Introduction to the Main Gene Sequencing Methods for Companion Diagnostics

Companion diagnostics require the determination of drug receptor action sites through analysis methods such as gene sequencing of samples. Currently, commonly used sample types are tissues and body fluids.

The technology for molecular analysis of tissues is called tissue biopsy. Specifically, it refers to the removal of diseased tissues from the patient's body by cutting, clamping or puncturing in response to the needs of diagnosis and treatment, and performing molecular analysis on them.

For tumor companion diagnosis, tumor tissue biopsy is the gold standard for obtaining tumor DNA . The technology of analyzing liquid is called liquid biopsy, which specifically refers to the molecular analysis of analytes (CTC, cfDNA, ctDNA, etc.) in the patient's body fluids to guide the patient's personalized treatment and prognosis.

Comparison of the Advantages and Disadvantages of Tissue Biopsy and Liquid Biopsy

From the perspective of clinical application, tissue biopsy and liquid biopsy each have their own advantages and disadvantages. Although liquid biopsy-based tumor companion diagnostics have been booming in recent years, it does not mean that they can replace tissue biopsy technology. The cooperation and complementarity between the two may greatly improve diagnostic efficiency and reduce overtreatment. With the continuous development of liquid biopsy technology, liquid biopsy-based tumor companion diagnostics may further replace tissue biopsy technology in the future and become the main force of companion diagnostic testing.

Main Gene Sequencing Methods for Companion Diagnostics

Companion diagnostics is the determination of drug receptor action sites through gene sequencing and other means. With the application and promotion of targeted drugs, companion diagnostics has gradually established an important position in guiding treatment. Among the detection technologies used in companion diagnostics, PCR, NGS, and FISH are the most widely used technologies in the market. They each have their own advantages and disadvantages and are distributed in major companion diagnostic companies.

1. PCR

PCR (polymerase chain reaction) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as a special DNA replication outside the body. Its biggest feature is that it can greatly increase trace amounts of DNA. In the presence of a DNA template, primers, dNTPs, and a suitable buffer (Mg2+) in the reaction mixture, a pair of oligonucleotide primers are amplified under the catalysis of a thermostable DNA polymerase. This amplification is based on the denaturation, annealing, and extension three-step reaction between the template DNA and the primers. The cycle is repeated to amplify the target DNA fragment.

Schematic Diagram of PCR

The first PCR technology could only amplify the target gene and then analyze the product by agarose gel electrophoresis to achieve simple qualitative analysis. This is the first generation of PCR. In view of the fact that (1) the nucleic acid fuel used in the first generation of PCR caused great harm to the experimenters and the environment, (2) the detection after the PCR was completed was prone to contamination and false positive results, (3) the detection was time-consuming and the operation was cumbersome and prone to errors, and (4) it could only perform qualitative detection, the second generation of PCR was developed and became the mainstream of domestic PCR technology. The second generation of PCR is real-time PCR, also known as qPCR, which refers to the use of fluorescent dyes or fluorescently labeled specific probes (such as Taqman Probe) to add fluorescent groups to the PCR reaction system to label and track the polymerase chain reaction products, monitor the reaction process in real time, and analyze the fluorescent signals with software to achieve qualitative and quantitative analysis of gene detection. qPCR is commonly used in infectious disease pathogen detection, disease resistance gene research, drug efficacy assessment, and genetic disease diagnosis. As the number of reaction cycles increases, the amplified target gene fragment grows exponentially. The Ct value is obtained by real-time detection of the corresponding fluorescence signal intensity that changes with amplification. By using a standard sample with a known template concentration as a control, the number of target genes in the sample to be tested can be obtained. Since the rPCR quantitative process is indirectly reflected by the Ct value, it is called the second generation PCR.

qPCR Principle Diagram

Application of Fluorescent Dyes: SYBR green: In the PCR reaction system, add excess SYBR fluorescent dye. After the SYBR fluorescent dye is specifically incorporated into the DNA double strand, it emits a fluorescent signal, while the SYBR dye molecules 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 PCR products.

Application of Specific Labeled Probes: Taqman Probe: When adding a pair of primers during PCR amplification, a specific fluorescent probe is added. 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 will enzymatically degrade 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.

Fluorescent Dyes vs Fluorescent Probes

With the further development of PCR system, a digital PCR technology with high sensitivity and specificity has begun to be widely used in clinical tumor detection and scientific research. Digital PCR (dPCR) is an emerging nucleic acid detection technology . By allocating each nucleic acid molecule to an independent space, it avoids the interference of selective amplification on the amplification results, and can achieve absolute quantitation of nucleic acid templates, detection of rare mutations, copy number variation, DNA methylation, gene rearrangement and other detection functions. Because digital PCR can achieve direct quantitative analysis, it is called the third generation of PCR.

By diluting the sample DNA into the corresponding detection wells, after PCR amplification, adding specific fluorescent probes to each well to hybridize with the product, and then directly counting the number of mutant and wild-type alleles in the sample. dPCR is often used to detect a small number of mutant cells in a large number of normal cell populations, and is mainly used for mutation analysis, allele loss, cancer detection of promiscuous DNA, etc.

2. NGS

NGS (high-throughput sequencing technology) refers to the chemical modification of template DNA molecules, anchoring them on nanopores or microcarrier chips, and using the principle of base complementary pairing to collect fluorescent labeling signals or chemical reaction signals during the DNA polymerase chain reaction or DNA ligase reaction to interpret the base sequence. It can complete the determination of hundreds of thousands to millions of sequences at a time.

NGS is a DNA sequencing technology developed based on PCR and gene chips. The first generation of sequencing is synthesis termination sequencing, while the second generation of sequencing pioneered the introduction of reversible termination ends, thus achieving sequencing while synthesis. The second generation of sequencing determines the DNA sequence by capturing the special markers (usually fluorescent molecular markers) carried by the newly added bases during DNA replication.

In the second-generation sequencing, a single DNA molecule must be amplified into a gene cluster composed of the same DNA and then replicated synchronously to enhance the intensity of the fluorescent signal and thus read the DNA sequence; as the read length increases, the synergy of gene cluster replication decreases, resulting in a decrease in the quality of base sequencing . The second-generation sequencing is suitable for amplicon sequencing (such as the variable regions of 16S, 18S, and ITS), while the genomic and metagenomic DNA needs to be broken into small fragments using the shotgun method and then spliced using bioinformatics methods after sequencing.

3. FISH

FISH (fluorescence in situ hybridization) is a widely clinically recognized method for detecting changes in gene copy number. If the target DNA on the chromosome or DNA fiber section being tested is homologous and complementary to the nucleic acid probe used, the two can form a hybrid of the target DNA and the nucleic acid probe through denaturation-annealing-renaturation. A certain nucleotide of the nucleic acid probe is labeled with a reporter molecule such as biotin or digoxigenin. The immunochemical reaction between the reporter molecule and the specific avidin labeled with fluorescein can be used to perform qualitative, quantitative or relative positioning analysis on the DNA to be tested through fluorescence microscopy. FISH technology can be used to detect gene amplification, deletion and rearrangement, such as for the chromosome localization of known genes or sequences, the study of uncloned genes or genetic markers and chromosome aberrations.

The advantages of FISH technology are:

① Fluorescent reagents and probes are economical and safe;

②The probe is stable and can be used within two years after one labeling;

③Short experimental cycle, rapid results, good specificity and accurate positioning;

④FISH can locate DNA sequences as long as 1kb, and its sensitivity is comparable to that of radioactive probes;

⑤Multicolor FISH can detect multiple sequences simultaneously by displaying different colors in the same nucleus;

⑥ It can display the changes in the number or structure of metaphase chromosomes on the slide, and it can also display the structure of interphase chromosome DNA in the suspension.

However, FISH cannot achieve 100% hybridization, especially when using shorter cDNA probes, the efficiency is significantly reduced.

Although NGS, as an emerging sequencing technology, has developed rapidly in recent years, the companion diagnostic sequencing services and products on the market are still dominated by PCR technology, and compared with PCR and NGS, FISH technology is generally less used.