Main Gene Sequencing Methods For Companion Diagnostic

Companion diagnostic (CD) requires the identification of drug receptor sites of action by means of genetic sequencing and other analysis of samples, which are currently commonly used for tissues and body fluids.

The technique of tissues molecular analysis is called tissue biopsy, which specifically refers to the removal of diseased tissues from the patient's body by excision, forceps or puncture for molecular analysis in response to the need for diagnosis and treatment.

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

Comparison of The Advantages and Disadvantages of Tissue Biopsy and Liquid Biopsy Techniques

From the perspective of clinical application, tissue biopsy and liquid biopsy have their own advantages and disadvantages. Although liquid biopsy-based tumor companion diagnostic has been developing in recent years, it does not mean that it can replace tissue biopsy technology, but the cooperation between them may greatly improve the diagnostic efficiency and reduce over-treatment. With the continuous development of liquid biopsy technology, liquid biopsy-based tumor companion diagnostic?may further replace tissue biopsy technology and become the main force of companion diagnostic testing in the future.

Main?Gene Sequencing Methods for Companion Diagnostic

Companion diagnostic is the identification of drug receptor sites by means of gene sequencing, etc. With the promotion of targeted drugs, companion diagnostic has gradually established an important position in guiding treatment. Among the testing technologies used in companion diagnostic, PCR, NGS and FISH are the most widely used technologies in the market at present, each of which has its own advantages and disadvantages and is distributed in major companion diagnostic enterprises.

1.PCR

PCR (polymerase chain reaction) is a molecular biology technique for the amplification of specific DNA fragments and can be thought of as a special DNA replication outside the organism, whose most important feature is the ability to increase micro DNA dramatically. In the presence of a reaction mixture of DNA template, primers, dNTPs, and an appropriate buffer (Mg2+), a nucleic acid fragment defined by a pair of oligonucleotide primers is amplified in a cycle of a three-step reaction of denaturation, annealing, and extension between the template DNA and the primers, allowing the target DNA fragment to be amplified, catalyzed by a heat-stable DNA polymerase.

Initially, PCR technology could only amplify target genes and then analyze the products by agarose gel electrophoresis to achieve simple qualitative analysis, which was the first generation PCR. Given that (1) the nucleic acid fuel used in the first generation PCR caused significant harm to laboratory personnel and the environment, (2) after the completion of PCR, the cap opening test was prone to contamination and false positive results, (3) the test took a long time and was difficult to operate, and was prone to errors, and (4) only qualitative testing could be performed, the second generation PCR was able to develop and become the mainstream PCR technology in China at present. The second generation PCR is real time PCR, also known as qPCR.?It refers to the use of fluorescent dyes or specific probes labeled with fluorescence (such as Taqman Probe) to add fluorescent groups to the PCR reaction system to label and track the products of polymerase chain reaction. The reaction process is monitored in real time, and the fluorescence signal is analyzed using software to achieve qualitative and quantitative analysis of gene detection. QPCR is commonly used for detecting infectious disease pathogens, studying disease resistance genes, assessing drug efficacy, and diagnosing genetic diseases. With the increase of reaction cycle times, the amplified target gene segment shows exponential growth. Through real-time detection of the corresponding fluorescence signal intensity that changes with amplification, the Ct value can be obtained. Using the standard sample with known template concentration as the control, the target gene number of the standard to be tested can be obtained. Due to the indirect reflection of Ct values in the quantitative process of rPCR, it is called second-generation PCR.

Application of fluorescent dye: SYBR green: In the PCR reaction system, an excess of SYBR fluorescent dye is added. After the SYBR fluorescent dye is specifically incorporated into the DNA double strand, it emits a fluorescence signal, while SYBR dye molecules that are not incorporated into the strand do not emit any fluorescence signal, ensuring that the increase in fluorescence signal is completely synchronized with the increase in PCR products.?

Application of specific labeled probes: Taqman Probe: PCR amplification is performed by adding a pair of primers together with a specific fluorescent probe, which is an oligonucleotide labeled with a reporter fluorophore and a quenched fluorophore at each end. When the probe is intact, the fluorescence signal emitted by the reporter group is absorbed by the quenched group; during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme degrades the probe enzymatically, separating the reporter and quenched fluorescence groups so that the fluorescence signal can be received by the fluorescence monitoring system, i.e., for each DNA strand amplified, a fluorescent This allows the fluorescence monitoring system to pick up the fluorescence signal, i.e., for every DNA strand amplified, a fluorescent molecule is formed, achieving complete synchronization between the accumulation of fluorescence signal and PCR product formation.

With the further development of PCR already for detection, a digital PCR technology with high sensitivity and specificity has started to be widely used in tumor clinical detection and scientific research. Digital PCR (dPCR) is an emerging nucleic acid detection technology?that can achieve absolute quantification of nucleic acid templates, rare mutation detection, copy number variation, DNA methylation, gene rearrangement and other detection functions by assigning each nucleic acid molecule to an independent space and avoiding the interference of selective amplification on amplification results. Since digital PCR can achieve direct quantitative analysis, it is called the third generation PCR.

The sample DNA is diluted into the corresponding wells, amplified by PCR, and specific fluorescent probes are added to each well to hybridize with the product, and then the number of mutant and wild-type alleles in the sample is counted directly. dPCR is commonly used to detect a small number of mutant cells in a large population of normal cells, mainly for mutation analysis, allelic deletion, cancer detection of mixed DNA, etc.

2.NGS

NGS (high-throughput sequencing technology) refers to the chemical modification of template DNA molecules, anchoring them in nanopores or microcarrier chips, and using the principle of base complementary pairing to achieve base sequence interpretation by collecting fluorescent labeling signals or chemical reaction signals during the DNA polymerase chain reaction or DNA ligase reaction, which can complete the determination of hundreds of thousands to millions of sequences at one time.??

NGS is a DNA sequencing technology developed based on PCR and gene chips. While first-generation sequencing is synthetic termination sequencing, second-generation sequencing pioneered the introduction of reversible termination ends to enable sequencing while synthesizing. Second-generation sequencing determines the sequence of DNA by capturing the special markers (usually fluorescent molecular markers) carried by newly added bases during DNA replication.??

3.FISH

FISH (fluorescence in situ hybridization) is a widely accepted clinical method for detecting gene copy number changes. If the target DNA on the chromosome or DNA fibre section being tested is homologous and complementary to the nucleic acid probe used, the two are denatured-annealed-renatured to form a hybrid between the target DNA and the nucleic acid probe. Labeling a specific nucleotide of a nucleic acid probe with a reporter molecule such as biotin or digoxin can utilize the immunochemical reaction between the reporter molecule and the specific affinity molecule labeled with fluorescein to perform qualitative, quantitative, or relative localization analysis of the tested DNA through fluorescence microscopy. FISH can be used to detect gene amplifications, deletions and rearrangements, such as chromosomal localization of known genes or sequences, uncloned genes or genetic markers and chromosomal aberrations.

The advantages of FISH technology include:

①Fluorescent 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 availability of results, good specificity and accurate localization;

④FISH can localize DNA sequences up to 1kb in length with a sensitivity comparable to that of radioactive probes;

⑤Multicolor FISH allows simultaneous detection of multiple sequences by displaying different colors in the same nucleus;

⑥Both changes in the number or structure of intermediate chromosomes can be shown on slides, and the structure of interphase chromosomal DNA can be shown in suspension.?

However, FISH does not achieve 100% hybridization, especially when shorter cDNA probes are applied, and the efficiency is significantly reduced.

Although NGS as an emerging sequencing technology has been developing rapidly in recent years, PCR technology is still the main companion diagnostic sequencing service and product in the market, and FISH technology is generally less applied?compared to PCR and NGS.