Polymerase Chain Reaction (PCR)

• Polymerase Chain Reaction, or PCR, is the focus
• Transcribed by Lloyd T. Caballero

The Human Genome Project

• Started in 1990, involving approximately 30 different institutions
• The project aimed to fully sequence the entire human genome sequence
• Goals include sequencing chemical bases, identifying all genes, and developing genetic analysis tools
• Proved important for understanding disease development, prevention, treatment, and technological and analytical advancements
• Established an open approach to data sharing and awareness of ethical issues
• Impacted medicine by establishing somatic cell genetic disease as a genetic disease category
• Helped define mutation as a basis of cancer and altered medical practices
• Had impacts in other fields inspiring initiatives like the International HapMap Project and The Cancer Genome Atlas
• Initial completion was in April 2003, mission was accomplished in May 2021
• Total size is approximately 3.2 billion base pairs

PCR Issues

• Issues of specificity with other sequences in the genome not of real interest
• Amplification required due to very small DNA amounts
• PCR makes studying specific sequences more efficient

Kary Mullis

• Dr. Kary Mullis discovered a doubling method for test target DNA using 21 copies
• Repeating N times would yield 2N
• Achieved by adding an oligonucleotide primer with 4 dNTPs and DNA polymerase in a chain reaction
• Yields millions of copies with N = 30 or 40
• Kary B. Mullis is a biochemist, won the 1993 Nobel Prize in Chemistry
• Discovered a way to analyze DNA easily and cheaply

PCR Steps

• PCR exponentially amplifies DNA, a molecular biology technique
• Begins with a single DNA strand as a template
• Includes denaturation, annealing, and extension

PCR Cycle

• The first cycle starts with denaturation, where double-stranded DNA separates into single strands with heat
• Annealing follows, where primers bind to complementary sequences on single-stranded DNA
• In the extension phase, DNA polymerase synthesizes new DNA strands using primers
• The first cycle yields two DNA copies
• In the second cycle, newly synthesized strands serve as templates
• Repeating the process leads to 4 copies of the target DNA sequence, and 8 after the third
• DNA strands accumulate exponentially

Exponential Amplification

• The process is repeated for multiple cycles, typically approximately 30 in a standard PCR reaction
• About copies of the target DNA sequence are generated by the 30th cycle
• PCR achieves exponential DNA through denaturation, annealing, and extension

Denaturation

• Denaturation refers to the protein unfolding, modifying its standard three-dimensional structure
• Proteins denature by chemical action, heat, or agitation, which causes them to unfold
• Double-stranded DNA separates into 2 single strands
• Temperature of 94-96°C, several seconds to minutes or longer template or longer time

Annealing

• Annealing is most critical step, for specificity
• Primers hybridize to their complementary DNA sequences
• Temperatures maintained around 50-70°C
• Starting point is determined by using the melting temperature (Tm)
• Proper reaction temperature is important for primer binding whilst avoiding undesired duplexes
• Tm values should be similar

Melting Temperature (Tm)

• The melting temperature is controlled to allow correct primer attachment
• Amplification proceeds at the selected temperature
• Monitors duplex hybridization, which simplifies sequence verification and genotyping
• Primers with Tm in the range of 52-58°C produce the best results
• Primers with Tm above 65°C have a tendency for secondary annealing
• Leading strand 5' to 3', while lagging strand proceeds 3' to 5'
• Leading strand is synthesized continuously, while lagging strand is synthesized in small fragments called Okazaki fragments.

Extension

• Extension is performed by DNA polymerase at 68-72°C, resulting in 2x template number
• The polymerase synthesizes a copy of the template DNA
• Taq polymerase helps in the amplification stage of a PCR reaction
• Is responsible for heat resistance due to synthesizing new DNA strands
• Extracted from the bacteria "Thermus aquaticus"

PCR Components

• Primers are single-stranded DNA fragments measuring 20-30 bp long that works like primers
• Determines the specificity of the reaction
• Sequences are complimentary to regions flanking the region of interest
• The forward primer hybridizes with the minus strand and is 5’ to the sequences amplified
• A reverse primer hybridizes with the plus strand and is 3’ to the sequences amplified
• Forward primer helps gene copy and allows the template to bind correctly
• Primer sequence (%GC) and length of strand affect how they bind to the template
• DNA templates are extracted from nucleotide routine clinical analyses that need 100 ng – 1 µg

Dexoyribonucleotide Bases

• The building blocks of DNA
• Consists of dNTPs with dATP (adenine), dTTP (thymine), dGTP (guanine), and dCTP (cytosine)
• Usual requirement: 0.1-0.5 mM of each

DNA Polymerase

• Derived from E. coli
• Taq Polymerase: thermostable with good fidelity extracted from Thermus aquaticus
• Tth polymerase: from Thermus thermophilus, with reverse transcriptase activity

PCR Buffer

• Buffers promote enzyme activity to help primers bind to the template
• Improves PCR to specify components, including salts, stabilizers, enhancers, and Taq polymerase
• Tris buffers help offer the required pH to ensure optimal enzyme activity for accurate amplification at a pH 8-9.5

Monovalent Cations

• KCI and (NH4)2SO4 affecting temperature
• Salt concentration increases and longer DNA sequences slow the denature process

Divalent Cations

• MgCl2 in a concentration with 1 NTP that are needed by the DNA pol

Oligodeoxynucleotide (ODN)

• A short DNA molecule used in research, testing and therapeutic applications

Methods

• ODNs using solid-phase chemical synthesis, connecting A, T, G, and C nucleic acids
• Physical separations using UV light for decontamination to induce base damage
• Use 10% bleach chemical solution

Other Chemistries

• DUPT-UNG system with Uracil-N-glycosylase (UNG) degrades
• Concerning Primers - Avoid misprimes with good design

Misprimes

• Aberrant binding of the primer and carriers
• Result in other amplifications

Primer Dimers

• PCR artifacts that are doubled by the sizes of primers, leading to possible binding
• Problems caused by them require good preparation and hot-starts
• Solutions are good preparation with optimal conditions
• These are harmful to the workers, with inefficient and light limitations

Thermal Cycler

• Multiple water baths or heat blocks set rapidly and automatically
• Precise for different stages of PCR controls to facilitate

Controls

• Enzyme is active, buffers, primers, and machine
• Reagent blank with "Contamination"
• Negative template

Real Time PCR

• Used in gene expression studies for detecting targeted copy number and tumor load
• A cycle occurs when samples fluoresce at equal rates
• SYBR Green I and TaqMan can be used as assays

SYBR Green I Assay

• Replaces Ethidium bromide (EtBr) that has robust fluorescence
• Safer, more specific probe produces high quality results
• Toxicity is a related Key difference