Polymerase Chain Reaction (PCR)

• Polymerase Chain Reaction, or PCR, is covered by the notes
• Second Semester, AY 2024-2025 at San Pedro College

The Human Genome Project/History of PCR

• Started in 1990 with approximately 30 different institutions participating
• The goal was to fully sequence the entire human genome
• Objectives included sequencing chemical bases, identifying all genes, and developing genetic analysis tools
• It was important because it helped scientists understand, prevent, and treat diseases
• The project advanced technology, analytical tools, open data sharing, supportive policies, and ethical awareness
• The project helped define somatic cell genetic disease and mutation-based cancer, and alter the practice of medicine
• It inspired large-scale data initiatives like the International HapMap Project and The Cancer Genome Atlas
• Initial completion was in April 2003, mission accomplished in May 2021
• Total size: ~3.2 billion bp
• Issues identified included specificity (large genome) and amplification (small DNA samples)
• PCR makes studying specific sequences possible
• Dr. Kary Mullis discovered a way to double test target DNA with 21 copies
• Repeating N times yields 2N
• Adding an oligonucleotide primer with 4 dNTPs and DNA polymerase results in millions of copies if N = 30 or 40
• Kary Mullis, eccentric, used drugs, left science, and drove on the California Highway
• He won the 1993 Nobel Prize in Chemistry for discovering a way to analyze DNA easily and cheaply
• His work paved the way for major advances in medical diagnostics, molecular biology, & forensic science

PCR Steps

• Illustrates the Polymerase Chain Reaction (PCR) Process
• A technique used to amplify DNA
• Template DNA (single copy) is the first step
• The process begins with a single DNA strand as the template for amplification

PCR Cycles

• Has a First Cycle
• Denaturation heats double-stranded DNA to separate it into single strands
• Annealing involves primers binding to complementary sequences on single-stranded DNA
• Extension uses DNA polymerase to synthesize new DNA strands, creating 2 copies of the original DNA
• Second Cycle: Newly synthesized DNA strands serve as templates
• The process repeats, leading to 4 copies of the target DNA sequence
• Third Cycle: Repeats, producing 8 copies
• DNA strands accumulate exponentially

Exponential Amplification

• The process continues for multiple cycles, typically around 30 in a standard PCR reaction
• After 30 cycles, approximately copies of the target DNA sequence are generated
• Demonstrates the power of PCR in DNA amplification
• PCR achieves exponential DNA replication through repeated cycles of denaturation, annealing, and extension

Denaturation

• Denaturation defines the unfolding or breaking up of a protein, modifying its standard three-dimensional structure
• Proteins may be denatured by chemical action, heat, or agitation
• This causes a protein unfolds or its polypeptide chains become disordered, leaving molecules non-functional
• Double-stranded DNA is separated into two single strands
• Temperature: 94-96°C
• Time: varies from seconds to minutes
• Larger template requires longer time

Annealing

• Most critical step for specificity
• Primers hybridize to their complementary DNA sequences
• Temperature ranges from 50-70°C
• The starting point is determined using the Tm of the primer sequences
• Melting Temperature (Tm) in PCR controls the temperature at which primers attach to DNA, crucial for experiments
• It's important for Annealing
• The reaction temperature needs to be low enough for primers to bind to the DNA template
• Needs to be high enough to prevent the formation of undesired duplexes

Amplification

• All primers in a reaction should have similar values
• Tm allows each amplification to proceed at the selected temperature

Sequence Verification

• Melting analysis post-PCR monitors duplex hybridization as temperature changes
• It's a method for sequence verification and genotyping
• Tm range of 52-58°C generally produces the best results, primers above 65°C tend to secondary anneal

Strands

• 5'-'3 leading strand and 3'-5' lagging strand exist
• The leading strand synthesizes DNA continuously in the 5' to 3' direction
• Lagging strand synthesizes in small fragments known as Okazaki fragments

Extending

• Performed by DNA polymerase
• Polymerase synthesizes a copy of the template DNA and catalyzes formation of the phosphodiester bonds between dNTPs and the 3' end of the primer
• Temperature: 68-72°C
• End result: 2x template number
• Taq polymerase belongs to the amplification stage of a PCR reaction
• Heat-resistant nature is crucial for PCR amplification
• Taq polymerase is a heat-stable enzyme from "Thermus aquaticus"
• It amplifies specific DNA sequences by repeatedly copying them
• It functions at high temperatures without denaturing, allowing multiple cycles of DNA amplification

PCR Components - Primers

• Single-stranded DNA fragments, 20-30 bp long, function like primers in vivo
• Determine the specificity of PCR, containing sequences complementary to sites flanking the region of interest

Forward Primer

• 5’ to the sequences amplified, hybridizes with the minus strand, creates a copy same as target gene, the plus strand sequence
• Binds to target dependent on primer sequence, length of strand, and melting temp (Tm)

Reverse Primer

• 3’ to the sequences amplified, Hybridizes with the plus strand

DNA Template

• Derived from nucleotide extraction routine clinical analyses require 100 ng – 1 μg, must be in the very best condition
• Must be free of contaminants
• Have no nicks or breaks

Deoxynucleotide Bases

• Building blocks of DNA include dNTPs like dATP (adenine), dTTP (thymine), dGTP (guanine), and dCTP (cytosine)
• The requirement is 0.1-0.5 mM of each

DNA Polymerase

• The first polymerase was isolated from E. coli and had to be added after each round of denaturation
• A more advanced polymerase called Taq is derived from thermus aquaticus, is thermostable, with good fidelity, is accurate in copying the template
• Thermus aquaticus can tolerate high temperatures
• Taq polymerase belongs to the amplification stage of a PCR reaction and is heat-resistant
• Tth polymerase is derived from Thermus thermophilus, possesses reverse transcriptase activity, and is utilized in RT-PCR (RNA template)

PCR Buffer

• Provides optimal conditions for enzyme activity
• pH buffers and salts that provide cations, consists of salts, stabilizers, and enhancers for maintaining the pH of a PCR reaction
• The maintains pH by neutralizing acidic or basic compounds in the sample
• Enzyme activity relies on it
• Buffer provides potassium/ammonium ions for primer binding to a template DNA
• Improves PCR specificity and destabilizes imperfect binding by salts like magnesium chloride/potassium chloride

Tris Buffer

• Provides pH for optimal enzyme activity and accurate amplification
• Effective in pH 8 – 9.5

Monovalent Cations

• KCI and (NH4)2SO4
• They affect denaturing and annealing temperatures
• Higher salt concentration means that DNA sequences denature slower

Divalent Cations

• Including MgCl2, where Mg2+ is needed by DNA polymerase
• 1 NTP = 1 Mg atom
• If too low, amplicons are reduced
• If too high, non-specific products increase
• The typical reaction is below

Oligodeoxynucleotide

• ODN is a DNA molecule used in molecular biology research, genetic testing, and therapeutic applications
• Manufactured in a lab using solid-phase chemical synthesis

Physical Separation

• Physical separation relies on UV light is employed during which induces base damage catalyzing single/double-stranded breaks in DNA
• Enhanced effectiveness with psoralens
• UV is an intercalating agent that prevents denaturation and amplification of the treated DNA

Chemical Methods

• 10% bleach is widely used
• 7mm hypochlorite wipes removes most DNA contaminations
• dUTP-UNG, where Uracil-N-glycosylase (UNG) degrades any nucleic acid

Primers - Misprimes and Problem Solving Techniques

• PCR amplification requires precise and efficient primer binding to target DNA
• Issues such as mispriming can reduce yield and generate non-target products
• To overcome these problems, techniques exist such as:
• Misprimes: Aberrant binding, carrying the primer sequence
• Primer Dimers: Duplication, product will double in size
• To fix, prepare and design primers accordingly
• Hot-Start, pre-heat to fix issues and stop polymerase

Nested PCR

• Increases sensitivity & specificity
• A Disadvantage is it is time consuming

Reverse Transcriptase PCR

• Also known as "RT-PCR"
• RNA becomes the start material
• The starting material is converted to DNA by reverse transcriptase
• Used in gene expression studies

Real-Time PCR

• Also known as "rt-PCR"
• Used to detect sequence using fluorescence

SYBR Green I Assay

• Replaces Ethidium bromide (EtBr)
• Highly specific and robust
• Less toxic and cheaper than TaqMan
• SYBR Green and Ethidium Bromide are used to stain DNA and is a much safer alternative
• Key differences in toxicity, Ethidium Bromide is a potent mutagen and excitation wavelength requires more use on UV light sources.

Binding Affinity

• SYBR Green has a higher binding to DNA, leading to more sensitivity