Molecular Biology

Polymerase Chain Reaction & Lateral Flow

Polymerase Chain Reaction – A Short Summary

 

Polymerase Chain Reaction (PCR) is the oldest, best under­stood and most diverse DNA ampli­fi­cation technique. However, it plays a rather subor­dinate role in combi­nation with lateral flow based readout formats. Only about 11% of the NALFIA related publi­ca­tions refer to the use of PCR. The reasons for this are frequently mentioned in scien­tific publi­ca­tions. Polymerase Chain Reaction is often charac­terized as: laborious, time-​​and cost-​​intensive, needs special equipment and  highly trained personnel. Here, various points are explained and discussed in order to criti­cally question these arguments. This is a little teaser:

 

Especially in the context of NALFIAs, the PCR is may be the most under­rated
ampli­fi­cation technique with the biggest untapped potential.“

History of the Polymerase Chain Reaction

The concept of the polymerase chain reaction was initially described by Kary Mullis in the early 1980s. About ten years later, PCR has become one of the most important tools in molecular biology. In 1993, Kary Mullis received the well-​​deserved nobel prize in chemistry for inventing PCR. Today, more than 40 years after the first publi­ca­tions of this DNA ampli­fi­cation technique, PCR forms the basis for countless molecular biological methods and is used in manyfold in everyday life. A very concise example is the field of molecular diagnostics, which go far beyond human medical issues.

1983

Invention

PCR method was invented by Kary Mullis

1985

Landmark Paper

First description of the PCR-​​technique by Randall Saiki et al. (Science)

1988

Themal Cycler

First commer­cially available PCR machine intro­duced

1988

Taq Polymerase

Heatstable DNA-​​polymerase from Thermus aquaticus patented by Mullis

1991

RNA-​​Detection

Combi­nation of reverse transcription and PCR for the detection or RNA (RT-​​PCR)

1993

Nobel Prize

Micheael Smith & Kary Mullis received nobel prize in chemistry

1993

quanti­tative PCR

Quanti­tative Analysis with Real-​​Time PCR

2000

PCR & Lateral Flow

One of the first descrip­tions for the combi­nation of PCR & Lateral Flow Analysis by Kozwich et al. (Appl. Environ. Microbiol.)

1983

Invention

PCR method was invented by Kary Mullis

1988

Themal Cycler

First commer­cially available PCR machine intro­duced

1991

RNA-​​Detection

Combi­nation of reverse transcription and PCR for the detection or RNA (RT-​​PCR)

1993

quanti­tative PCR

Quanti­tative Analysis with Real-​​Time PCR

1985

Landmark Paper

First description of the PCR-​​technique by Randall Saiki et al. (Science)

1988

Taq Polymerase

Heatstable DNA-​​polymerase from Thermus aquaticus patented by Mullis

1993

Nobel Prize

Micheael Smith & Kary Mullis received nobel prize in chemistry

2000

PCR & Lateral Flow

One of the first descrip­tions for the combi­nation of PCR & Lateral Flow Analysis by Kozwich et al. (Appl. Environ. Microbiol.)

General Mechanism of the Polymerase Chain Reaction

The mechanism of the Polymerase Chain Reaction has been described over and over again. We try to keep it as simple as possible. First of all, you have to know what the PCR is. The Polymerase Chain Reaction is an enzyme-​​driven in vitro DNA-​​amplification technique, which allows the specific and sensitive detection of low abundant template DNA. The Polymerase Chain Reaction is subdi­vided into three charac­ter­istic, temperature-​​dependent steps:

  1. Denat­u­ration – melting double stranded template DNA into single strands (>90°C)
  2. Annealing – specific binding of primers to ss-​​template-​​DNA (50 – 70°C)
  3. Elongation – DNA-​​synthesis is catalized by heat stable DNA-​​polymerase, which elongates primers (68 – 72°C)

The cyclical repetition of these three essential steps allows exponential ampli­fi­cation a defined DNA fragment. Within 30 cycles, more than 1 billion DNA copies can be generated from a single template molecule. The following video „from 1 to 1 billion DNA copies – PCR explained in one minute“ shows the principle of PCR.

General Mechanism of the Polymerase Chain Reaction
Figure 2. Ampli­fi­cation mechanism of the Polymerase Chain Reaction

Modifi­cation of a PCR for Lateral Flow based Readout

In order to visualize a PCR product on a universal Lateral Flow Device (LFD), labels must be incor­potated somehow into the double strand. Various labeling strategies are applicable, which should be carefully considered before starting assay devel­opment. The labeling strategy is directly linked to the selected PCR method­ology. In addition to the incor­po­ration of the labels, other method­ological modifi­ca­tions can be used, which can be partic­u­larly important for PCR-​​LFA. An example for this is the PCR temper­ature protocol. It is an important tool within a PCR devel­opment project and can therefore make a signif­icant contri­bution to speci­ficity and sensi­tivity of the assay.

Terminal labeling of PCR primers may be the easiest and most intiutive way to detect a PCR product on a universal Lateral Flow Device. Labeled primers are efficiently incor­po­rated into amplicons by most DNA-​​polymerases during the ampli­fi­cation reaction. These short, dual labeled PCR products can be detected 100 to 1000 times more sensitive with Lateral Flow Analysis compared to standard agarose gelelec­trophoresis.

Polymerase Chain Reaction-based DNA-Amplification and Lateral Flow
Figure 3. Lateral Flow relevant labels are incor­po­rated during DNA-​​amplification reaction. Dual labeled amplicons are detectable via Lateral Flow Analysis.

The temper­ature protocol of a PCR should be under­stood as a charac­ter­istic and important tool. Compared to isothermal ampli­fi­cation methods, the inter­action of the PCR compo­sition and the temper­ature profile can be used in advance for the devel­opment of an excellent detection perfor­mance.

Method­ological Varia­tions of Polymerase Chain Reaction

PCR is probably the most multi­faceted and flexible DNA ampli­fi­cation technique. PCR-​​LFA can partic­u­larly benefit from this. The choice of the right PCR method for the appli­cation should be considered carefully. The condi­tions under which the method should finally be used are absolutely decisive for the choice of the appro­priate detection technique.

Advantage: Multi­plexing

Multi­plexing is the ability to amplify multiple, defined products in one reaction. PCR in particular is charac­terized by the fact that it allows a strong multi­plexing. The simple assay design in combi­nation with the multiplex compat­i­bility under­scores the value of the Polymerase Chain Reaction in general and especially in the field of Point-​​of-​​Care diagnostics.

Direct PCR and Internal Ampli­fi­cation control (IAC)

A wide variety of sample matrices can be used in PCR, if a suitable and robust enzyme system has been selected. This is the prereq­uisite for the direct appli­cation of a biological material in a PCR, saving the demand for a DNA or RNA extraction prior to do the PCR reaction. This is an important feature for the devel­opment of point-​​of-​​care compatible rapid tests. Direct analysis from blood is just one example of practical relevance. The direct PCR format can benefit from the multiplex ability by imple­menting an internal ampli­fi­cation control (IAC). This method­ological approach allows a clear differ­en­ti­ation between negative results and matrix-​​dependent PCR inhibition. The Milenia HybriDetect 2T allows rapid evalu­ation of a duplex PCR.

Importance of an internal amplification control using the direct Polymerase Chain Reaction approach
Figure 4. Impor­tance of an internal ampli­fi­cation control using the direct PCR approach

Advantage: Speci­ficity – Detection of SNPs

PCR is often used in molecular diagnostics when extremely specific detection is required. An example for this is the identi­fi­cation of single nucleotide polymor­phisms (SNPs). A SNP is a substi­tution of a single nucleotide at a certain position in a genome. Mostly SNPs are detected by special sequencing-​​, array– or PCR techniques. But the combi­nation of PCR and lateral flow analysis is partic­u­larly suitable for a rapid and Point-​​of-​​Care compatible SNP analysis. For example, the amplification-​​refractory mutation system (ARMS) – PCR has proven to be a useful tool for detection of SNPs over decades. We could also demon­strate, that simple hybridization can be very useful for genotyping or multiplex SNP-​​detection.

Limita­tions

Apper­ently, the PCR has certain limita­tions compared to other DNA-​​amplification techniques. A differ­en­tiated view reveals that many PCR-​​associated weak points can be compen­sated. The choice of appro­priate reaction compo­nents, the equipment and the overall simpli­fi­cation of the processing allow point-​​of-​​care compatible analysis. In addition, the PCR impresses with important charac­ter­istics like simple assay design, method­ological diversity, multi­plexing and excellent speci­ficity. When choosing the right DNA ampli­fi­cation method for the devel­opment of an individual rapid test, PCR should not be under­es­ti­mated.

Method Comparison Table
Method combined with LFASensi­tivitySpeci­ficitySpeedreverse Transcription (one pot)RobustnessMethod. VarietySimple Assay DesignMulti­plexing AbilityPrevention Carryover Cont.
PCR
LAMP
RPA
CRISPR (Label-​​Sep.)
CRISPR (Label-​​Inc.)