA in real time by immobilizing a single DNA polymerase on a chip.
What is nanopore DNA sequencing?
A method that directly analyzes long DNA or RNA fragments in real time using nanopores.
What are microfluidic systems?
Systems that manipulate small fluid volumes for automated and efficient sequencing.
Why is real-time PCR valuable?
It allows quantitative analysis, such as gene expression studies and viral load monitoring.
What is agarose gel electrophoresis used for?
Separating nucleic acid fragments (DNA or RNA) by size.
