Polymerase Chain Reactions (PCR)

• Amplification techniques generate enough copies of a single gene sequence
• Amplification increases the yield of a nucleic acid sequence of interest for subsequent analyses
• The methods developed amplify nucleic acids based on whether the target nucleic acid itself, a probe specific for the target sequence, or the signal used to detect the target nucleic acid is amplified

Polymerase Chain Reactions (PCR)

• The first application of PCR was for diagnosis of sickle cell anemia

Historical Review

• Karl Mullis first used PCR in 1983 in California
• Polymerase-catalyzed chain reaction is a former name for PCR
• The first successful amplification was a short fragment of Escherichia coli plasmid pBR322

Historical Review

• DNA polymerase drives replication and catalyzes the addition of nucleotides to the growing strand
• Primer use adds subsequent bases

PCR components

Primer

• Primers direct DNA synthesis to desired regions and determine the specificity of the entire sequence to amplify
• Primers are chemically manufactured on a DNA synthesizer
• Melting temperature (Tm) determines the starting point of the primer as it affects the binding of the primer to the sequence
  • Melting point is 50-70°C
  • High GC (Guanine Cytosine) content needs high temperatures to melt
• Primers contain sequences homologous to sites flanking the region to be analyzed

2 Types of Primer

• FORWARD primers bind the 3' end of the template (Leading strand)
• REVERSE primers bind the 3' end of the complementary strand (Lagging strand)

DNA Template

• The DNA template is the sample
• dsDNA or ssDNA is derived from a patient's sample or microorganism causing infection
• Routine analysis uses 100ng-1ug of DNA as a template for amplification
• Templates may be derived from the patient's genomic or mitochondrial DNA, viruses, bacteria, fungi, or parasites

Deoxyribonucleotide Bases

• Nucleotide triphosphates are the building blocks of DNA
• Standard procedures require 0.1–0.5 mM concentrations of each nucleotide
• dNTPs are deoxynucleotidetriphosphates
  • Adenine – deoxyadenosinetriphosphate (dATP)
  • Guanine – deoxyguanosinetriphosphate (dGTP)
  • Thymine – deoxythymidinetriphosphate (dTTP)
  • Cytosine – deoxycytidinetriphosphate (dCTP)

DNA Polymerase

• Thermostable enzymes retain their active form even in high temperatures
  • Taq polymerase comes from Thermus aquaticus
  • Tth polymerase comes from Thermus thermophilus

Stoffel Fragment

• Used when the template has high GC content
  • Modified Taq polymerase lacks N-terminal 289 amino acid
  • Has a broader range of magnesium chloride concentration than Taq
  • Ideal for amplifying regions with high GC content

PCR Buffer

• Maintains pH by providing magnesium ions
  • PCR and Magnesium ions are directly proportional
• The ideal pH is 8.3- 8.8 pH
• Provides optimal conditions for enzyme activity; increasing salt concentration makes products longer
• DNA products denature more slowly than shorted DNA products
• Maintains the proper pH of the buffer
  • Potassium chloride (20-100mM)
  • Ammonium sulfate (15-30 mM)
  • Magnesium chloride (1-4 mM)
  • Tris-HCl (10 mM) – pH8-9.5 for proper buffering

Accessory Buffer

- Bovine serum albumin (10-100ug/mL)
- Dithiothreitol (0.01 mM)
- Formamide (1-10%)
- Chaotropic agents: triton X-100, glycerol, dimethyl sulfoxide (1-10%)

PCR Controls

• Running controls is essential for maintaining and ensuring the accuracy of the assay

Positive Control

• Ensures that the enzyme is active, buffer is optimal, and primers are priming right sequences
• Known sample with a known sequence
• Same sample is to be detected

Negative Control Without DNA

• Also called a contamination control or reagent blank
• Ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run
• No sequence is produced

Negative Control With DNA

• Lacks the target sequence (negative template control)
• Ensures that the primers are not annealing to unintended sequences of DNA
• There is a sequence produced but not the target sequence

Thermal Cyclers

• Designed to ramp through the required incubation temperature rapidly and automatically in several cycles
• Rapid PCR uses a small sample volume
• Real-Time PCR has fluorescent detectors to measure the products

Basic Procedure

Denaturation

• In vivo, DNA helicase is used
• In vitro, heat is applied
• dsDNA is denatured into two single strands
• Temperature: 90 – 96°C
  • Optimal temperature is 94-96°C
• Time: 20-60 seconds

Annealing

• Most critical step for the specificity of the PCR
• Attachment of primers to the strand to be amplified
• Temperature: 50-70°C
• Time: 20-90 seconds
• If the primer does not match the region of interest, then the result is negative

Extension

• DNA polymerase catalyzes the formation of phosphodiester bonds to replicate DNA by extending the primers (in vitro DNA synthesis)
• For every cycle of one dsDNA, it will be replicated into two dsDNA
• Temperature: 68 to 75°C
  • Optimal temperature is 68-72°C
• Time: 10-60 seconds

Target Amplifications

Multiple PCR Method

• More than one primer pair can be added to a PCR for multiple amplifications that are primed simultaneously resulting in the formation of multiple products
• Used for typing or identification analyses
• For example, organisms that cause sexually transmitted diseases (HIV, syphilis, gonorrhea) can be targeted in multiplex PCR using one genital swab

Nested PCR Method

• Increased sensitivity by using two pairs of primers to amplify a single target in a separate run
• A low level of target and the presence of interfering sequences can prevent a regular PCR from working with the reliability required for clinical applications
• Used with specimen that have a limited quantity and quality
  • Run the 1st primer for the 1st run of PCR -Run the 2nd primer for the 1st primer again

Reverse Transcriptase PCR Method

• Amplification is modified to include the initial incubation at 45-60°C for 30-60 minutes which makes reverse transcriptase able to convert any RNA sample to cDNA template
• Reverse transcriptase is an enzyme that converts any RNA sample to become dsDNA in the form of cDNA
• Enzyme is inactivated at 80°C for 30-60 minutes once the reaction is fulfilled
• Used in gene expression analysis

Real-Time/Quantitative PCR Method (RT-PCR or QPCR)

• Requires dsDNA-specific dyes such as ethidium bromide or SYBR green that is added to the sample before the PCR test and provides a fluorescent signal as evidence of amplification
  • Exponential phase: fluorescence occurs during reaction
• Is the gold standard for SARS-Cov-2 in combination with Reverse Transcriptase-PCR

PCR Modifications

• PCRs today have been adapted for various applications and several modifications are used in the clinical laboratory
• There are three groups of methods that have been developed to amplify nucleic acids including:
  • Target amplification
  • Probe amplification
  • Signal amplification