Use of PCR Flashcards
lecture 3
Why is PCR considered a major breakthrough in molecular research?
PCR allows for the amplification of scarce DNA, manipulation of genetic material, forensic DNA analysis, detection of pathogens, diagnosis of genetic diseases, and various applications in biotechnology.
What are key tips for performing good PCR?
Use clean gloves and lab coats.
Maintain dedicated lab areas and clean benches.
Avoid contaminating templates and reagents.
Include proper controls (negative and positive).
Use UV to ensure contamination-free equipment.
Accurately pipette and handle tubes carefully.
What is the difference between DNA and RNA as PCR templates?
DNA can be used directly in PCR.
RNA requires reverse transcription (RT) to convert it into complementary DNA (cDNA) before amplification.
What is reverse transcription PCR (RT-PCR)?
RT-PCR involves:
Using reverse transcriptase to convert RNA to cDNA.
Amplifying the cDNA using standard or quantitative PCR.
What is the difference between endpoint PCR and real-time PCR (qPCR)?
Endpoint PCR: Semi-quantitative; results are analyzed at the end of the reaction.
qPCR: Quantitative; fluorescence is measured during the exponential phase of DNA amplification, providing precise data on template quantity.
What are the main ingredients for reverse transcription?
Template RNA
Primers (e.g., oligo(dT), random hexamers)
dNTPs
Reverse transcriptase enzyme
Buffer
What are key components of a qPCR reaction?
Clean and purified starting material.
Thermos lightcycler for fluorescence detection.
Fluorescent dye or probe (e.g., SYBR Green or TaqMan).
Master mix containing primers, dNTPs, Mg²⁺, buffer, and DNA polymerase.
How does SYBR Green work in qPCR?
SYBR Green binds to the grooves of double-stranded DNA (dsDNA) and fluoresces, with fluorescence intensity increasing as more DNA is amplified.
How does TaqMan work in qPCR?
TaqMan uses fluorescent probes with a reporter and quencher. When the probe is degraded during amplification, fluorescence increases proportional to the DNA quantity.
What is the cycle threshold (Ct) in qPCR?
Ct is the cycle number at which fluorescence surpasses the threshold line. Lower Ct values indicate higher initial cDNA amounts (more starting material).
Why are reference genes important in qPCR?
Reference genes, or housekeeping genes, have constant expression and are unaffected by experimental conditions. They validate RNA extraction efficiency and ensure accurate quantification.
What are examples of reference genes used in qPCR?
Common examples include:
Beta-actin
GAPDH
Albumin
18S rRNA
TATA-binding protein
What are the differences in applications between endpoint PCR and qPCR?
Endpoint PCR: Used for sequencing, genotyping, and cloning.
qPCR: Used for quantifying gene expression, viral loads, assay validation, SNP genotyping, and RNA interference experiments.
What is a standard curve in qPCR, and why is it important?
A standard curve is created using serial dilutions of known DNA concentrations. It helps quantify unknown samples and normalize data to reference genes.
What does “normalization of qPCR data” mean?
Normalization adjusts the target gene expression levels to reference gene expression to ensure accurate comparisons between samples.
