Polymerase Chain Reaction (PCR)

 Introduction

• PCR is a method widely used in molecular biology to make millions to billions of copies of a specific DNA sample, allowing scientists to amplify and study it in detail.
• It's a crucial tool in various fields including genetics, forensics, and medical diagnostics

History of PCR

• The man behind the PCR: Kary Banks Mullis.
• This technique was developed in 1985 and was awarded the Nobel Prize in 1993.
• In 1983, while driving along US Route 101 in Northern California, Kary Mullis. a scientist who was working for Cetus Corporation, conceived the idea for the polymerase chain reaction.
• IN 1985, at a conference in October, the polymerase chain reaction was introduced to the scientific community. For his invention, Cetus rewarded Kary Mullis with a $10,000 bonus.
• Later, during a corporate reorganization, Cetus sold the patent for the PCR process to Hoffmann-LaRoche, a pharmaceutical company, for $300 million.

Credit: https://www.mun.ca/biology/scarr/PCR_simplified.html

PCR (Polymerase Chain Reaction)

• PCR, or polymerase chain reaction, targets and amplifies a specific region of a DNA strand. 
• This in vitro technique generates large quantities of a specified DNA, which is particularly useful when only a small amount of DNA is available, such as a drop of blood, semen strains, single hair, vaginal swab, etc.
• Two methods currently exist for amplifying the DNA or making copies. 
• The first one is the cloning. The particular cloning takes time for long enough clones to reach maturity. 
• And the second one is the PCR. It works on even a single molecule quickly.

Requirement Of PCR

• DNA Template
• Primers
• Taq Polymerase
• Dioxynucleoside Triphosphate (dNTPs)
• Buffer solution
• Divalent Cation (Mg2+)

Steps Involved In PCR

• Denaturation
• The reaction mixer is heated to a temperature between 90 to 98 degree Celsius so that the double-stranded DNA is denatured into single strands by disrupting the hydrogen bonds between complementary bases 
• The duration of this step is 1 to 2 minutes.
• Annealing
• The temperature of the reaction mixer is cold to 42 to 60 degrees Celsius.
• Primers are jiggling around caused by thermal agitation or Brownian motion.
• Primer's base pairs with the complementary sequence in the DNA.
• Hydrogen bones reform.
• Annealing fancy word for renaturing.
• Extension
• Temperature set to 72°C for optimal polymerase activity.
• Primers extended by polymerase, linking complementary bases.
• Elongation stage: polymerase adds dNTPs from 5' to 3', reading template from 3' to 5'.
• Bases added to new strand complementarily to the template.
• Completion of first cycle; process repeats.
• PCR machine, an automated thermocycler, repeats the cycle 30-40 times for amplification.

Example of a PCR program:

• Initial Denaturation: 95°C for 5 minutes - This step denatures the DNA, separating the two strands.
• Denaturation: 95°C for 30 seconds - Further denaturation of the DNA, separating the strands.
• Annealing: 55°C for 30 seconds - Primers anneal to the complementary sequences on the DNA strands.
• Extension: 72°C for 1 minute - DNA polymerase extends the primers by adding nucleotides to the template strand.
• Final Extension: 72°C for 5 minutes - Final extension to ensure all DNA fragments are fully extended.
• Hold: 4°C - The reaction is held at 4°C until it is stopped or analyzed.
• This cycle is typically repeated 30-40 times to amplify the DNA exponentially. Each cycle results in a doubling of the target DNA sequence.

Advantages Of PCR

• Small amount of DNA required per test.
• Result obtained more quickly - usually within 1 day for PCR
• Usually not necessary to use radioactive materials (32P) for PCR
• PCR is much more precise in determining the sizes of alleles - essential for some disorders.
• PCR can be used to detect point mutations.

Applications Of PCR

• Molecular Identification
• Molecular Archeology
• Molecular Epidemiology
• Molecular Ecology
• DNA Fingerprinting
• Classification of Organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug Discovery
• Genetic Matching
• Detection of Pathogens
• Sequencing
• Bioinformatics
• Genomic Cloning
• Human Genome Project
• Genetic Engineering
• Site-directed Mutagenesis
• Gene Expression Studies

Factors For Optimal PCR

• PCR Primers
• A correctly designed pair of primers is required.
• Primer dimer and hairpin formation should be prevented
• Length of Primer
• DNA Polymerase
• Thermus Aquaticus -170"F
• Taq Polymerase is heat resistant.
• It lacks proofreading exonuclease activity.
• Other polymerases can be used, eg, 
• tRNA DNA Polymerase from Thermotoga maritama.
• Pfu DNA Polymerase from Pyrococcus furiosus.
• Annealing Temperature
• Very important since the success and specificity of PCR depend on it because DNA-DNA hybridization is a temperature-dependent process.
• If the annealing temperature is too high, pairing between primer and template DNA will not take place then PCR will fail.
• Ideal annealing temperature must be low enough to enable hybridization between primer and template but high enough to prevent amplification of nontarget sites.
• Should be usually 1-2'C or 5'C lower than the melting temperature of the template-primer duplex.
• Melting Temperature
• Temperature at which 2 strands of the duplex dissociate.
• It can be determined experimentally or calculated from the formula
• Tm = [4(G+C)] + [(2(A+T)]
• G/C content
• Ideally a primer should have a near-random mix of nucleotides, 50% GC Content.
• There should be no PolyG or PolyC stretches that can promote non-specific annealing.

Variants of PCR:

• Gradient PCR
• Gradient PCR is a variation of the polymerase chain reaction (PCR) technique that involves running the reaction at a range of temperatures simultaneously across multiple tubes or wells within the same thermal cycler. 
• This technique is used to identify the optimal annealing temperature for a specific primer pair by testing a range of temperatures in a single experiment. By doing so, gradient PCR helps to optimize PCR conditions and improve the specificity and efficiency of amplification for a particular target sequence.
• Imagine you're baking cookies, but you're not sure what temperature is just right for them to turn out perfectly golden brown. Gradient PCR is like trying different oven temperatures all at once. You set up your PCR machine to test the same DNA sample at different temperatures in different tubes. This helps you find the exact temperature where your DNA amplification works best, ensuring accurate and efficient results. It's like finding the sweet spot for your cookies but for PCR!

• Multiplex PCR
• Multiplex PCR is like doing multiple tasks at once. Instead of just copying one piece of DNA, it copies several different pieces all together in the same test. This helps save time and resources because you only need to run one test instead of many separate ones. It's really handy for things like identifying different genetic markers or studying multiple. 
• Imagine you have a bunch of different keys, and you want to make copies of each one. Normally, you'd make copies of each key separately. But with multiplex PCR, it's like having a special machine that can copy all your keys at once!
• In biology, instead of keys, we have DNA sequences we want to copy. Multiplex PCR lets us copy several different DNA sequences all in one go. We use different sets of "primers" (like special markers) to copy each DNA sequence we're interested in. This saves time and resources, making the process more efficient.
• Criteria for Successful Multiplex PCR
• Optimize Primer Design: Ensure primers for all target sequences work at the same annealing temperature.
• Ensure Specificity: Design primers to be highly specific for their target sequences.
• Consistent Amplification Size: Aim for similar amplification sizes across all target sequences.
• Optimize Polymerization Time: Adjust polymerization time to ensure efficient amplification of all targets

• Allele-specific PCR
• Defining Allele-Specific PCR: It's a technique tailored to a particular allele, which is a variant form of a gene.
• Targeting SNP Differences: If one allele has an SNP (Single Nucleotide Polymorphism) and the other is normal, we design specific primers for each allele.
• Primer Modification: We tweak one base at the 3' end of each primer so that one matches the normal allele and the other matches the mutant allele.
• Simultaneous PCR: Both alleles are analyzed together in a single PCR reaction.
• Amplification Outcome: If the mutant allele is present, it's amplified; if the normal allele is present, it's amplified. This way, we can identify which allele is present in the sample.

• ARMS PCR
• The allele-specific PCR is also called as the (amplification refractory mutation system) ARMS-PCR because of the use of two different primers for two different alleles. Here the word "refractory" is very important (Refractory - resistant to something).
• As we discussed, two sets of primers are designed, the mutant set of the primer is refractory (resistant) to the normal PCR and the normal set of the primers is refractory to the mutant PCR reaction.
• That is why it is called an amplification refractory mutation system.
• The name ARMS-PCR is given by its actual developer C. R. Newton.
• PRINCIPLE:
• ARMS-PCR works by modifying primers for different alleles.
• The 3' end of the primers is altered so that one set amplifies the normal allele and the other set amplifies the mutant allele.
• A single base mismatch is introduced at the 3' end of the primer.
• This mismatch allows each primer to selectively amplify one allele, distinguishing between the normal and mutant alleles.

• Asymmetric PCR
• Imagine you have a double-stranded DNA molecule, but you're only interested in one of the strands. Asymmetric PCR lets you amplify just that one strand more than the other.
• Here's how it works:
• You mix the DNA with two primers in a PCR reaction, but you use a lot more of one primer than the other.
• The primer in higher concentration grabs onto its matching sequence on the DNA more often, kicking off the PCR process.
• As PCR continues, you end up with a lot more copies of the strand that matches the higher-concentration primer.
• So, in the end, you get a bunch of copies of just the strand you're interested in, while the other strand doesn't get amplified as much.

• Helicase-dependent amplification
• Constant Temperature: Maintains a steady temperature throughout the process.
• Helicase Enzyme: Uses DNA Helicase to unwind DNA strands for amplification.
• Intersequence Specific PCR (ISSR)
• APCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment length
• Inverse PCR
• A method used to allow when only one internal sequence is known.
• This is especially useful in identifying flanking sequences (determination of genomic sequences) of various genomic inserts.
• Anchored PCR
• In Anchored PCR, if we only know one end of a gene segment, we use a primer that matches that end. This helps make many copies of just one strand of the gene.

Introduction to RT-qPCR

• RNA as the Starting Material
• Quantitative Reverse Transcription PCR (RT-qPCR) is a method used when starting with RNA. Here's how it works:
• RNA to cDNA: First, RNA is converted into complementary DNA (cDNA) using reverse transcriptase.
• qPCR Reaction: The cDNA is then used as the template for the qPCR reaction.
• RT-qPCR is valuable in many areas like gene expression analysis, RNAi validation, pathogen detection, and disease research.

• What are RT-PCR, qPCR and RT-qPCR
• RT-PCR is a term specifically for reverse transcription PCR, not real-time PCR. Here's how it works:
• RNA to cDNA: Reverse transcription PCR lets us use RNA to make complementary DNA (cDNA) using reverse transcriptase.
• cDNA Amplification: The cDNA is then amplified by a DNA polymerase, creating more cDNA for analysis.
• It's a helpful tool for converting RNA into DNA for further study.
• This technique is commonly used for cloning genes or as the first step in RT-qPCR.
• qPCR is real-time PCR that measures DNA amplification using fluorescent probes, allowing quantitation. qPCR can detect pathogens and determine DNA copy numbers.
• RT-qPCR combines RT-PCR with qPCR to quickly measure changes in gene expression by using cDNA.

• Sample preparation
• The most important step in qPCR and RT-qPCR is sample preparation. If the starting material is contaminated or degraded, accurate results won't be obtained.
• In RT-qPCR, the last step of sample prep is making cDNA using RT-PCR. This can be done with oligo(dT) primers, which attach to the RNA's polyA tail, or with random hexamers, which attach at different points along the RNA.
• A mix of both primers is often used to amplify both polyA-containing RNA (like mRNA) and non-polyA RNA (like tRNA, rRNA).
• RT-qPCR combines RT-PCR with qPCR to measure RNA levels using cDNA, allowing quick detection of gene expression changes.

• Detection method for qPCR
• When choosing a detection method for qPCR, you have two main options:
• Fluorescent Dye Method: Uses a dye like SYBR® Green that binds to all double-stranded DNA. More DNA means more fluorescence.
• Hydrolysis Probe Method: Uses probes like TaqMan® that have a fluorescent tag and a quencher. When DNA polymerase extends the primer, the probe is cut, releasing fluorescence.

• Quantitation and data analysis
• In qPCR, the amplification curve shows three phases: initiation, exponential growth, and plateau.
• Quantitation relies on the threshold level, where fluorescence significantly increases compared to the baseline.
• The Ct or Cq value is recorded when the sample crosses this threshold, providing quantification.
• A reference gene (RG) is crucial for accurate quantitation, serving as a stable control unaffected by biological conditions. Popular RGs include GAPDH and β-actin, but they must be carefully matched to the experiment to ensure reliability.

Covid-19: the new frontier for real-time PCR assays

• RT-PCR has long been used in public health to detect respiratory viruses. With the Covid-19 outbreak, real-time RT-PCR has become crucial for high-throughput screening.
• Real-time RT-PCR allows quick detection of SARS-CoV-2, the virus causing Covid-19, by targeting specific genes like RdRp.
• Tests have been rapidly developed using the SARS-CoV-2 genome, allowing prompt design of primers and probes specific to the virus.
• This method, similar to TaqMan probes, is widely implemented by organizations like the WHO, PHE, and NHS for efficient screening during public health emergencies.
• A group in Hong Kong has developed a test using two one-step RT-qPCR assays for SARS-CoV-2 genes ORF1b and N. These tests can be run on large automated machines, allowing fast and accurate detection with high sensitivity. This method is valuable not only for research but also for rapid diagnosis during public health emergencies.