Polymerase Chain Reaction

Polymerase Chain Reaction

Powerful molecular biology technique used to amplify specific DNA sequences

Invented by Kary Mullis in 1983

Function of temperature cycles in PCR

Facilitate:

-DNA denaturation

-Primer annealing

-DNA synthesis

What is the purpose of PCR?

To gain an abundance of target DNA sequence which can be further analysed

Importance of PCR

-Molecular diagnostics and research

-Provides sensitive and specific method for DNA amplification

-Allow for detection, quantification and analysis of DNA or RNA sequences

Use of PCR to identify pathogens

PCR-based tests for viral, bacterial and fungal infections

Use of PCR to detect genetic disorders

PCR-based screening for inherited diseases and genetic mutations

Use of PCR for DNA profiling

Forensic analysis

PCR amplification of DNA from crime scene samples for identification

Use of PCR for paternity testing

PCR based analysis of genetic markers to determine parentage

Function of DNA polymerase in PCR

Synthesis new DNA strands

What is the most commonly used DNA polymerase in PCR?

Taq polymerase

Why is Taq polymerase used in PCR?

Derived from hot springs in bacterium Thermus aquaticus

Means it has a high heat-stable property so can withstand denaturing

What is the temperature optimum for Taq polymerase?

72-75 °C

Suitable for PCR cycles involving heating and cooling

Proofreading activity of PCR

Lacks proofreading activity

Makes it error-prone but faster and suitable for routine applications

What DNA polymerase would be used to achieve a higher fidelity?

Pfu polymerase

Initial stage of PCR cycle

Separates double-stranded DNA template into single strands

Exposes DNA sequence

What is the process of heating in PCR?

High temperatures (94-98°C)

Breaks hydrogen bonds between complementary DNA stands

Separates double-stranded DNA into two single strands

Process of execution in PCR

Heating PCR reaction to denatuartion temperature for 15-30 seconds=depends on DNA template length

Causes loss of secondary structure and DNA-protein interactions

Process of annealing in PCR

Temperature is lowered to allow primers to bind to complemenatry sequences

What steps form 1 PCR cycle?

Denaturation and annealing

Function of primers in PCR

Provide starting point for DNA replication

Characteristics of primers

Short, synthetic complementary DNA sequences that target DNA regions

Designed based on DNA sequence being targeting

Uses of primers in PCR

-Forward and reverse primers define region of DNA being amplified

-Bind to complementary sequences on 5' and 3' ends of DNA strands during denaturation

Initiation of DNA replication

Primers provide starting point for DNA polymerase to initiate DNA replication

DNA polymerase extends from primers

What does the extension of primers by DNA polymerase lead to?

Amplification of target DNA sequence during each PCR cycle

Specificity and design of primers

Need to be designed for desired DNA segment being amplified

What factors need to be considered when designing primers?

-Length

-Melting temperature

-Avoiding self-complementarity or primer-dimer formation

Purpose of extension step in PCR

Synthesise new DNA strands using target DNA region as a template

Process of extension in PCR

Follows denaturation and annealing

DNA polymerase extends from annealed primers, adding complementary nucleotides to synthesis new DNA strands

DNA synthesis in PCR

DNA polymerase catalyses formation of phosphodiester bonds between incoming nucleotides and growing DNA strands

At what temperature does DNA synthesis occur at in PCR?

68-72°C

DNA replication in PCR

Allows DNA polymerase to move along DNA template

Extends the primers and copying target DNA sequence

Result of extension step in PCR

Production of two complete double-stranded DNA molecules from each single-stranded DNA template

Gene expression analysis

Process in molecular biology to understand gene activation and transcription into RNA molecules

Reflects gene expression levels

Reverse Transcription PCR (RT-PCR)

Measures RNA amount by converting RNA into complementary DNA (cDNA) using reverse transcriptase

Then amplifies cDNA with PCR

When is reverse transcription PCR used?

Used to measure gene expression levels and relative abundance of specific mRNA transcripts

Quantitative Real-Time PCR (qPCR)

Highly sensitive method for quantifying gene expression levels by real time monitoring of DNA amplification during PCR

What can Quantitative Real-Time PCR (qPCR) utilise?

Fluorescent probes or DNA-binding dyes

Emit signals proportional to amplified DNA amount for relative expression level determination

Digital PCR (dPCR)

Allows absolute quantification of nucleic acids without standard curves or reference genes

Function of Digital PCR (dPCR)

Partitions sample into individual reactions, amplifies DNA and detects target sequence present in absolute concentration determination

When can Digital PCR (dPCR) be used?

Biomedical research and clinical diagnostics:

-Studying gene expression

-Identify disease biomarkers

-Experimental intervention effects

-Develop target therapies

Principle of Quantitative PCR (qPCR)

Based on conventional PCR

Integrates fluorescent dyes or probes for real-time DNA amplification monitoring

What fluorescent probes or DNA-binding dyes does quantitative PCR (qPCR) use?

Probes=TaqMan probes, molecular beacons

DAN-binding dyes=SYBR Green

Data analysis of Quantitative PCR (qPCR)

Involves cycle threshold (Ct) values calculation

Inversely proportional to amount of target RNA in sample

Use comparative methods like ΔΔCt method

What can Quantitative PCR be used to understand?

Relative or absolute abundance of specific RNA molecules eg mRNA

Provides insight into gene regulation and activity

Step 1 of quantification of gene expression levels using qPCR

Reverse Transcription:

-RNa molecules converted into complementary DNA (cDNA) use reverse transcriptase

Step 2 of quantification of gene expression levels using qPCR

PCR Amplification:

-cDNA amplified using PCR with specific primers, DNA polymerase and fluorescent probes or DNA-binding dyes

Step 3 of quantification of gene expression levels using qPCR

Fluorescence Detection:

-Fluorescent probes or DNA-binding dyes emit fluorescence signals during PCR amplification

Step 4 of quantification of gene expression levels using qPCR

Real-time monitoring:

-Fluorescence signals measured at each cycle

-Allow detection of exponential phase of DNA amplification for precise quantification

Step 5 of quantification of gene expression levels using qPCR

Ct Value calculation:

-Cycle threshold (Ct) value determined

-Inversely proportional to the amount of target RNA molecule in sample

Step 6 of quantification of gene expression levels using qPCR

Data analysis:

-Relative gene expression levels compared sing methods like ΔΔCt

-Absolute quantification achieved using standard curves or external calibrators with known concentrations of the target RNA

Allele-Specific PCR for identifying DNA sequence variation

PCR-based technique used for detection and genotyping of DNA sequence variations

eg Single nucleotide polymorphisms (SNPs) and point mutations associated with hereditary disorders

Principle of allele-specific PCR

Relies on allele-specific primers

Selectively amplify and target DNA sequence containing specific variation of interest

Design of Allele-specific primers

Designed perfectly to match specific allele of interest at 3' end

Intentionally introduces mismatches to prevent amplification of undesired allele

PCR amplification of allele-specific PCR

Allele-specific primers selectively anneal to complementary DNA strands carrying specific allele

Allows for amplification of target sequence

Detection of amplification for allele-specific PCR

Amplification products can be analysed using gel electrophoresis or real-time PCR

Provide qualitative or quantitative information about target allele

Advantages of allele-specific PCR

-High specificity

-Sensitivity

-Cost-effective

High-specificity of allele-specific PCR

Provides high specificity in detecting and genotyping DNA sequence variations, minimisng false positive or false-negative results

Sensitivity of allele-specific PCR

Can detect even low amounts of target allele

Suitable for apllications where sensitivity is crucial

Cost-effectiveness of allele-specific PCR

Simple and cost-effective compared to other genotyping methods

Genotyping allele-specific PCR

Widely used for genotyping DNA sequence variations associated with hereditary disorders, pharmacogenetics

Disease association studies using allele-specific PCR

Investigate association between specific DnA sequence variation and disease susceptibility or treatment response

Diagnostic testing using allele-specific PCR

Employed in clinical diagnostics to identify disease-causing mutations or mutations associated with drug resistance

Multiplex PCR

Simultaneous amplification of multiple target DNA sequences in a single reaction

Advantages of Multiplex PCR

-Saves time

-Less effort and resources than individual PCR

Principle of Multiplex PCR

Use multiple primer sets

Each specific to a different target sequence

Primer design for multiplex PCR

Crucial for successful multiplex PCR

Focus on:

-specificity

-Primer length

-Melting temperature

-Avoiding interactions

Optimisation of Multiplex PCR

Requires careful balance of reaction conditions for efficient amplification of all target sequences simultaneously

PCR controls of Multiplex PCR

Inclusion of positive and negative controls to monitor the success and absence of contamination

Function of Thermal Cycler in PCR

Precisely controls and cycles the temperature of reaction mixture to facilitate DNA amplification

Importance of Temperature cycling in PCR

-Denaturation=breaks hydrogen bonds between DNA strands, ensures DNA template is single-stranded

-Annealing=primers bind to single-stranded DNA template

Extension (elongation) of Thermal Cycler in PCR

DNA polymerase extends the primers

Synthesises new DNA strands

Significance of temperature cycling

Facilitate key steps of DNA amplification

Enables separation of DNA strands, primer binding, DNA synthesis and exponential amplification of target DNA sequence