PCR Flashcards
(19 cards)
What is PCR and why is it important in molecular diagnostics?
PCR (Polymerase Chain Reaction) is a technique used to amplify specific DNA sequences. It is crucial in molecular diagnostics because it allows detection and analysis of minute amounts of DNA by amplifying them to detectable levels, aiding in identifying infections, genetic mutations, and more.
Who invented PCR and why is thermostable DNA polymerase essential to its function?
PCR was invented by Kary Mullis. Thermostable DNA polymerase is essential because PCR involves repeated heating to ~95°C to denature DNA, and the enzyme must withstand these temperatures to synthesize DNA.
What are the essential reagents for a PCR reaction?
Essential reagents include: DNA template, primers (forward and reverse), thermostable DNA polymerase, dNTPs (nucleotides), buffer (with correct pH and cofactors like Mg2+), and water.
Describe the three main steps in a PCR cycle and their purpose.
- Denaturation (~95°C): Separates DNA strands. 2. Annealing (~50-65°C): Primers bind to specific DNA sequences. 3. Extension (~72°C): DNA polymerase extends primers to synthesize new DNA strands.
How does exponential amplification occur in PCR?
Each cycle of PCR doubles the amount of DNA. Starting from 1 copy, 30 cycles can theoretically produce over a billion copies due to exponential growth (2^n).
What is the role of primers in PCR?
Primers provide the starting point for DNA polymerase to begin synthesis. They are designed to bind to specific sequences flanking the target DNA region, conferring specificity to the reaction.
Why is contamination a significant concern in PCR, and how is it addressed?
Due to PCR’s high sensitivity, even trace amounts of contaminant DNA can be amplified, causing false positives. Controls (negative, positive, and internal) are used to monitor and validate results.
What are the types and purposes of PCR controls?
- Negative control: Contains all reagents except the DNA template; checks for contamination. 2. Positive control: Contains known template; checks reaction performance. 3. Internal control: Amplifies a known sequence alongside the test target; ensures reaction conditions are functional.
How is PCR product size determined and validated?
PCR product size is determined by comparing it against molecular weight markers using gel electrophoresis. Bands indicate the size and presence of amplified DNA fragments.
What is quantitative PCR (qPCR) and how does it differ from standard PCR?
qPCR quantifies DNA in real-time using fluorescence, allowing determination of initial DNA concentration. Standard PCR only indicates presence/absence and quantity of DNA post-reaction.
How does SYBR Green work in qPCR?
SYBR Green intercalates into double-stranded DNA. Its fluorescence increases proportionally with DNA quantity, enabling real-time monitoring of PCR product formation.
What is reverse transcription PCR (RT-PCR) and when is it used?
RT-PCR converts RNA to cDNA using reverse transcriptase, followed by PCR. It is used when starting material is RNA, such as in viral RNA detection (e.g., HIV, SARS-CoV-2).
What are common diagnostic uses of PCR?
PCR is used to detect pathogens (e.g., HSV, Chlamydia), antibiotic resistance genes (e.g., mecA for MRSA), and mutations causing genetic disorders (e.g., CF, sickle cell disease).
What are single gene mutations and how can PCR detect them?
Single gene mutations include point mutations and indels. PCR can detect them via allele-specific PCR or sequencing of amplified products.
What is allele-specific PCR and how does it work?
Allele-specific PCR uses primers that match either the normal or mutant sequence. Only the matching allele is amplified, allowing detection of specific mutations.
What is Restriction Fragment Length Polymorphism (RFLP) analysis and how is it used with PCR?
RFLP uses restriction enzymes to cut DNA at specific sequences. PCR amplifies the target DNA first; then mutations affecting restriction sites change the fragment lengths, detected via gel electrophoresis.
What types of genetic disorders can PCR help diagnose?
- Single gene disorders (e.g., CFTR in cystic fibrosis) 2. Chromosomal abnormalities (e.g., trisomy 21) 3. Multifactorial disorders (e.g., some cancers, Alzheimer’s).
What is CAPS and how is it related to RFLP?
CAPS stands for Cleaved Amplified Polymorphic Sequences. It is a type of RFLP where PCR amplifies a region followed by restriction enzyme digestion to detect polymorphisms.
How does gel electrophoresis help in analyzing PCR results?
It separates DNA fragments based on size. When PCR products are loaded and run through an electric field, they migrate through the gel and form bands that are visualized under UV light.
