L4-Molecular Methods-Jill Brooks-250225 Flashcards
(21 cards)
What does PCR stand for?
Polymerase Chain Reaction
A technique used to amplify DNA sequences.
Who won the Nobel Prize for Chemistry in 1993?
Kary Mullis
Known for the invention of PCR.
What is the role of a PCR machine?
Amplifies DNA in vitro
Also known as a thermocycler.
What are the components needed for a PCR reaction?
- Buffer containing MgCl2 (1-4mM)
- DNA template (10pg – 1mg)
- Primers (0.1-1 μM)
- dNTPs (0.2mM of each)
- Taq DNA polymerase (1.25u)
These components are essential for the PCR process.
What are the four phases associated with a PCR reaction?
- Sub-background
- Exponential
- Linear
- Plateau
These phases describe the stages of PCR product accumulation.
What is the purpose of a No Template Control (NTC)?
Checks for contamination
Contains all reagents except DNA template.
What is the technique for quantitative measurement of DNA or RNA called?
Quantitative PCR (qPCR)
Allows for real-time measurement during amplification.
What does Ct stand for in qPCR?
Cycle threshold
The cycle number at which fluorescence passes a defined threshold.
What is the main application of Sanger sequencing?
Determines DNA/RNA sequence
Uses dideoxyribonucleotides to stop synthesis.
What is the significance of the Human Genome Project (HGP)?
Mapped and sequenced all genes in the human genome
A major biomedical research project that began in 1990.
What does NGS stand for?
Next Generation Sequencing
Allows for rapid sequencing of DNA.
What is the read length typically achieved by Sanger sequencing?
600 - 900 base pairs
Known for high accuracy.
What is the role of clonal amplification in NGS?
Creates clusters of identical DNA fragments
Essential for sequencing by synthesis.
Fill in the blank: Taq polymerase only works on _______.
DNA templates
It is not effective for RNA.
What is an important control in qPCR?
TaqMan Probe
A specific probe used for real-time PCR.
What are the two main types of PCR mentioned?
- RT-PCR (Reverse Transcription PCR)
- qPCR (Real-Time PCR)
Both are used for different applications of nucleic acid analysis.
What is the typical coverage required for many applications in NGS?
30x
Higher coverage (1000x) is needed for detecting rare mutations.
What is the purpose of primer design in RT-PCR?
To prevent amplification of genomic DNA
Primers are designed to cross exon-exon boundaries.
True or False: RNA is very stable and easily amplified.
False
RNA is unstable and prone to degradation.
What does the 100,000 Genomes Project aim to achieve?
Sequence 100,000 genomes from NHS patients
Focuses on rare diseases and cancer.
What types of data does The Cancer Genome Atlas (TCGA) Program provide?
Genomic, epigenomic, transcriptomic, and proteomic data
Characterizes over 20,000 primary cancer samples.
