What Is Oligonucleotide Synthesis? PCR-based amplification

Learn how scientists create custom DNA and RNA strands for use in research, diagnostics, and drug development.

This first article will cover method 3. PCR-based amplification

Oligonucleotide synthesis represents a foundational technique in molecular biology, biotechnology, and genomics, enabling the creation of custom-designed short sequences of nucleotides—DNA or RNA fragments—crucial for numerous applications such as genetic testing, diagnostics, gene editing, synthetic biology, and therapeutic development. The ability to synthesize these molecules with high specificity and precision has revolutionized the study of nucleic acids and facilitated a broad range of innovations across multiple scientific disciplines. Typically ranging between 5 and 100 nucleotides in length, oligonucleotides serve as primers for PCR, molecular probes, antisense therapies, and components in gene assembly, underscoring their indispensable role in both experimental and applied molecular sciences.

As this article series unfolds, we’ll explore each method in detail—covering workflows, reagents, advantages, limitations, and real-world applications across biotech, pharma, and academic research. This first article will cover method 3.

PCR-based amplification

Polymerase Chain Reaction (PCR) is one of the most transformative technologies in molecular biology, allowing for the rapid and exponential amplification of specific DNA sequences. Originally developed in the 1980s, PCR has become a cornerstone technique for DNA cloning, genetic analysis, diagnostics, and numerous other applications. The essence of PCR is its ability to take a small initial sample of DNA and generate millions to billions of copies of a specific sequence within a matter of hours.

While PCR is primarily known as a method for amplification, it also plays a crucial role in DNA synthesis and modification through creative variations like overlap extension PCR. This opens up new possibilities for gene assembly, site-directed mutagenesis, and synthetic biology.

Let’s break down the PCR process in technical detail and explore its applications in both DNA amplification and synthesis.

Key Components of PCR

Before diving into the mechanism of PCR, it is essential to understand the core components of the reaction, which are carefully selected to ensure efficient DNA amplification:

Template DNA: This is the DNA sequence to be amplified. The template can be any source of DNA, from genomic DNA to plasmid DNA, even as little as a single molecule.

Primers: Primers are short, single-stranded oligonucleotides (usually 18-30 nucleotides long) that flank the target DNA region. PCR uses two primers: a forward primer and a reverse primer, which are designed to anneal to the complementary sequences on opposite strands of the template DNA. They serve as starting points for the DNA polymerase to begin replication.

DNA Polymerase: The enzyme responsible for synthesizing new DNA strands. Most commonly, Taq polymerase is used, a thermostable enzyme derived from Thermus aquaticus, which can withstand the high temperatures of PCR. There are also high-fidelity polymerases available, such as Pfu polymerase, that possess proofreading activity, reducing error rates during amplification.

dNTPs (Deoxynucleotide Triphosphates): These are the building blocks of DNA (dATP, dCTP, dGTP, dTTP). They provide the necessary nucleotides for the polymerase to assemble the new DNA strand.

Buffer: A buffer solution maintains the optimal pH and ionic strength for the enzyme's activity. The buffer typically contains Mg²⁺, which is a critical cofactor for DNA polymerase.

Thermal Cycler: PCR is carried out in a thermal cycler, which precisely controls the temperatures required for the different steps of the reaction. It rapidly heats and cools the reaction mixture to cycle through denaturation, annealing, and extension.

Step-by-Step Mechanism of PCR

PCR operates through a series of repetitive thermal cycles, each consisting of three primary steps:

Denaturation (94-98°C)

In the denaturation step, the double-stranded DNA (dsDNA) template is heated to a high temperature (typically between 94°C and 98°C), causing the hydrogen bonds between the complementary bases to break. This results in the separation of the DNA into single-stranded DNA (ssDNA) molecules.

Denaturation typically lasts for 20-30 seconds and is crucial for ensuring that the template DNA is accessible for primer binding in the next step.

Annealing (50-65°C)

In the annealing step, the reaction is cooled to a temperature that allows the primers to bind (anneal) to their complementary sequences on the ssDNA template. The exact temperature depends on the melting temperature (Tm) of the primers, which is influenced by their length and GC content.

Annealing temperatures are generally 5°C below the Tm of the primers.

Primers bind to their target regions on the template DNA, with the forward primer binding to the 3’ end of the sense strand and the reverse primer binding to the 3’ end of the antisense strand.

Annealing typically lasts for 20-40 seconds.

Extension (72°C)

In the extension (or elongation) step, the temperature is raised to 72°C, the optimal temperature for the activity of Taq polymerase. The polymerase extends the primers by adding nucleotides to the 3’-OH end, using the single-stranded template as a guide.

The polymerase adds dNTPs in the 5’ to 3’ direction, synthesizing a new complementary DNA strand.

The extension time depends on the length of the target DNA fragment and the speed of the polymerase. For Taq polymerase, the extension rate is typically 1000 base pairs per minute.

After the extension step, a complete copy of the target region is synthesized, doubling the number of DNA molecules in the reaction.

This denaturation-annealing-extension cycle is repeated typically for 25-40 cycles, resulting in exponential amplification of the target DNA sequence.

Final Elongation (72°C)

After the last cycle, an additional final extension step is often added to ensure that any incomplete DNA strands are fully extended. This step typically lasts for 5-10 minutes at 72°C.

PCR Amplification: Exponential Growth of DNA Copies

The beauty of PCR lies in its exponential amplification of the target sequence. In each cycle, the number of DNA molecules approximately doubles. After n cycles, the total number of copies can be represented by the equation:

Number of copies=2n\text{Number of copies} = 2^nNumber of copies=2n

Thus, after 30 cycles, a single molecule of DNA can theoretically produce over a billion copies (2³⁰ ≈ 10⁹). However, in practice, efficiency slightly decreases with each cycle due to limitations like enzyme degradation, reagent depletion, or incomplete reactions.

Creative Uses of PCR in DNA Synthesis

In addition to its role in amplifying existing DNA sequences, PCR can also be creatively used to synthesize new DNA sequences or introduce specific changes into DNA. Some of the key techniques include overlap extension PCR, site-directed mutagenesis, and gene assembly.

Overlap Extension PCR (OE-PCR)

Overlap extension PCR (OE-PCR) is a powerful technique used to assemble two or more DNA fragments into a longer sequence. It is frequently used in gene synthesis and protein engineering to fuse genes, create chimeric proteins, or introduce mutations into specific regions of DNA.

The key innovation in OE-PCR is that the fragments to be joined contain overlapping regions of sequence homology at their ends, which allows them to anneal to each other during PCR. Here’s a detailed step-by-step breakdown of how OE-PCR works:

Step 1: Amplification of DNA Fragments

First, two or more DNA fragments are amplified using standard PCR, but the primers are designed so that the ends of the fragments overlap. For example, the 5' end of Fragment A will share homology with the 3' end of Fragment B.

Step 2: Overlap Extension

The overlapping ends of the amplified fragments are complementary, so they can anneal to each other in the next round of PCR without additional primers.

The annealed fragments serve as templates for DNA polymerase, which fills in the gaps to create a full-length DNA sequence that combines the two original fragments.

Step 3: Amplification of the Full-Length Product

After the overlap extension step, the newly synthesized full-length product can be amplified using external primers that flank the ends of the two original fragments. This ensures that the product is fully extended and ready for downstream applications.

This technique is extremely useful for:

Gene synthesis: Assembling multiple fragments of a gene into one long, continuous sequence.

Mutagenesis: Introducing specific mutations or deletions by designing primers that incorporate the desired changes into the overlapping regions.

Fusion proteins: Joining two or more protein-coding sequences to create chimeric proteins or novel protein constructs.

Site-Directed Mutagenesis via PCR

Site-directed mutagenesis is another creative use of PCR, where specific changes are introduced into a DNA sequence at precise locations. This technique allows researchers to alter single nucleotides, insertions, or deletions to study the function of specific regions of DNA or protein domains.

Here’s how site-directed mutagenesis works:

Designing Mutant Primers: Mutant primers are designed to contain the desired base change (or multiple changes) at a specific site within the sequence. These primers still anneal to the template DNA but contain a mismatch at the target position.

PCR Amplification: The mutant primers are used in a PCR reaction with the template DNA. The polymerase extends the primer, incorporating the mutation into the newly synthesized strand.

Amplification of Mutant DNA: After a few cycles, the mutant DNA becomes the predominant product, which can then be cloned or sequenced to confirm the introduction of the desired mutation.

Gene Synthesis and Assembly

PCR can also be used in gene synthesis by assembling multiple short oligonucleotides into a complete gene. This is achieved by designing overlapping oligonucleotides that represent the entire sequence of the gene. These oligonucleotides are then assembled using a combination of overlap extension PCR and normal amplification PCR.

Steps involved in gene synthesis using PCR:

Design of Oligonucleotides: Short, overlapping oligonucleotides are designed to cover the entire gene sequence. Each oligonucleotide overlaps with its neighboring oligonucleotide by around 20-30 bases.

Assembly by PCR: The overlapping oligonucleotides are mixed and undergo PCR. The overlapping regions anneal to one another, and the DNA polymerase fills in the gaps, assembling the full-length gene.

Amplification of Full-Length Gene: After assembly, external primers flanking the ends of the gene are used to amplify the entire construct.

Gene synthesis via PCR enables researchers to design completely novel genes or optimize codons for expression in different organisms.

Key Considerations and Challenges in PCR

While PCR is a powerful tool, several technical challenges and considerations must be taken into account:

Primer Design: Primers are critical for the specificity and success of the PCR. Poorly designed primers can lead to non-specific amplification or primer-dimers, where primers anneal to each other instead of the template. Primer design software often helps ensure proper design.

Error Rates: DNA polymerases like Taq polymerase lack proofreading activity, which can lead to errors in the amplified sequences, especially in long products. High-fidelity polymerases such as Pfu or Q5 can be used to minimize errors, but they are generally more expensive and slower than Taq.

Template Complexity: Highly complex templates, such as GC-rich regions or secondary structures, can cause PCR failure or incomplete amplification. Additives like DMSO or betaine can be used to mitigate these issues.

Contamination: PCR is highly sensitive, and even trace amounts of contaminating DNA can result in false positives. Stringent laboratory practices, such as using separate work areas and dedicated pipettes, are essential to avoid contamination.

Applications of PCR Beyond Amplification

PCR has applications far beyond simple DNA amplification:

Cloning: Amplified DNA sequences can be cloned into vectors for expression in cells or organisms.

Diagnostics: PCR is used in pathogen detection, such as in the diagnosis of viral infections (e.g., COVID-19 tests), bacterial infections, and genetic diseases.

Forensics: PCR is a fundamental tool in forensic science for analyzing DNA from crime scenes, often using STR analysis (short tandem repeat).

Quantitative PCR (qPCR): This variation of PCR allows for the quantification of DNA or RNA in real-time, often used in gene expression studies.

The Polymerase Chain Reaction (PCR) is one of the most versatile and widely used techniques in molecular biology. While its primary function is to amplify specific DNA sequences, PCR can also be used for gene synthesis, mutagenesis, and other creative applications like overlap extension. With proper primer design and careful optimization, PCR remains a powerful and efficient method for generating large quantities of DNA from small starting material, and for modifying or assembling new genetic sequences.