Lecture 3 : Molecular Methods Flashcards
LO
Understand PCR (theory and prac)
DNA and RNA sequencing
How to set up PCR reaction
- Gather Reagents
- Nuclease-free water
- PCR buffer (with Mg²⁺)
- dNTPs (deoxynucleotide triphosphates)
- Forward and reverse primers
- Template DNA
- DNA polymerase (e.g., Taq polymerase)
- Prepare the Master Mix (if running multiple reactions) - Combine water, buffer, dNTPs, primers, and polymerase in a single tube to ensure consistency across samples.
- Aliquot the Master Mix
- Dispense the appropriate volume into PCR tubes or a PCR plate. - Add Template DNA
- Pipette the correct amount of DNA into each reaction tube. - Mix and Spin Down
- Gently mix by pipetting or flicking the tube. Avoid bubbles.
- Briefly spin down in a microcentrifuge. - Run the PCR Program
- Place tubes in a thermal cycler and set the cycling conditions (e.g., denaturation, annealing, extension). - Post-PCR Handling
- Store at 4°C if using soon or freeze for long-term storage.
- Analyze results via gel electrophoresis.
First Round of PCR
- Initial Denaturation (Usually 94–98°C for 1–5 minutes)
- Breaks hydrogen bonds between DNA strands, fully separating them to allow primer binding. - First Cycle of PCR
- Denaturation (94–98°C for 15–30 seconds): Separates DNA strands.
- Annealing (50–65°C for 15–60 seconds): Primers bind to complementary sequences on the template DNA.
- Extension (72°C for 30 seconds–1 minute per kb): DNA polymerase synthesizes the new strand.
Note : Each round of PCR causes DNA to exponentially increase in size
What is the point in PCR
To amplify DNA, to essentially make a bunch of copies from a small starting amount.
Useful in :
- Genetic research
- Forensic science
- Cloning
- Other, any shit
What are the 4 phases of PCR
- Lag Phase (Baseline Phase)
- Initial cycles where little detectable product is made.
- DNA amplification begins, but the amount is too low to be measured. - Exponential Phase
- DNA doubles with each cycle, leading to rapid and efficient amplification.
- Reaction efficiency is at its highest, and product accumulation is predictable. - Linear Phase
- Amplification slows as reagents (primers, dNTPs, polymerase) become limiting.
- Efficiency decreases due to competition for resources and enzyme activity decline. - Plateau Phase
- PCR reaches saturation; amplification stops as reagents are exhausted.
- No significant increase in product, and signal may begin to degrade.
Why do each of these phases happen?
Lag Phase – DNA concentration is too low for detection; primers bind, and polymerase starts working.
Exponential Phase – Reagents are abundant, allowing DNA to double each cycle with high efficiency.
Linear Phase – Amplification slows as reagents (primers, dNTPs, polymerase) become limited.
Plateau Phase – Reaction stops as reagents are depleted, and polymerase loses activity.
What is endpoint analysis
Endpoint analysis refers to evaluating the PCR product after the amplification process has finished (typically after 30–40 cycles). This analysis is used to assess the presence and size of the amplified DNA.
How it’s done:
After PCR, the amplified DNA is analyzed using techniques like gel electrophoresis, where the DNA is separated by size and visualized, often with a dye that binds to DNA.
Key Features:
- It is a qualitative analysis (detecting whether the target DNA is present or not).
- It assesses the presence/absence of the PCR product and sometimes the relative size of the amplified fragments.
Controls in Endpoint Analysis:
- Positive Control: Contains a known template DNA that should amplify, ensuring the PCR reagents and conditions are working properly.
- Negative Control: Contains all PCR components except the template DNA, ensuring there is no contamination or non-specific amplification.
- Template DNA: The sample DNA from which the target sequence is amplified.
Limitations:
- Endpoint analysis is typically performed after PCR reaches the Plateau Phase, where reagent depletion can cause inconsistent results.
- It provides limited information on the exact quantity of DNA, as it focuses on the presence and size of the amplified product rather than measuring amplification efficiency.
What is real-time PCR?
Real-Time PCR (qPCR) with Fluorescence is a method to monitor PCR amplification in real-time as it occurs. It uses fluorescent dyes or probes to measure the accumulation of PCR products after each cycle.
- How it works: As the DNA amplifies, a fluorescent signal is emitted. The intensity of fluorescence increases as the amount of DNA increases.
- Fluorescent Dyes: Bind to double-stranded DNA and emit fluorescence when bound (e.g., SYBR Green).
- Fluorescent Probes: Sequence-specific probes (e.g., TaqMan probes) bind to the DNA and emit fluorescence when cleaved by the polymerase during amplification.
- Why it’s useful: Provides quantitative data by measuring the fluorescence at each cycle, allowing for accurate determination of initial template concentration.
What is sanger sequencing
Sanger sequencing, also known as chain-termination sequencing, is a method used to determine the exact sequence of nucleotides in a DNA molecule.
How it works:
- DNA Template: The DNA to be sequenced is denatured into single strands.
- Primers and DNA Polymerase: A short primer binds to the template, and a DNA polymerase begins adding nucleotides.
- Dideoxynucleotides (ddNTPs): Modified nucleotides (ddATP, ddTTP, ddCTP, ddGTP) are included in the reaction. When a ddNTP is incorporated, it terminates the chain, preventing further elongation.
- Fragment Separation: The resulting fragments of varying lengths are separated by size using gel electrophoresis or capillary electrophoresis.
- Sequence Reading: The termination points correspond to the nucleotide bases (A, T, C, G), which are then detected and read as a sequence.
Key Features:
- Generates high-accuracy DNA sequence data.
- Typically used for short DNA fragments or single gene sequencing.
Limitations:
- Less efficient for sequencing large genomes due to lower throughput compared to newer methods (like next-generation sequencing)
What is new generation sequencing
Next-Generation Sequencing (NGS) refers to a group of modern sequencing technologies that allow for massively parallel sequencing, enabling the sequencing of millions to billions of DNA fragments simultaneously. This provides high throughput and faster, cheaper results compared to older methods like Sanger sequencing.
How it works:
Sample Preparation: DNA is fragmented into smaller pieces, which are then attached to a solid surface or bead.
Amplification: The DNA fragments are amplified (e.g., via PCR or bridge amplification) to create clusters of identical sequences.
Sequencing by Synthesis: Each cluster is sequenced in parallel, with nucleotides being added to each fragment. As each nucleotide is incorporated, a signal (usually fluorescence) is detected and recorded.
Data Analysis: The signals are translated into nucleotide sequences, and computational tools are used to align the fragments and assemble the full sequence.
Key Features:
Can sequence entire genomes or transcriptomes quickly and cost-effectively.
Generates large volumes of data, allowing for deeper coverage and more accurate sequencing of complex genomes.
Popular Platforms:
Illumina (most commonly used for high-throughput sequencing)
Ion Torrent
PacBio
Oxford Nanopore
Applications:
Whole genome sequencing (WGS)
Exome sequencing (targeting protein-coding regions)
RNA sequencing (RNA-seq)
Metagenomics (analyzing microbial communities)
Cancer genomics (identifying mutations)
Advantages:
High throughput and cost-effective for large-scale sequencing.
Enables the study of complex genetic data and rare mutations.
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