Difference Between NGS and PCR – A Comparative Analysis

In 2024, the infectious disease diagnostics segment holds the largest share of the molecular diagnostics applications market, accounting for 49.3%. This dominance is fueled by the growing incidence of respiratory infections, sexually transmitted diseases (STDs), and viral outbreaks such as COVID-19. 

The demand for rapid and accurate diagnostic solutions has accelerated the adoption of Polymerase Chain Reaction (PCR) and next-generation sequencing (NGS) technologies, enabling precise pathogen identification and improved disease management. 

Meanwhile, the oncology segment is projected to experience the fastest growth, with a CAGR of 10.0%. Due to the increasing need for early cancer detection, personalized therapies, and biomarker-based treatments, it is predicted to achieve a valuation of $32 billion.

Given this evolving environment, understanding the core technologies driving these advancements is essential. This article will provide a comparative analysis of NGS and PCR, highlighting their key differences in methodology, sensitivity, speed, clinical utility, and more.

Overview of Next-generation Sequencing (NGS)and Polymerase Chain Reaction (PCR)

Next-generation sequencing (NGS) and polymerase chain reaction (PCR) are key molecular technologies. NGS allows fast, large-scale reading of DNA and RNA for deep genetic analysis, while PCR focuses on quickly copying specific DNA sequences for detecting mutations and infections. 

Both play important roles in modern diagnostics and research. Let’s explore both of them individually.

NGS Overview

Next-generation sequencing (NGS) is a powerful technology that can quickly read and analyze large amounts of DNA and RNA. It can study millions of genetic pieces at once, helping find mutations, changes, and complex patterns with great speed and accuracy. 

NGS has changed the field of genomics by allowing full analysis of entire genomes, exomes, or specific gene sections. It plays a big role in research, disease diagnosis, and personalized medicine.

PCR Overview

Polymerase Chain Reaction (PCR) has contributed hugely to molecular biology since its development by Kary Mullis in the 1980s. Polymerase chain reaction (PCR) is a basic but important technique in molecular biology that copies specific DNA sequences. By going through repeated heating and cooling steps, PCR can create millions of copies of a targeted DNA fragment. 

It is widely used for detecting mutations, finding infections, and genetic testing because it is simple, sensitive, and reliable. Even though it is an older method than NGS, PCR is still a key tool in research and clinical diagnostics.

Now that you understand NGS and PCR, let’s compare how they work, starting with their core technological capabilities.

Technological Capabilities: NGS vs PCR

NGS and PCR differ significantly in how they analyze genetic material. NGS offers high-throughput sequencing with deep genomic coverage, which is ideal for detecting complex mutations and large-scale variations. 

Conversely, PCR provides fast and targeted DNA amplification, making it highly effective for specific mutation detection and pathogen identification. 

Each technology has unique strengths that suit different diagnostic and research needs. Let’s start the key difference exploration with NGS first.

Next Generation Technology (NGS) Capabilities

Next-generation sequencing (NGS) has fundamentally changed the field of genomics research. It can sequence millions of DNA fragments simultaneously, providing a detailed view of genome architecture, genetic variations, gene activity, and regulatory changes. 

Recent technological advancements have improved sequencing speed, accuracy, and cost-efficiency, opening new possibilities for disease research, personalized medicine, and clinical applications. Over the past two decades, DNA sequencing technologies have evolved rapidly, categorized into three generations:

1. First-Generation Sequencing

The first sequencing breakthrough came with methods like Sanger sequencing, which laid the groundwork for modern DNA analysis. Sanger’s chain termination method, later automated with instruments like the ABI 370, allowed researchers to produce relatively short DNA reads with high accuracy. Although now largely replaced, first-generation sequencing remains an important milestone.

2. Second-Generation (NGS) Technologies

Second-generation platforms, including Illumina, Ion Torrent, and 454 sequencing, introduced massive parallelization, enabling high-throughput, cost-effective DNA analysis. These technologies enabled whole-genome sequencing (WGS), whole-exome sequencing (WES), and targeted sequencing, significantly advancing disease research, variant discovery, and personalized genomics.

3. Third-Generation Sequencing

Third-generation methods, such as Pacific Biosciences’ SMRT sequencing and Oxford Nanopore’s technology, provide ultra-long reads, allowing for better genome assembly, real-time analysis, and the detection of complex structural variations. These methods eliminate amplification biases and can directly detect epigenetic modifications like DNA methylation.

Short-Read vs. Long-Read Sequencing: 

• Short-read sequencing provides high accuracy and is ideal for mutation detection and quantifying gene expression, but may struggle with large structural variants. 
• Long-read sequencing excels at resolving complex genomic regions and capturing structural variations, offering reads up to tens of kilobases.

While Next-generation sequencing (NGS) offers unmatched clarity for complex genomic analysis, it’s equally important to understand the strengths of traditional methods like PCR.

Polymerase Chain Reaction (PCR) Technology Capabilities

Polymerase Chain Reaction (PCR) is a powerful molecular biology technology that enables the rapid amplification of specific DNA sequences. By mimicking natural DNA replication in a controlled laboratory setting, PCR can generate billions of copies of a target DNA region from minimal starting material. 

Its high sensitivity, capable of detecting even a few DNA molecules, has transformed fields such as genetics, diagnostics, forensics, and biotechnology. At its core, PCR technology combines key scientific tools: precision temperature control through thermal cyclers, heat-stable DNA polymerases like Taq polymerase, carefully designed primers that target specific DNA sequences, and the use of nucleotides as raw materials for strand synthesis. 

Together, these technological elements allow PCR to automate and accelerate DNA copying outside living cells.

Core Components and Mechanism

PCR relies on four key components:

• DNA Template: Contains the target sequence for amplification. High temperatures (~95°C) denature the double-stranded DNA, separating it into single strands.
  
• Primers: Short, single-stranded DNA sequences that flank the target region. Both forward and reverse primers are essential to initiate replication.
  
• DNA Polymerase: A heat-resistant enzyme (e.g., Taq polymerase) that synthesizes new DNA strands by extending from the primers.
  
• dNTPs (Deoxynucleotide Triphosphates): The building blocks (A, T, G, and C) used to construct new DNA strands.

PCR operates through repeated cycles of:

• Denaturation: DNA strands separate at ~95°C.
  
• Annealing: Primers bind to target sequences at 50–65°C.
  
• Extension: DNA polymerase synthesizes new strands at ~75–80°C.

Each cycle doubles the amount of DNA, leading to exponential amplification. Typically, 30–40 cycles are performed before the reaction plateaus due to reagent depletion or enzyme inhibition. Variants like multiplex PCR further extend capabilities by amplifying multiple target sequences simultaneously within a single reaction.

Advancements: Real-Time PCR (qPCR)

Traditional PCR methods were limited to detecting amplified DNA using gel electrophoresis at the end of the process. The introduction of Real-Time PCR (qPCR) enhanced this approach by enabling real-time monitoring of DNA amplification through fluorescent markers.

Key features of qPCR include:

• Use of fluorophores such as SYBR Green for non-specific binding detection.
  
• Application of hybridization probes for sequence-specific detection using reporter and quencher molecules.
  
• Sensitive photodetectors in qPCR machines track fluorescence levels during each amplification cycle, allowing precise quantification of nucleic acids.

qPCR has become a standard tool for quantitative genetic analysis, including applications in pathogen detection, mutation analysis, and gene expression studies.

Reverse Transcription PCR (RT-PCR)

PCR technology expanded further with the development of Reverse Transcription PCR (RT-PCR), enabling the analysis of RNA. In RT-PCR:

• Reverse transcriptase first converts RNA into complementary DNA (cDNA).
  
• Standard PCR then amplifies the resulting cDNA, allowing researchers to study gene expression from mRNA samples.

Combined with qPCR (RT-qPCR), it provides powerful quantitative insights into gene expression levels across various biological and clinical samples, making it indispensable for virology, oncology, and personalized medicine.

As the demand for faster and higher-volume analyses grew, NGS and PCR technology have continued evolving with innovations to improve scalability and throughput.

Scalability and Throughput NGS vs PCR

When comparing Next-Generation Sequencing (NGS) and Polymerase Chain Reaction (PCR), two critical factors define their suitability for modern genomic applications: scalability and throughput.

Next-Generation Sequencing (NGS): High Scalability and Massive Throughput

NGS has redefined genomic analysis by offering unparalleled scalability and high-throughput capabilities. Modern NGS platforms, such as Illumina, PacBio, and Oxford Nanopore, can sequence millions to billions of DNA fragments simultaneously in a single run. This massively parallel processing allows researchers to:

• Perform whole-genome sequencing (WGS), whole-exome sequencing (WES), transcriptomics, epigenomics, and metagenomics.
  
• Scale projects from targeted gene panels to large population studies with minimal adjustments.
  
• NGS platforms support both short-read (e.g., Illumina) and long-read sequencing (e.g., PacBio, Oxford Nanopore), enabling, enabling detailed analysis of genetic variations, structural variants, and epigenetic modifications.
  
• Achieve real-time data generation and analysis, especially with portable platforms like Nanopore.

NGS platforms’ flexibility ensures they can adapt to diverse research and clinical needs, from small diagnostic labs to large-scale genomic initiatives. Additionally, advancements in automation, data handling, and cost-efficiency continue to enhance NGS scalability, making it ideal for large-scale and complex genomic projects..

PCR: Limited Scalability, Targeted Throughput

While PCR remains a cornerstone for targeted DNA amplification, its throughput and scalability are inherently limited:

• PCR is designed to amplify specific DNA sequences, making it ideal for focused applications like detecting known mutations, pathogen identification, or gene expression studies (via RT-PCR).
  
• Each PCR reaction targets a limited number of sequences, and while Real-Time PCR (qPCR) allows for quantification, it is still constrained to analyzing a few genes per run.
  
• Scaling PCR for high-throughput demands requires multiple parallel reactions, increasing manual workload, time, and reagent costs.
  
• PCR requires prior knowledge of target sequences, making it unsuitable for discovering novel mutations or untargeted regions.

NGS vs PCR: A Comparative Snapshot

This table compares NGS and PCR, outlining their strengths, limitations, and typical applications. NGS offers broad genomic insights, while PCR excels in targeted, rapid detection. 

Features	Next-Generation Sequencing (NGS)	Polymerase Chain Reaction (PCR)
Throughput	Millions to billions of reads/runs	Limited to specific targets per reaction
Scalability	Easily scalable from small panels to whole genomes	Requires manual scaling via multiple reactions
Data Output	Comprehensive genomic, transcriptomic, and epigenetic data	Targeted amplification data
Use Cases	Large-scale research, diagnostics, and precision medicine	Mutation detection, pathogen screening, gene quantification
Time Per Run	NGS typically takes 6–48 hours depending on platform	PCR often takes 1–3 hours.
Flexibility	High (can detect known and unknown sequences)	Low (requires known target sequences)

While PCR excels in speed and specificity for targeted applications, NGS dominates in scenarios requiring comprehensive analysis, high data volume, and scalable solutions. NGS offers a future-proof platform with unmatched throughput and adaptability for large-scale genomics, discovery research, and precision medicine. PCR, however, remains invaluable for routine diagnostics and focused genetic studies where rapid, targeted detection is essential.

Now that you have examined the scalability and throughput of NGS and PCR, let’s explore how their sensitivity and dynamic range impact the accuracy and depth of genetic analysis. 

Sensitivity and Dynamic Range NGS vs PCR

Next-Generation Sequencing (NGS) and Polymerase Chain Reaction (PCR) are both critical technologies in genetic mutation detection. However, they differ significantly in their sensitivity and dynamic range, which play a key role in determining their effectiveness for various applications, particularly in clinical diagnostics and research.  Let’s first explore the NGS sensitive and dynamic range. 

What is the Dynamic range? Dynamic range refers to the span between the lowest and highest detectable concentrations, which is critical when tracking both trace mutations and dominant alleles in a sample.

NGS Sensitivity in Mutation Detection

NGS provides high sensitivity, particularly for detecting low-frequency mutations. The sensitivity of NGS can vary depending on the mutation type and the background noise levels in the sample.

• KRAS Mutation Detection:
  • NGS demonstrated the ability to detect KRAS mutant alleles at concentrations as low as 0.02% to 0.56%, depending on the specific mutation type.
  • For example, G12D mutation: Detection limit as low as 0.02%. G12V mutation: Higher detection limit at 0.56%.
    
• Limit of Detection (LOD):
  • Background “noise” in wild-type DNA was carefully evaluated to determine the LOD (Limit of Detection), calculated as the mean noise level plus three standard deviations (3 SD).
  • NGS sensitivity outperforms traditional DNA sequencing methods, like Sanger sequencing (LOD ~20%), and is comparable to other NGS platforms such as Ion Torrent (LOD ~2%).
    
  • No false positives were detected below the defined LOD, indicating that NGS maintains high specificity within validated mutation thresholds.

NGS Dynamic Range of Quantification

NGS excels in quantifying genetic mutations across a broad concentration spectrum, making it an ideal tool for various clinical and research applications.

• Quantification of KRAS Mutations:
  • Serial dilutions of mutant alleles (ranging from 1% to 50%) showed a strong correlation between the prepared values and the NGS measurements.
  • The concordance rate exceeded 0.93, and the linearity was confirmed by an R² value ≥ 0.998, indicating high precision and accuracy.
    
• Applications:
  • This broad dynamic range allows NGS to quantify both low- and high-frequency mutations, making it suitable for tumor heterogeneity studies, minimal residual disease monitoring, and detailed allele frequency measurements.
  • However, mutations below 1% allele frequency pose challenges for consistent quantification, although NGS remains highly reliable above this threshold.

Now that we’ve covered NGS, let’s examine how PCR-based techniques perform, especially in low-concentration mutation detection.

PCR Sensitivity in Mutation Detection

PCR-based techniques, including digital PCR (dPCR), are widely used for mutation detection and quantification, but they face limitations, particularly at low concentrations.

• Sensitivity Challenges: Traditional digital PCR systems require large numbers of wells for high sensitivity, which can make them impractical for certain applications.
• Multivolume Digital PCR (MV dPCR): MV digital PCR enhances sensitivity by utilizing wells of varying volumes, where larger wells improve the detection of low-concentration targets.
  • The Lower Detection Limit (LDL) of MV dPCR is approximately 120 molecules/mL, making it suitable for applications such as HIV viral load detection, minimal residual disease monitoring, and early cancer diagnostics.
  • MV digital PCR eliminates the need for complex serial dilutions, reducing sample handling errors and cross-contamination risks.

PCR Dynamic Range of Quantification

The dynamic range of PCR refers to its ability to quantify mutations across different concentrations, from low to high.

• MV Digital PCR Advantages: MV digital PCR decouples resolution and dynamic range, allowing for independent adjustment based on application needs.
  • Dynamic Range: MV dPCR offers a range spanning over 4.5 orders of magnitude, covering concentrations from 520 molecules/mL to 3,980,000 molecules/mL.
  • The system achieves high precision and efficiency with fewer wells (e.g., 160 wells vs. 12,000 in traditional systems), reducing reagent consumption by over 67% compared to serial dilution methods.
  • Multiplex Testing: MV dPCR also supports multiplex testing, allowing for detection of multiple mutations in a single test.

Overall, both NGS and PCR play vital roles in genetic mutation detection, each with distinct strengths and trade-offs that suit different clinical and research needs. 

Applications and Use Cases of NGS and PCR

While NGS is known for its high-throughput sequencing capabilities, enabling comprehensive genetic analysis across a wide range of mutations, PCR excels in providing rapid, sensitive, and quantitative detection of specific DNA sequences. 

Together, these technologies play a critical role in disease diagnostics, genetic screening, and personalized medicine. Here’s how NGS and PCR are applied in diverse settings, including cancer diagnostics, infectious disease monitoring, and genetic research.

Applications of Next-Generation Sequencing (NGS) in Clinical and Research Settings

NGS is crucial for applications ranging from cancer diagnostics to genetic screening, rare disease research, and personalized medicine, offering detailed insights into the genetic makeup of individuals. Let’s explore some of the major applications of NGS below:-

1. Cancer Diagnostics and Personalized Medicine

Next-Generation Sequencing (NGS) has transformed cancer diagnostics by enabling the simultaneous detection of multiple gene mutations within a single test. This is crucial in oncology, where tumors often exhibit complex mutation patterns due to clonal evolution and tumor heterogeneity. NGS supports personalized medicine by identifying actionable mutations that guide targeted therapies and immunotherapy decisions. 

Additionally, it plays a key role in assessing tumor mutation burden (TMB), which helps predict patient response to immune checkpoint inhibitors.

2. Hereditary Disease and Cancer Risk Assessment

NGS is widely used for detecting germline mutations associated with hereditary cancers and rare genetic disorders. By offering comprehensive screening through whole-genome or exome sequencing, NGS aids in early diagnosis, especially when conventional genetic tests fail. 

It allows clinicians to assess cancer risk in high-risk populations by identifying mutations in genes like BRCA1/2, TP53, and PALB2, supporting preventive care and personalized surveillance strategies.

3. Infectious Disease and Virology

NGS has become a critical tool in infectious disease diagnostics and virology research. It enables the discovery of novel or unexpected viral pathogens through metagenomic approaches, especially in unexplained clinical syndromes or outbreaks. NGS also facilitates full viral genome sequencing for surveillance and epidemiological studies. 

In contrast, its ability to detect viral quasispecies helps in understanding mutation dynamics, immune evasion, and disease progression in infections like HIV and HCV.

4. Antiviral Drug Resistance Monitoring

In the management of chronic viral infections such as HIV, HBV, and HCV, NGS offers high sensitivity in detecting low-frequency drug-resistant variants that are often missed by traditional sequencing methods. 

This capability is essential for tailoring effective antiviral therapies, preventing treatment failures, and managing resistance development. Deep sequencing allows clinicians to make informed decisions by revealing minor resistant strains before they become dominant.

5. Microbiome and Virome Studies

NGS plays a pivotal role in characterizing the human microbiome and virome, uncovering complex microbial and viral communities residing in various body niches. These studies have linked microbiome imbalances to numerous health conditions. 

Additionally, NGS-based metagenomics is used to explore environmental viromes, providing insights into viral diversity in ecosystems like oceans, wastewater, and soil, with implications for public health and environmental monitoring.

6. Quality Control in Vaccine Production

In vaccine manufacturing, particularly for live-attenuated vaccines, NGS ensures genetic stability by detecting reversion mutations and potential contaminant viruses. 

This high-resolution monitoring safeguards vaccine safety and efficacy, offering a more sensitive alternative to traditional quality control methods.

7. High-Throughput Diagnostic Virology

NGS plays a transformative role in diagnostic virology by enabling comprehensive, high-throughput detection of viral pathogens, particularly in complex or unexplained infections where conventional tests are limited. Its sensitivity allows for the identification of low-abundance, drug-resistant mutations in chronic viral infections and supports rapid, unbiased analysis during outbreaks, making it a cornerstone of modern infectious disease diagnostics.

Applications of PCR in Clinical Diagnostics and Microbiology

Polymerase Chain Reaction (PCR) has contributed hugely to clinical microbiology by offering fast, sensitive, and highly specific detection of pathogens. Moving beyond traditional culture-based methods, PCR enables clinicians to identify infectious agents at the genetic level, even in challenging scenarios where conventional diagnostics fall short. 

Below are key applications where PCR plays a critical role in modern infectious disease management.

1. Rapid Detection of Infectious Agents

PCR has revolutionized the speed at which clinicians can detect infectious agents. Unlike traditional culture methods that may take days or even weeks, PCR-based diagnostics can identify microbial DNA or RNA within hours. 

This rapid turnaround is crucial in acute infections such as meningitis or sepsis, where early identification can guide timely and targeted therapy, potentially saving lives and reducing reliance on broad-spectrum antibiotics.

2. Enhanced Sensitivity for Difficult-to-Culture Pathogens

One of PCR’s greatest strengths lies in its ability to detect pathogens that are fastidious, slow-growing, or entirely uncultivable using standard laboratory methods. Microorganisms like Mycobacterium leprae, Treponema pallidum, or certain viruses evade traditional cultivation but can be identified through PCR by amplifying their unique genetic material. This makes PCR indispensable for diagnosing infections where conventional methods routinely fail.

3. Broad-Range and Multiplex Pathogen Detection

PCR offers flexibility through broad-range assays that can detect a wide spectrum of bacteria or viruses by targeting conserved genetic regions, such as the 16S rRNA gene in bacteria. Additionally, multiplex PCR allows simultaneous detection of multiple pathogens in a single reaction, which is particularly useful for syndromic testing like identifying the causative agent of meningitis or respiratory infections from a predefined panel of microbes.

4. Genetic Identification and Phylogenetic Analysis

Unlike phenotypic methods, PCR provides genetic-level identification, offering higher specificity and reliability. By amplifying and sequencing conserved yet variable genomic regions, clinicians can accurately identify microbial species and even infer evolutionary relationships. This genetic “fingerprint” is invaluable for diagnosing rare or novel pathogens and for epidemiological tracking of outbreaks.

5. Quantitative PCR for Monitoring Disease Progression

Real-time PCR (qPCR) not only detects pathogens but also quantifies microbial load in patient samples. This quantitative capability is essential for monitoring chronic infections, such as HIV or hepatitis, where viral load measurements guide treatment decisions and assess therapeutic efficacy over time.

6. Application in Histology and Difficult Specimens

PCR complements histological analysis by detecting microbial DNA directly from tissue samples, including formalin-fixed paraffin-embedded (FFPE) specimens. This is particularly useful when visual detection through microscopy is inconclusive due to low organism burden or ambiguous staining, providing a molecular confirmation of infection.

7. Safe Identification of High-Risk Pathogens

For highly infectious agents like Francisella tularensis or Coccidioides immitis, PCR offers a safer diagnostic alternative. Since nucleic acids can be extracted after pathogen inactivation, laboratory personnel are not exposed to live dangerous organisms, reducing biosafety risks while ensuring accurate diagnosis.

As you have already covered the applications, it’s clear that both NGS and PCR have distinct strengths in diagnostics and research. Next, let’s explore the key factors that guide the choice between these two technologies.

Factors Influencing Technology Choice

When selecting between advanced molecular techniques, the decision often depends on the scope, objective, and resources of the study or clinical application. Both DNA sequencing methods, like NGS, and gene amplification techniques, such as PCR, offer distinct advantages based on specific needs.

1. NGS for Genetic Testing and Comprehensive Genomic Profiling

Next-Generation Sequencing (NGS for genetic testing) is the preferred choice when comprehensive genomic profiling is required. Its ability to sequence millions of fragments simultaneously makes it ideal for discovering novel mutations, analyzing tumor heterogeneity, or conducting whole-genome and exome studies. 

NGS is invaluable in research settings and personalized medicine, where broad, in-depth analysis across numerous genes is critical.

2. PCR for Targeted Analysis Using Gene Amplification Techniques

Gene amplification techniques like PCR are more practical when the focus is on detecting known mutations or specific pathogens. PCR offers rapid, cost-effective, and highly sensitive detection, making it suitable for routine diagnostics where targets are predefined.

For institutions with limited budgets or when fast turnaround is essential, PCR remains a go-to method among DNA sequencing methods for targeted applications.

Overall, NGS excels in exploratory and large-scale genomic studies, while PCR is ideal for focused, hypothesis-driven analyses. 

Conclusion

Both Next-Generation Sequencing (NGS) and Polymerase Chain Reaction (PCR) play essential roles in modern genomics. NGS is well-suited for large-scale, detailed genetic analysis, while PCR offers speed, specificity, and cost-efficiency for targeted applications. Choosing between them depends on your research goals, whether you’re conducting broad discovery or focused validation.

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FAQs

1. What is the difference between PCR and gene sequencing?

PCR (Polymerase Chain Reaction) is a technique used to amplify specific DNA segments, creating millions of copies. Gene sequencing, however, is used to determine the exact nucleotide order in a DNA strand. PCR multiplies DNA; gene sequencing reads its sequence.

2. What are the advantages of NGS over PCR?

Next-Generation Sequencing (NGS) offers high-throughput, parallel sequencing of millions of DNA fragments, enabling comprehensive genomic analysis. Unlike PCR, which targets known DNA regions, NGS can detect unknown mutations, genetic variations, and provides broader, more detailed data in a cost-effective manner for large-scale studies.

3. What is the difference between NGS and DNA sequencing?

NGS (Next-Generation Sequencing) is an advanced form of DNA sequencing that sequences millions of fragments simultaneously. Traditional DNA sequencing methods, like Sanger sequencing, process one fragment at a time. NGS is faster, scalable, and suited for whole-genome or complex genetic analyses.

4. What is the difference between cycle sequencing and PCR?

Cycle sequencing is a technique based on PCR, used in Sanger sequencing to both amplify DNA and incorporate chain-terminating nucleotides for reading sequences. PCR is solely for amplifying DNA without revealing sequence information. Cycle sequencing provides both amplification and sequencing, while PCR focuses only on duplication.