【 Collection 】 Comprehensive Analysis of Molecular Diagnostic Techniques: PCR Technology, Isothermal Amplification Technology of Nucleic Acids, Sequencing Technology

1. TaqMan Fluorescent probe

During PCR amplification, a pair of primers and a specific fluorescent probe are added simultaneously. This probe is an oligonucleotide, with a reporter fluorescent group and a quenching fluorescent group labeled at both ends, respectively.

When the probe is intact, the fluorescence signal emitted by the reporter group is absorbed by the quencher group. During PCR amplification, the 5'-3' exonuctase activity of Taq enzyme cleaves and degrades the probe, separating the reporter fluorescent group from the quenched fluorescent group. Thus, the fluorescence monitoring system can receive fluorescence signals. That is, for each DNA strand amplified, one fluorescent molecule is formed, achieving complete synchronization between the accumulation of fluorescence signals and the formation of PCR products.

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 DNA double strand, it emits a fluorescence signal. However, the SYBR dye molecules not incorporated into the strand do not emit any fluorescence signal, thus ensuring that the increase in the fluorescence signal is completely synchronous 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 dissolution curve.

3. Molecular beacons

It is a stem-ring double-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, causing the fluorescent group and the quenching group to be closely adjacent and no fluorescence will be produced.

After the PCR product is generated, during the annealing process, the middle part of the molecular beacon pairs with a specific DNA sequence, and the fluorescent gene separates from the quenched gene to produce fluorescence.

The main disadvantages of the second-generation PCR

① The sensitivity is still lacking, and the detection of low-copy specimens is inaccurate.

② The results are easily disturbed due to the influence of background values.

When there are PCR inhibitors in the reaction system, the test results are prone to interference.

Four. Third-generation Digital PCR

DigitalPCR (DigitalPCR, dPCR, Dig-PCR) calculates the copy number of the target sequence through endpoint detection, enabling precise absolute quantitative detection without the need for internal references and standard curves.

Digital PCR uses endpoint detection and is not dependent on Ct values (cycle thresholds). Therefore, the impact of amplification efficiency on digital PCR reactions is reduced, and the tolerance to PCR reaction inhibitors is enhanced, featuring high accuracy and reproducibility.

Due to its high sensitivity and accuracy, it is not easily interfered with by PCR reaction inhibitors and can achieve true absolute quantification without standard substances, thus becoming a research and application hotspot.

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

Microfluidic digital PCR, Microfluidic digital PCR, mdPCR

Based on microfluidic technology, the DNA template is separated. Microfluidic technology can achieve the nanoscale upgrade of the sample or the generation of smaller droplets. However, the droplets need to be combined with the PCR reaction system through a special adsorption method. mdPCR has gradually been replaced by other methods.

2. Droplet-based digital PCR, ddPCR

The sample was subjected to microdroplet treatment by using the water-in-oil microdroplet generation technology, dividing the reaction system containing nucleic acid molecules into tens of thousands of nanoscale microdroplets, each of which either did not contain the nucleic acid target molecule to be detected or contained one to several nucleic acid target molecules to be detected.

3. Chip-baseddigital PCR, cdPCR

By using integrated fluid path technology, numerous microtubes and microcavities are etched on silicon wafers or quartz glass. Different control valves are used to control the flow of the solution within them. The sample liquid is divided into uniform-sized nanoscales and placed in reaction Wells for digital PCR reactions, achieving absolute quantification.

The main disadvantages of the third-generation PCR

① The instruments and reagents are expensive.

② The quality requirements for the template are relatively high. If the amount of template exceeds that of the microsystem, quantification cannot be achieved; if it is too small, the accuracy of quantification will decrease.

False positives can also occur when non-specific amplification is present.

V. Various extended Techniques of PCR

1. touchdown PCR: The temperature gradually drops in the first few cycles.

2. Reverse transcription PCR (RT-PCR) : It uses cDNA reverse transcribed from mRNA as the template. Also, since the increment is from the phenotypic gene, the cDNA product generated does not contain introns (meaningless segments in the gene), and is often applied in molecular cloning technology.

3. hotstart PCR: This reaction is carried out using highly heat-activated nucleic acid polymerase to reduce non-specific products.

4. nested PCR: First, amplify several cycles with low-specificity primers to increase the number of templates, and then amplify with high-specificity primers.

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

6. reconditioning PCR: Dilute the PCR product 10 times and then re-place it in primers and DNTPS of the original concentration for three cycles to eliminate heterodimers in the product.

7.dsRNA synthesis (dsRNA replicator) : The combined use of high-fidelity DNA Polymerase, T7RNA polymerase and Phi6 RNA replicase; Transcription from double-stranded DNA to the corresponding double-stranded RNA (dsRNA). It can be applied to RNAi experimental operations.

8.COLD-PCR (co-amplification at lower denaturation temperature PCR): A PCR application technique used to detect mutations or specific alleles.

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

PCR is the most widely used nucleic acid amplification technology, which is widely applied due to its sensitivity and specificity. However, PCR requires repeated thermal denaturation and cannot escape the limitation of relying on instruments and equipment, thus limiting its application in clinical field detection.

Since the early 1990s, many laboratories have begun to develop isothermal amplification techniques that do not require thermal denaturation. Currently, technologies such as loop-mediated isothermal amplification, chain substitution isothermal amplification, rolling loop isothermal amplification, and nucleic acid sequence-dependent isothermal amplification have been developed.

I. Loop-mediated isothermal amplification

loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technology first proposed by Notomi et al. of Eiken Corporation in Japan in 2000.

Based on this technology, Rongyan has also been involved in a patent dispute with a domestic company.

The amplification principle is based on the fact that DNA is in a dynamic equilibrium state around 65℃. When any primer extends base pairing towards the complementary site of the double-stranded DNA, 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 continuously self-circulate the synthesis of strand displacement DNA.

First, identify six specific regions F3, F2, F1, B1, B2, and B3 on the target gene, and then design four primers based on these six specific regions (as shown in the following figure) :

Forward Dinner primer (FIP), composed of F1c and F2. backwardinner primer (BIP), composed of B1c and B2, is separated by TTTT in the middle. The primers F3 and B3 are respectively composed of the F3 and B3 regions on the target gene.

In the LAMP reaction system, the concentration of the internal primers is several times that of the external primers. The internal primers first combine with the template strand to synthesize complementary strands, forming a DNA double strand. Subsequently, the external primers bind to this template strand to form a DNA double strand. Under the action of BstDNA polymerase, the complementary strand synthesized from the internal primers is released. This complementary strand undergoes a series of reactions and eventually forms a DNA single strand with a dumbbell structure.

Using the single strand of dumbbell-structured DNA itself as a template, transitional stem-ring structured DNA with one end open is continuously formed. Guided by internal and external primers, the transitional stem-ring structured DNA undergoes continuous strand displacement and extension reactions, and finally forms DNA mixtures of different lengths with multiple stem-ring structures.

The advantages of LAMP

(1) It has a high amplification efficiency, capable of effectively amplifying 1 to 10 copies of the target gene within 1 hour, with an amplification efficiency 10 to 100 times that of ordinary PCR.

(2) It has a short reaction time, strong specificity and does not require special equipment.

Ii. Strand displacement amplification

strand displacement amplification (SDA) is an in vitro isothermal amplification technology of DNA based on enzymatic reactions, which was first proposed by American scholar Walker in 1992. The basic system of SDA includes a restriction endonuclease, a DNA polymerase with strand displacement activity, two pairs of primers, DNTPS, as well as calcium and magnesium ions and a buffer system.

Strand displacement amplification is based on the principle that the target DNA has chemically modified restriction endonuclease recognition sequences at both ends. The endonuclease opens a gap in the DNA strand at its recognition site, and the DNA polymerase extends the 3 'end of the gap and replaces the next DNA strand.

The single-stranded DNA that has been replaced can bind to primers and be extended into double-stranded DNA polymerase. This process is repeated continuously, enabling the target sequence to be amplified efficiently.

The advantages and disadvantages of chain displacement amplification technology

The advantages of SDA:

It features high amplification efficiency, short reaction time, strong specificity and does not require special equipment.

The shortcomings of SDA

The products are not homogeneous. In the SDA cycle, some single and double-stranded products are always produced. When detected by electrophoresis, tailing is bound to occur.

Iii. Rolling Loop nucleic Acid amplification

rolling circle amplification (RCA) was proposed by drawing on the way pathogenic organisms replicate DNA in a rolling loop. It refers to the synthesis of rolling loop DNA at a constant temperature using single-stranded circular DNA as a template under the action of special DNA polymerase (such as Phi29). 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 10^5 times, while that of exponential RCA can reach 10^9 times. a simple distinction is made as shown in the following figure. Linear amplification A only uses one primer, while exponential amplification b requires two primers.

Linear RCA, also known as single primer RCA, is a single primer that binds to circular DNA and is extended under the action of DNA polymerase. The product is a linear single strand with a large number of repetitive sequences thousands of times the length of a single ring.

Because the products of linear RCA are always attached to the starting primers, the ease of signal fixation is one of its major advantages.

Exponential RCA, also known as hyper-branched RCA, in exponential RCA, one primer amplifies the RCA product, the second primer hybridizes and extends with the RCA product, and displaces the downstream primer extension chain already bound to the RCA product. This process of extension and displacement is repeated. Generate tree-like RCA amplification products.

The advantages and disadvantages of rolling loop nucleic acid amplification

The advantages of RCA

High sensitivity, good specificity and easy to operate.

The shortcomings of RCA

Background issues during signal detection. During the RCA reaction process, the unlooped locked probes and the template DNA or RNA of the unbound probes may generate some background signals.

Four. Amplification techniques relying on nucleic acid sequences

nucleicacid sequence-based amplification (NASBA) is a new technology developed on the basis of PCR. It is a continuous and isothermal nucleicacid amplification technique guided by a pair of primers carrying the T7 promoter sequence. The template RNA can be amplified by approximately 10^9 times in about 2 hours, which is 1,000 times higher than the conventional PCR method, and no special instruments are required.

This technology was applied to the rapid diagnosis of diseases as soon as it emerged. Currently, many companies' RNA detection kits use this method. Although reverse transcription PCR technology can also be used for RNA amplification, NASBA has its own advantages: it can be carried out under relatively constant temperature conditions and is more stable and accurate than traditional PCR technology. The reaction takes place at 41 degrees Celsius and requires AMV (avian myeloblastosis virus) reverse transcriptase, RNase H, T7 RNA polymerase and a pair of primers to complete.

The process mainly includes:

The forward primers contain complementary sequences of the T7 promoter. During the reaction, the forward primers bind to the RNA chain and are catalyzed by the AMV enzyme to form DNA-RNA double strands.

② RNase H digests the RNA in the hybrid double-stranded DNA while retaining the single-stranded DNA.

③ Under the action of reverse primers and AMV enzyme, a double-stranded DNA containing the T7 promoter sequence is formed.

④ Under the action of T7 RNA polymerase, the transcription process is completed, generating a large amount of target RNA.

The 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 technique has high specificity and sensitivity.

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

Disadvantages of NASBA

(1) The reaction components are rather complex.

(2) Three enzymes are required, which makes the reaction cost relatively high.

References

Jiang Su, Li Yirong, Principles and Applications of Isothermal Amplification Technology

2. Peng Tao, Isothermal Amplification Technology of Nucleic Acids and Its Applications

3. Zhao Luyao et al., Research on Rapid Detection Technology Based on Ring-mediated Isothermal Amplification of Nucleic Acids

4.England biolabs, Loop Mediated Isothermal Amplification.

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

6. Wu Xiaoliang et al., Research Progress on Rolling Loop Amplification Technology of DNA.

Gene sequencing technology originated in 1977, when sanger's invention of DNA dideoxy end-termination sequencing marked the beginning. Sanger won the Nobel Prize in Chemistry twice, in 1958 and 1980, for insulin sequencing and DNA sequencing. He is the fourth person to win the Nobel Prize twice and the only one to have won it twice.

Sanger

Sanger sequencing involves adding four dideoxyribonucleic acids (DDNTPS) to the chain being synthesized. Since the dideoxyribonucleic acid lacks one oxygen atom, the reaction is terminated once it is added to the DNA chain.

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

Subsequently, the products were separated by gel electrophoresis, divided into four lanes, each corresponding to a base, and then the band development results were read.

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

The Human Genome Project in 2001 was completed using sanger sequencing. Since the establishment of the Human Genome Project in 1990, scientists around the world spent 11 years and 3 billion US dollars to complete it.

After entering the 21st century, with the development of physical and chemical technologies, fluorescent groups with the same excitation wavelength but different emission wavelengths began to be used to label ddNTP. ATGC generates light of different colors corresponding to different fluorescent groups and is read by computers, greatly improving the speed and efficiency of sequencing.

First and second-generation sequencing

The second-generation sequencing technology, also known as high-throughput sequencing (HTS) technology, can achieve large-scale parallel sequencing compared with the first-generation sequencing. Its basic principle is to divide the genome into short fragments, sequence the short fragments and then stitch them together. Compared with the first-generation sequencing technology, it has advantages such as high throughput and low cost. Currently, for the detection of the same amount of data, its cost is approximately 0.01% of that of the first-generation sequencing technology, which has greatly promoted the application of sequencing technology in clinical detection.

In 2005, 454 Company launched the Genome Sequencer 20 System(GS 20) based on pyrosequencing, initiating the process of high-throughput sequencing. In 2007, Roche acquired 454 and launched a series of NGS systems with superior performance, significantly enhancing sequencing throughput and accuracy. Despite its read length advantage, the 454 platform's promotion was always limited by sequencing throughput and cost. With the same data volume, its cost was approximately 100 times that of illumina. Therefore, Roche terminated its 454NGS sequencing-related business at the end of 2016.

In 2006, Solexa Company launched the Genome Analyzer system, which includes technologies such as DNA clusters, bridge PCR and reversible blocking. This makes the GA system have obvious advantages in terms of high throughput, low cost and wide application range. In 2007, Illumina acquired Solexa and released its second-generation sequencer. After years of development, second-generation sequencing has entered a mature stage. Currently, in the market, second-generation sequencing platforms can be classified into four types based on sequencing technology: synth-in-sequencing (Illumina), semiconductor sequencing (ThermoFisher), combined probe anchored aggregation sequencing (BGI), and pyrophosphate sequencing.

Illumina performs synthesis and sequencing simultaneously

The process of Illumina sequencing mainly includes sample preparation, cluster generation, sequencing and data analysis.

The first step is sample preparation and library construction

Nucleic acids are extracted using DNA or RNA extraction kits, and then they are immediately broken to a length of about 90-250bp by ultrasound or the entire DNA is controlled within a certain length range.

For subsequent amplification and sequencing, specific sequences need to be added to these DNA fragments. As shown in the following figure, they are respectively the regions complementary to the flow cell primers (P5, P7), the regions complementary to the Read 1 and read 2 sequencing primers (Rd1SP, Rd2 SP), and the tag sequence region Index.

The DNA collection with added adapter sequences is called a DNA library, thus completing the library construction. This step can be accomplished using commercial library preparation kits.

The second step is Cluster Generation

Clustering is the process by which the above-mentioned DNA fragments are amplified, and this process is completed in the Flow cell. The flow cell is a thick glass plate with 8 channels, and short DNA fragments that can complement and bind to the library linter P5 or P7 are randomly implanted in each channel. First, the primers and the fixed DNA fragments of the flow cell are complementary and paired, fixed on the channel surface. Then, under the action of DNA polymerase, the DNA strands are complementary and extended to form DNA double strands. Through denaturation, the single strand is eluted, and the remaining single strand will link with the fixed joint beside it, forming a single-strand bridge.

Similarly, single-stranded Bridges extend and pair under the action of DNA polymerase to form double-stranded Bridges. Through denaturation, two single strands are formed, and these two single strands respectively bind to the adjacent fixed primers to form two single-stranded Bridges. Repeat this cycle and eventually form millions of DNA clusters. All DNA fragments in the above process will be amplified. After amplification, the reverse links will be cut and eluted, leaving only the forward strands. To prevent complementary binding and the re-formation of single-stranded Bridges, the 3 'end is blocked.

The third step is sequencing

First, fluorescently labeled DNTPS and enzymes are added to the flow cell, and the synthesis of subchains begins from the primers. Because the 3 'end azide group of dNTP hinders the extension of the subchain, only one base can be measured in each cycle. After the synthesis of one base is completed, the excess DNTPS and enzymes are washed away, and the fluorescence signal is obtained by laser scanning.

Subsequently, reagents are added to remove the azide group and the fluorescent group. Then, fluorescently labeled DNTPS and enzymes are introduced into the flow cell, and a base is synthesized starting from the primers. Keep repeating this process to complete the first read.

All the bases of the DNA fragments will be read simultaneously. Meanwhile, different indices are added to distinguish each sample and the positive and negative strands. After the first read is completed, the copied chain will be washed away, the index fragment primer will be introduced and hybridized with the template, and then washed away after the sequence read is completed. After comparing the sequence read in this way with the index known at the beginning, the measured sequence can be labeled, which is convenient for subsequent analysis.

Paired end sequencing has become the mainstream nowadays. To complete double-end sequencing, the template strand 3 'needs to be protected first, the template folded, and the index fragment introduced. Under the participation of polymerase, a double-strand bridge is formed, then denaturated and restored to a single strand. The forward strand is then removed and washed away, leaving the reverse strand. Similar to the forward strand, after multiple cycles, the reading is completed.

The fourth step is data analysis

After sequencing is completed, millions of reads will be generated, classifying sequences from different samples based on the indices constructed during sample preparation. For each sample, bases with similar extensions are clustered together. Forward and reverse read pairs generate a continuous sequence. These sequences are matched with the reference genome to achieve the construction of complete sequences.

Ii. Third-generation sequencing technology

PacBio SMRT single-molecule sequencing technology

Pacific Biosciences was founded in 2004, went public on the Nasdaq in 2010, and released PacBioRS in 2011.

The third-generation gene sequencing technology represented by PacBio sequencing has gradually been applied to multiple scientific research fields. This platform is conducive to single-molecule Real-Time sequencing technology, also known as SMRT (Single Molecule Real-time) sequencing. It is a single-molecule reading technology based on nanopores and can quickly complete sequence reading without amplification.

2. Nanopore sequencing technology

In 2005, Oxford Nanopore Technologies was established, dedicated to the commercial transformation of nanopore sequencing technology. In 2014, the Nano space single-molecule sequencing technology was released.

The single-molecule sequencing technology developed by Oxford Nanopore is different from previous sequencing technologies. 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 stable current passing through the nanopores is generated by applying voltage on both sides of the membrane. When other objects pass through the nanopores, it will affect the magnitude of the current, thereby generating recognizable changes in the electrical signal.

During sequencing, the double-stranded DNA is uncoiled into single-stranded DNA under the traction of motor proteins and passes through nanopore proteins (also known as Reader proteins). Due to the differences in the structure and size of the four bases of ATCG, characteristic ionic current changes will occur in the current. By identifying these changes in electrical signals, the purpose of reading the base sequence can be achieved.

The emergence of next-generation sequencing has greatly solved the throughput problem. While significantly increasing the sequencing speed and accuracy, it has greatly reduced the sequencing cost, but the reading length is relatively short. The third-generation sequencing, characterized mainly by single-molecule sequencing, is evolving towards single-molecule, long-read, low-cost and miniaturized directions, bringing about another revolution in the sequencing field.