An Analysis of Molecular Diagnostic Technologies Such as PCR, Isothermal Nucleic Acid Amplification and Sequencing

Molecular diagnostic technology uses molecular biological methods to detect the expression and structure of genetic material in the human body and various pathogens, so as to achieve the purpose of predicting and diagnosing diseases.

In recent years, with the upgrading and iteration of molecular diagnostic technology, the clinical application of molecular diagnosis has become more extensive and in-depth, and the molecular diagnostic market has entered a period of rapid development.

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01 PCR Technology

PCR (polymerase chain reaction) is one of the in vitro DNA amplification technologies and has a history of more than 30 years.

Basic Principle of PCR

PCR can amplify the target DNA fragment more than one million times. Its principle is to use the parent chain DNA as a template and specific primers as the extension starting point under the catalysis of DNA polymerase, through denaturation, annealing, extension and other steps, to replicate the sub-chain DNA complementary to the parent chain template DNA in vitro.

The standard PCR process consists of three steps:

1. Denaturation : Use high temperature to separate the double-stranded DNA . The hydrogen bonds between the double-stranded DNA are broken at high temperatures ( 93-98 °C).

2. Annealing : After the double-stranded DNA is separated, the temperature is lowered to allow the primer to bind to the single-stranded DNA .

3. Extension : DNA polymerase starts to synthesize a complementary chain along the DNA chain from the primer bound when the temperature is lowered. Once the extension is completed, one cycle is completed and the number of DNA fragments doubles.

By repeating these three steps 25-35 times, the number of DNA fragments will increase exponentially.

PCR lies in that different primers can be designed for different target genes, so that the target gene fragment can be amplified to millions in a short period of time.

So far, PCR can be divided into three categories, namely PCR , real-time PCR?and digital PCR.

First Generation PCR

using a common PCR amplifier, and then the product is detected using agarose gel electrophoresis, which can only perform qualitative analysis.

The main disadvantages of the first generation PCR:

Nonspecific amplification and false-positive results are prone to occur.

The detection is time-consuming and the operation is cumbersome.

Only qualitative testing can be done.

Second Generation?Real-Time PCR

Real-time PCR also known as qPCR, adds fluorescent probes that can indicate the reaction process to the reaction system, monitors the accumulation of amplification products through the accumulation of fluorescent signals, judges the results through fluorescence curves, and can be quantified with the help of Cq values and standard curves.

qPCR technology is most widely used in clinical practice because the operation process is carried out in a closed system, which reduces the probability of contamination, and can perform quantitative detection by monitoring fluorescent signals. It has become the dominant technology in PCR.

Real-time PCR can be divided into: TaqMan fluorescent probes, molecular beacons and fluorescent dyes.

1) TaqMan Fluorescent Probe:

PCR amplification, a specific fluorescent probe is added together with a pair of primers. The probe is an oligonucleotide with a reporter fluorescent group and a quencher fluorescent group labeled at both ends.

When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5' - 3' exonuclease activity of the Taq enzyme cleaves and degrades the probe, separating the reporter fluorescent group and the quencher fluorescent group, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DNA chain is amplified, a fluorescent molecule is formed, achieving complete synchronization between the accumulation of the fluorescent signal and the formation of the PCR product.

2)?SYBR Fluorescent Dye:

In the PCR reaction system, an excess of SYBR fluorescent dye is added . After the SYBR fluorescent dye is non-specifically incorporated into the double-stranded DNA , it emits a fluorescent signal, while the SYBR dye molecules that are not incorporated into the chain will not emit any fluorescent signal, thus ensuring that the increase in the fluorescent signal is completely synchronized with the increase in the PCR product. SYBR only binds to double-stranded DNA , so the specificity of the PCR reaction can be determined through the melting curve.

3) Molecular Beacon

It is a stem-loop dual-labeled oligonucleotide probe that forms a hairpin structure of about 8 bases at the 5 and 3 ends . The nucleic acid sequences at both ends are complementary and paired, resulting in the fluorescent group and the quenching group being in close proximity, and no fluorescence is generated.

PCR product is generated, during the annealing process, the middle part of the molecular beacon pairs with the specific DNA sequence, and the fluorescent gene and the quenching gene are separated to produce fluorescence.

The main disadvantages of the second generation PCR:

The sensitivity is still lacking, and the detection of low-copy samples is inaccurate.

There is background value influence and the results are easily disturbed.

PCR inhibitors in the reaction system, the detection results are easily disturbed.

Third?Generation Digital PCR

Digital PCR (dPCR, Dig-PCR) calculates the copy number of the target sequence through endpoint detection, and can perform accurate absolute quantitative detection without the use of internal references and standard curves.

Digital PCR uses endpoint detection and is not dependent on the Ct value (cycle threshold), so the digital PCR reaction is less affected by amplification efficiency, has improved tolerance to PCR reaction inhibitors, and has high accuracy and reproducibility.

Because of its high sensitivity and high precision, it is not easily interfered by PCR reaction inhibitors and can achieve true absolute quantification without the need for standards, making it a hot topic in research and application.

According to the different forms of reaction units, they can be mainly divided into three types of systems: microfluidic, chip and droplet.

1) Microfluidic digital PCR, mdPCR.

Based on microfluidics technology, DNA templates are separated. Microfluidics technology can achieve the generation of nanoliter or smaller droplets of samples, but the droplets require special adsorption methods and then combine with the PCR reaction system. mdPCR has gradually been replaced by other methods.

2) Droplet-based digital PCR, ddPCR.

The sample is processed into droplets using oil-in-water droplet generation technology, and the reaction system containing nucleic acid molecules is divided into thousands of nanoliter droplets, each of which either contains no nucleic acid target molecules to be detected, or contains one to several nucleic acid target molecules to be detected.

3) Chip-based digital PCR, cdPCR.

Many microtubes and microcavities are engraved on silicon wafers or quartz glass using integrated fluid pathway technology. The flow of solutions in them is controlled by different control valves, and the sample liquid is divided into nanoliters of uniform size and placed in reaction wells for digital PCR reaction to achieve absolute quantification.

To summarize, the sample and PCR reaction solution are added to the microfluidic chip and placed in the instrument. A single-layer tiled droplet array is generated by physical methods. Each droplet is then amplified, and then six-channel fluorescence signals are collected. The positive and negative droplets are counted, and the absolute copy number concentration of the target gene is calculated through Poisson distribution.

The main disadvantages of the third generation PCR:

The equipment and reagents are expensive.

The template quality requirement is high. If the amount of template exceeds the amount of the microsystem, quantification will be impossible. If the amount of template is too little, the quantitative accuracy will be reduced.

False positives can also occur when there is nonspecific amplification.

PCR Extension Techniques

1. Touchdown PCR: The temperature gradually decreases in the first few cycles.

2. Reverse transcription PCR ( RT-PCR ): Using cDNA reverse transcribed from mRNA as a template, and because it is incremented from the phenotypic gene, the cDNA product produced does not contain introns (meaningless sections in the gene) and is often used in molecular cloning technology.

3. Hotstart PCR: Use highly heat-activated nucleic acid polymerase to carry out the reaction to reduce non-specific products.

4. Nested PCR: First use low-specificity primers to amplify for several cycles to increase the amount of template, and then use high-specificity primers to amplify.

5. Multiplex PCR: Using multiple sets of primers in the same tube.

6. Reconditioning PCR: The PCR product is diluted 10 times and then the original concentration of primers and dNTPs are added back and cycled for 3 times to eliminate heterodimers in the product.

7. dsRNA replicator: Combine high-fidelity DNA polymersae , T7 RNA polymerase and Phi6 RNA replicase ; transcribe double-stranded DNA into corresponding double-stranded RNA ( dsRNA ). Can be applied to RNAi experimental operations.

8.COLD-PCR (co-amplification at lower denaturation temperature PCR): a PCR application technology used to detect mutations or special alleles .

Reference:

1. Liu Wantong et al., Progress in the clinical application of molecular diagnostic technology

2. Yang Liu et al., Research progress on gene detection technology for non-squamous non-small cell lung cancer

3.G.Terrance Walker,etc.Strand displacement amplification-an isothermal, in vitro DNA amplification technique

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02 Isothermal Nucleic Acid Amplification

PCR is the most widely used nucleic acid amplification technology and is widely used for its sensitivity and specificity. However, PCR requires repeated thermal denaturation and cannot get rid of the limitation of dependence on instruments, which limits its application in clinical field testing.

Since the early 1990s, many laboratories have begun to develop isothermal amplification technologies that do not require thermal denaturation. They have now developed loop-mediated isothermal amplification technology, strand displacement amplification technology, rolling circle amplification technology, and nucleic acid sequence-based amplification technology and other technologies.

Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification ( LAMP ) is based on the fact that DNA is in a dynamic equilibrium state at around 65?°C. When any primer extends to the complementary site of double-stranded DNA through base pairing, the other strand will dissociate and become a single strand.

At this temperature, DNA uses four specific primers and relies on a strand displacement DNA polymerase to cause the synthesis of strand displacement DNA to continue in a self-cycle.

Six specific regions of the target gene: F3 , F2 , F1 , B1 , B2 , B3 , and then design four primers based on these six specific regions (as shown below):

Forward inner primer (FIP) consists of F1c and F2 .

Backward inner primer (BIP) is composed of B1c and B2 , with TTTT as the spacer in the middle.

The outer primers F3 and B3 are composed of the F3 and B3 regions on the target gene respectively .

In the LAMP reaction system, the concentration of the inner primer is several times that of the outer primer. The inner primer first binds to the template strand to synthesize a complementary strand to form a double-stranded DNA. The outer primer then binds to the template strand to form a double-stranded DNA. Under the action of BstDNA polymerase, the complementary strand synthesized by the inner primer is released, and the complementary strand undergoes a series of reactions to eventually form a single-stranded DNA with a dumbbell structure.

Using the dumbbell-structured DNA single strand itself as a template , a transitional stem-loop structure DNA with an open end is continuously formed. The transitional stem-loop structure DNA is guided by internal and external primers to continuously undergo chain displacement extension reactions, and finally a DNA mixture of different lengths with multiple stem-loop structures is formed.

Advantages and Disadvantages of Loop-Mediated Isothermal Amplification

Advantages of LAMP:

( 1 ) High amplification efficiency. It can effectively amplify 1-10 copies of the target gene within 1h. The amplification efficiency is 10-100 times that of PCR.

( 2 ) The reaction time is short, the specificity is strong, and no special equipment is required.

Disadvantages of LAMP:

(1) The requirements for primers are particularly high.

(2) The amplified product cannot be used for cloning and sequencing but can only be used for judgment.

(3) Due to its high sensitivity, it is easy to form aerosols, causing false positives and affecting the test results.

Strand Displacement Amplification

Strand displacement amplification (SDA) includes a restriction endonuclease, a DNA polymerase with strand displacement activity, two pairs of primers, dNTP, calcium and magnesium ions, and a buffer system.

The principle of strand displacement amplification is based on the presence of chemically modified restriction endonuclease recognition sequences at both ends of the target DNA. The endonuclease nicks the DNA strand at its recognition site, and the DNA polymerase extends the 3' end of the nick and replaces the next DNA strand.

Replaced single-stranded DNA can bind to the primer and be extended into a double-stranded DNA by DNA polymerase. This process is repeated continuously, allowing the target sequence to be amplified efficiently.

Advantages and Disadvantages of Strand Displacement Amplification

Advantages of SDA:

The amplification efficiency is high, the reaction time is short, the specificity is strong, and no special equipment is required.

Disadvantages of SDA:

The products are not uniform. Some single-stranded and double-stranded products are always produced in the SDA cycle, and tailing will inevitably occur when detected by electrophoresis.

Rolling Circle Amplification

Rolling circle amplification ( RCA ) was proposed by referring to the rolling circle replication of DNA in pathogenic organisms . It refers to the use of?single-stranded circular DNA as a template at a constant temperature, under the action of a special DNA polymerase (such as Phi29), to carry out rolling circle DNA synthesis to achieve the amplification of the target gene.

RCA can be divided into two forms: linear amplification and exponential amplification. The efficiency of linear RCA can reach 105 times, while the efficiency of exponential RCA can reach 109 times.

To make a simple distinction, as shown in the figure below, linear amplification a uses only one primer, while exponential amplification b uses two primers.

Linear RCA is also called single-primer RCA . A primer binds to the circular DNA and is extended by DNA polymerase. The product is a linear single strand with a large number of repetitive sequences that are thousands of times the length of a single loop.

Since the product of linear RCA is always connected to the initiator primer, the signal is easy to fix, which is a major advantage.

Exponential RCA , also known as hyper?branched RCA , is a process in which one primer amplifies an RCA product, and the second primer hybridizes with the RCA product and extends, displacing the downstream primer extension chain that has already bound to the RCA product. Extension and displacement are repeated to produce a tree-like RCA amplification product.

Advantages and Disadvantages of Rolling Circle Amplification

Advantages of RCA:

High sensitivity, good specificity and easy operation.

Disadvantages of RCA:

Background issues during signal detection: During the RCA reaction, uncircularized padlock probes and template DNA or RNA that has not bound to the probe may generate some background signals.

Nucleic Acid Sequence-Based Amplification Technique

Nucleic?acid sequence-based amplification (NASBA) is a new technology developed on the basis of PCR. It is a continuous, isothermal nucleic acid amplification technology guided by a pair of primers with a T7 promoter sequence. It can amplify the template RNA by about 109?times in about 2 hours , which is 1000 times higher than PCR method, and does not require special instruments.

As soon as this technology appeared, it was used for rapid diagnosis of diseases, and many companies' RNA detection kits currently use this method.

Although reverse transcription PCR technology can also be used to amplify RNA , NASBA has its own advantages: it can be performed under relatively constant temperature conditions and is more stable and accurate than PCR technology.

The reaction is carried out at 41?°C?and requires AMV (avian myeloblastosis virus) reverse transcriptase, RNase H, T7 RNA polymerase and a pair of primers.

The process mainly includes:

The forward primer contains a T7 promoter complementary sequence. During the reaction, the forward primer binds to the RNA chain and is catalyzed by the AMV enzyme to form a DNA-RNA double chain.

RNase H digests the RNA in the double-stranded hybrid and retains the single-stranded DNA.

The reverse primer and AMV enzyme, a double-stranded DNA containing the T7 promoter sequence is formed.

The transcription process is completed under the action of T7 RNA polymerase to produce a large amount of target RNA.

Advantages of NASBA:

( 1 ) Its primers carry T7 promoter sequences, while foreign double-stranded DNA does not have T7 promoter sequences and cannot be amplified. Therefore, this technology has high specificity and sensitivity.

( 2 ) NASBA directly integrates the reverse transcription process into the amplification reaction, shortening the reaction time.

Disadvantages of NASBA:

( 1 ) The reaction components are relatively complex.

( 2 ) The need for three enzymes makes the reaction cost high.

References:

1. Jiang Su, Li Yirong, Principle and application of isothermal amplification technology

2. Peng Tao, Nucleic acid isothermal amplification technology and its application

3. Zhao Luyao et al., Research on rapid detection technology based on loop-mediated isothermal nucleic acid amplification

4.England biolabs, Loop Mediated Isothermal Amplification.

5.G.TerranceWalker etc. Strand displacement amplification is othermal, in vitro DNA amplification technique.

6. Wu Xiaoliang et al. Research progress on DNA rolling circle amplification technology.

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03?Sequencing Technology

Gene sequencing technology began in 1977 with the invention of the DNA dideoxy terminal termination sequencing method by Sanger.

Sanger sequencing uses four dideoxyribonucleic acids (ddNTPs) to be added to the chain being synthesized. Since dideoxyribonucleic acid lacks an oxygen atom, once it is added to the DNA chain, the reaction is terminated.

By constructing four reaction systems, adding four dideoxyribonucleic acids of AGCT , and adjusting the relative concentrations of deoxyribonucleic acid (dNTP) and ddNTP, the reaction amplification can obtain termination products of several hundred to thousands of bases.

The products are then separated by gel electrophoresis into four lanes, one for each base, and the band development results are read.

This method is known as the gold standard for genetic testing due to its high accuracy, but it is time-consuming and costly.

Entering the 21st century, with the development of physical and chemical technology, fluorescent groups with the same excitation wavelength but different emission wavelengths began to be used to label ddNTP. ATGC corresponds to different fluorescent groups to produce different colors of light which are read by computers, greatly improving the speed and efficiency of sequencing.

Next-Generation Sequencing

The second-generation sequencing technology is also called high-throughput sequencing (HTS) technology. Compared with the first-generation sequencing, it can achieve large-scale parallel sequencing. The basic principle is to divide the genome into short fragments, sequence the short fragments and then splice them.

Compared with the first-generation sequencing technology, It has advantages such as high throughput and low cost, the cost of detecting the same amount of data is currently about 0.01% of the first-generation sequencing technology, which has greatly promoted the application of sequencing technology in clinical testing.

After years of development, the second-generation sequencing has entered a mature stage. Currently, the second-generation sequencing platforms on the market can be divided into four categories based on sequencing technology: sequencing by synthesis, semiconductor sequencing, combined probe-anchored polymerase sequencing, and pyrophosphate sequencing.

Third-Generation Sequencing Technology

1. Single Molecule Real-Time?Technology

It is beneficial to single-molecule real-time sequencing technology, also known as SMRT ( Single Molecule Real-Time ) sequencing, which is a single-molecule reading technology based on nanopores and can quickly complete sequence reading without amplification.

2. Nanopore Sequencing Technology

Nano single-molecule sequencing technology is different from previous sequencing technologies in that it is based on electrical signals rather than optical signals.

The core of nanopore sequencing technology is a polymer membrane that integrates multiple transmembrane channel proteins (i.e., nanopore proteins). A voltage is applied on both sides of the membrane to generate a stable current that passes through the nanopore. When other objects pass through the nanopore, the magnitude of the current is affected, resulting in changes in recognizable electrical signals.

During sequencing, the double-stranded DNA is unwound into single-stranded DNA under the traction of the motor protein and passes through the nanopore protein (also called the reader protein). Due to the differences in the structure and size of the four ATCG bases, the current will produce characteristic ion current changes. By identifying the changes in this electrical signal, the purpose of reading the base sequence is achieved.

The emergence of second-generation sequencing has greatly solved the throughput problem, greatly improving sequencing speed and accuracy while greatly reducing sequencing costs, but the reading length is relatively short. The third-generation sequencing, which is mainly characterized by single-molecule sequencing, is developing in the direction of single molecule, long read length, low cost, and miniaturization, realizing another revolution in the sequencing field.

References:

1. Li Jinming, High-throughput Sequencing Technology