Genetic Sequencing and Polymerase Chain Reaction Technologies: Products and Emerging Trends

Sequencing methods have evolved to encompass high-throughput, high-resolution methods based on advanced capillary and microfluidic technologies

Genetic analysis and gene expression

Genetic analysis is the study of genes or DNA underlying an individual’s phenotype or physical state. Modern genetic analysis methods are used to identify variants or differences between native and diseased states -- in order to assess risk and to aid in therapeutic intervention. Gene expression is the activation of a certain gene or genes in a specific cell or group of cells. Gene expression leads to messenger RNA production which then results in protein synthesis – mRNA typically being the “readout” of gene expression. Again, differential analysis of gene expression can aid in understanding disease states and specific genes actively involved.

Genetic research and molecular genetics

Genetic research has come a long way from the early Mendelian Genetics - and with the help of Molecular Biology and Biochemistry - has evolved to include the molecular basis of gene structure, function, expression, and control – all integral parts of the extensive field of modern Molecular Genetics.

With that quick review, we can turn a focus on two main areas of molecular genetics research – genetic sequencing and expression analysis technologies.

What is genome sequencing?

  • In it’s basic form, genome sequencing is the process of determining the nucleotide code(s) underlying genes derived from genomic DNA – whether from human cells, model organisms, bacteria and viruses, or other sources.

Targeted Sequencing versus Whole Genome Sequencing

  • Whole genome sequencing is a comprehensive method in which the entire genome of an organism is analyzed.
  • Rapidly dropping costs and innovations in technology have fostered the production of large volumes of genetic data and the pursuit of genome-wide associated studies (GWAS).
  • Analysis of single nucleotide polymorphisms (SNPs) – single nucleotide changes which are by far the most common form of genetic variation within a species – is a critical component of these GWAS studies.
  • Comparison of SNP patterns and other genetic and biochemical markers fosters a greater depth of understanding regarding disease states, inherited markers, and gene mutations that drive cancer and disease susceptibility.
  • Targeted sequencing and deep sequencing have enabled follow on studies and fine-mapping of definitive SNPs, causative for a given disease or altered pathway.

DNA sequencing methods and technologies

Sequencing methods and instruments have evolved far beyond the early electrophoresis gel-based techniques to now encompass high-throughput, high-resolution methods based on capillary and microfluidic technologies. Major improvements include: depth of interrogation through parallel processing, accuracy, speed, and resource use.

Next Generation Sequencing Analysis

There are many advanced technologies and applications that fall under this new umbrella of Next Generation Sequencing (NGS) analysis.

  • The Applied Biosystems AB370 released in 1987 and the 3730xl in 1998 were early examples of automated capillary electrophoresis technology and became the primary workstations for the NIH and Celera-led Human Genome Project.
  • Advancement from these “first generation” instruments to “next generation sequencing” technologies meant huge increases in throughput, from ~80 kilobases per run to 1 gigabase per run. This NGS technology also introduced the concept of massively parallel sequencing, involving large-scale parallel reactions, short overlapping reads, and substantial data computational power.
  • The pace of sequencing throughput has accelerated immensely, with over 1000x increase in processing speed over the span of 2005 to 2014. At the same time, costs per run have plummeted to the point that individual human genomes can now be sequenced for ~$1000.
  • Prominent NGS instrument platforms include the suite of sequencing instruments from Illumina. The technology behind these systems builds off the fluorescence capillary electrophoresis concept in a technique called sequencing by synthesis (SBS). Massively parallel sequencing reactions are carried out with high accuracy, a high yield of error-free reads, with high precision.
  • A novel technology and a departure from the fluorescent CE concept is the Ion Torrent platform from Thermo Scientific. Based on semiconductor chip design, the Ion Torrent technology exploits the fact that addition of a dNTP to a DNA polymer results in release of a hydrogen ion. Use of scalable semiconductors to measure millions of hydrogen ions forms the basis for determining sequence of DNA samples.
  • Oxford Nanopore has pioneered an advanced sequencing technology based around nano-scale sensing of pore conductance. Nanopores embedded in membranes are subjected to ionic currents, and interaction of biomolecules (DNA/RNA/protein) can be detected by the change in conductance. In this way, unique conductance measurements of an entire sequence can be deduced from a single DNA sample. The systems can be tailored to suit specific analyte types as well as biological or solid material nanopore arrays.

Gene expression and PCR analysis technologies

The polymerase chain reaction method is used to quantify nucleic acids by amplifying a nucleic acid molecule with the enzyme DNA polymerase.

Digital PCR

  • Digital PCR builds upon traditional PCR and fluorescent probe-based detection methods to provide highly sensitive quantification of nucleic acides without the need for standard curves. Traditional quantitative PCR (qPCR) requires normalization to controls – either reference standard or standard curve.
  • A critical step in digital PCR, and a major departure from conventional PCR, is sample portioning – the division of each sample into discrete subunits prior to PCR amplification.
  • Following standard PCR setup procedures, the reaction sample is separated into thousands of partitions, each ideally containing a single DNA template.
  • Following PCR amplification, each partition is quantified as either having the target sequence or not, either a positive (1) or a negative (0) result.
  • The ratio of positives to negatives in each analyzed sample then forms the basis for quantification – which relies on Poisson statistics, rather than typical methods of counting PCR cycle numbers and estimating target abundance.

Droplet Digital PCR (ddPCR)

  • ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology.
  • A sample is fractionated into 20,000 droplets, and PCR amplification of the template molecules occurs in each individual droplet. ddPCR technology uses reagents and workflows similar to those used for most standard Taqman Probe-based assays.
  • The massive sample partitioning is a key aspect of the ddPCR technique.
  • The Biorad QX200 Droplet Digital PCR (ddPCR) System consists of two instruments, the QX200 Droplet Generator and the QX200 Droplet Reader, plus their associated software and consumables.
  • The QX200 Droplet Generator partitions samples (20 µl into 20,000 nanoliter-sized droplets) for PCR amplification.
  • Following amplification using a thermal cycler, droplets from each sample are analyzed individually on the QX200 Droplet Reader, where PCR-positive and PCR-negative droplets are counted to provide absolute quantification of target DNA in digital form.

View details on the Biorad QX200™ AutoDG™ Droplet Digital™ PCR System at

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