Release date: 2015-10-28
I. Molecular diagnostic techniques based on molecular hybridization
From the 1960s to the 1980s, molecular hybridization technology developed the most rapid 20 years. Because fashion can't artificially amplify target genes in samples, people can only capture and detect target sequences through probes with known gene sequences. Among them, the basic theory of liquid phase and solid phase hybridization, the probe immobilization technique and the artificial synthesis of cDNA probes, the initial technical reserve for in vitro diagnostic methods based on molecular hybridization.
(1) Southern blotting (Southernblot)
Southern invented the Southern blotting technique in 1975. The DNA was fragmented by restriction endonucleases, and the DNA fragments of different lengths were separated by gel electrophoresis and transferred to the cellulose acetate membrane by siphon or voltage. The DNA denaturation on the membrane is molecularly hybridized with the nuclide-labeled oligonucleotide probe, and after elution, the homologous sequence between the DNA fragment-probe to be detected is identified by autoradiography. This method ensures the specificity of detection by simultaneous DNA fragment digestion and molecular probe hybridization. Therefore, once introduced, it has become the most classical molecular detection method in the field of probe hybridization, and is widely used in various gene mutations, such as deletion, insertion, translocation, etc., and restriction fragment length (restriction fragment length) Identification of polymorphism, RFLP). Alwine is equivalent to the introduction of Northern blotting technology based on transfer hybridization in 1977, which also became the gold standard for RNA detection at that time.
(2) ARO-specific oligonucleotide reverse dot blot (ASO-RDB)
Hybridization detection of nucleic acid sequences using nucleic acid blotting techniques has extremely high specificity, but has the disadvantage of being extremely cumbersome to operate and having a long detection time. The sample spot spotting technique established in 1980 has got rid of the shortcomings of traditional DNA blotting that require sample immobilization by gel separation techniques. The first allele-specific oligonucleotide (ASO) was constructed by introducing a single base mutation into the plasmid vector, which made it possible to detect point mutations in the nucleic acid sequence. In 1986, Saiki [3] first combined the high sensitivity of PCR with the high specificity of ASO dot hybridization, enabling the use of ASO probes to classify specific gene polymorphisms. In order to perform high-throughput detection of multiple molecular markers of the same sample, Saiki [4] invented ASO-RDB by hybridizing biotin-labeled specific PCR amplification products to probes immobilized on the membrane. Color development, detection of genotyping and gene mutation. The method can fix a plurality of oligonucleotide probes on the same membrane strip, and can screen dozens or even hundreds of alleles of the sample DNA to be tested by one hybridization reaction, and has the advantages of simple operation, The rapid characteristics have once become the most commonly used technique for gene mutation detection, genotyping and pathogen screening.
(III) Fluorescence in situ hybridization (FISH)
FISH is derived from a nuclear-labeled in situ hybridization technique. In 1977, Rudkin first attempted in situ hybridization using a fluorescein-labeled probe. In the 1980s, the development of cytogenetics and non-isotopic labeling techniques pushed FISH into practical applications for clinical diagnosis. Compared to other molecular diagnostic techniques that only detect nucleic acid sequences, FISH combines the advantages of high specificity and histological localization of probes to detect specific normal or aberrant DNA sequences in intact cells or isolated chromosomes; Due to the use of high-energy fluorescein-labeled DNA probes, multiple fluorescein labels can be detected to simultaneously detect several targets.
Nowadays, FISH has been widely used in many aspects such as karyotype analysis, gene amplification, gene rearrangement, and pathogenic microorganism identification. By comparing FISH-derived techniques such as comparativegenomic hybridization (CGH) and spectral karyotyping (SKY), it is playing a role in more and more clinical diagnostic fields.
(4) Multiplexed probe amplification (MLPA)
MLPA technology was first reported in 2002 by Schouten et al. [6]. Each MLPA probe comprises two fluorescently labeled oligonucleotide fragments, one consisting of chemical synthesis and one by M13 phage derivatization; each probe comprises a 1-segment primer sequence and a 1-segment specific sequence. In the MLPA reaction, both oligonucleotide fragments are hybridized to the target sequence, and the two-part probe is ligated using a ligase. The ligation reaction is highly specific. Only when the two probes are completely hybridized to the target sequence, the ligase can join the two probes into one complete nucleic acid single strand; conversely, if the target sequence is not completely complementary to the probe sequence, even if only A difference in one base leads to incomplete hybridization, making the ligation reaction impossible. After the ligation reaction is complete, the ligated probes are amplified with a pair of fluorescently labeled universal primers, each probe being produced for a length that is unique. Finally, the nucleic acid sequence can be detected by separating the amplified product by capillary electrophoresis. Due to the clever use of the principle of amplification probes, MLPA technology can identify the copy number of 45 target sequences in one reaction.
This technique has the high throughput characteristics of probe-ligation specificity hybridization with multiplex amplification probes. After more than 10 years of development by MRC-Holland, MLPA technology has become a comprehensive molecular diagnostic system covering the diagnosis of various genetic diseases, the identification of multiple genetic sites for pharmacogenetics, the screening of mutations in tumor-related genes, and the quantification of DNA methylation. It is the most commonly used high-throughput method for detecting known sequence variations and gene copy number variations.
(5) Biochip
In 1991, Affymetrix's Fordor [7] used the photo-etching technology developed by it to prepare the first slide-based microarray, which marked the official realization of biochips as a practical molecular biology technology. Today, chip technology has been greatly developed. If it is classified by structure, it can be basically divided into two types: microarray-based hybrid chips and microfluidic-based reaction chips.
1. Microarray
(1) Solid phase chip: microarray-based geonomic DNA profiling (MGDP) chip: The microarray technology has been applied to MGDP detection for more than ten years. Its technology platform is mainly divided into two categories, namely Array-based combanthropy hybridization (aCGH) and genotype hybridization array (SNParray). As the name suggests, the aCGH chip uses a two-color alignment of the DNA to be tested and the reference DNA to show changes in copy number variation (CNV) between the two, while the single nucleotide polymorphism (SNP) chip does not need to be associated with the reference. Compared with DNA, the SNP information in the DNA to be tested is directly displayed by the hybridization signal intensity. With the continuous advancement of technology, high-resolution mixed gene array chips capable of simultaneously detecting SNPs and CNVs have appeared on the market. MGDP chips are mainly used for the auxiliary diagnosis and prenatal screening of children with genetic diseases such as stunting and congenital abnormalities. It has been verified that the MGDP chip for chromosome imbalance detection and FISH diagnosis can reach 100%, gene expression profiling array (GEParray): In 1999, Duggan et al first used cDNA chips to map mRNA expression profiles. With the increasing role of epigenetics in the development of diseases, microRNA chips and long-noncoding RNA (lncRNA) chips have also appeared. Similar to the MGDP chip, the GEP chip uses a cDNA library generated after reverse transcription to hybridize with a nucleic acid probe immobilized on a chip carrier to detect the expression of the intensity of the hybridized fluorescent signal.
Compared with genomic hybridization, GEP microarray detects transcriptome information that is more important in biological significance, and has special significance for disease diagnosis and prognosis. At present, the use of GEP chip for the diagnosis, classification and prognosis evaluation of acute myeloid leukemia, myelodysplastic syndrome and other blood diseases and neurodegeneration have achieved satisfactory results;
(2) Liquid phase chip: The traditional solid phase chip anchors the detection probe to the solid phase carrier to capture the target sequence, while Luminex's xMAP technology combines the different ratios of the two red fluorescent dyes to the polystyrene microsphere. Labeled with different fluorescent colors and encoded to obtain microspheres with hundreds of fluorescent numbers. Different specific hybridization probes are cross-linked to the coding microspheres by xTAG technology, enabling different probes to be distinguished by microsphere coding. The mixed probe-microsphere complex is hybridized with the sample to be tested, and the microsphere is passed through a red-green two-color flow cytometer driven by the flowing sheath liquid, wherein the red laser detection microsphere is encoded, and the green fluorescence detection is hybridized. The signal intensity of the fluorescent reporter group on the post-nuclear probe completes the simultaneous identification of multiple target sequences in a single sample. At present, the technology has been widely used in the diagnosis of hereditary diseases such as cystic fibrosis, multiple respiratory virus identification and human papillomavirus typing.
2. Microfluidic chip
In 1992, Harrison et al. first proposed the idea of ​​integrating capillary electrophoresis and sampling equipment onto a solid-phase glass carrier to construct a “micro-analysis systemâ€. Through the miniaturization and integration of analytical equipment, the traditional analytical laboratory to the on-chip experiment was completed. Lab-on-chip transformation. The microfluidic chip consists of micron-sized fluid tubes, reactors and other components. Compared with macro-scale analytical devices, its structure greatly increases the area/volume ratio of the fluid environment to maximize the use of liquids and objects. Surface-related special properties including laminar flow effect, capillary effect, rapid thermal conduction and diffusion effect, so that a series of experimental procedures such as sample injection, pretreatment, molecular biological reaction, and detection are performed on one chip.
At present, the use of microfluidic chips to guide the parallel detection of multiple gene loci is the main clinical application field.
Second, nucleic acid sequence determination
Sequencing reaction is the only technical means to directly obtain nucleic acid sequence information, and is an important branch of molecular diagnostic technology. Although molecular hybridization, molecular conformational variation or quantitative PCR technology has been greatly developed in recent years, its identification of nucleic acids only stays on the assumption of indirect inference, so the molecular diagnosis based on specific gene sequence detection, nucleic acid Sequencing is still the gold standard for technology.
(A) first-generation sequencing
In 1975, Sanger and Coulson published a method for DNA sequence determination using addition and subtraction. Maxam then proposed a model of chemical modification degradation in 1977, which kicked off the advent of the era of nucleic acid sequencing.
Sanger is equivalent to the end termination method proposed in the same year (Sanger sequencing method). The 2' and 3' hydroxyl-free dideoxynucleoside triphosphate (ddNTP) is used for sequencing primer extension reaction, and ddNTP cannot form phosphodiester bond in DNA synthesis reaction. The DNA synthesis reaction will be terminated. If a specific ddNTP-labeled ddNTP is added to each of the four independent DNA synthesis reaction systems, the product may be subjected to polyacrylamide gel electrophoresis (PAGE) and autoradiography after the synthesis reaction, according to the electrophoresis strip. A nucleotide sequence that determines the molecule to be tested. Based on the Sanger method, Appied Biosystems launched the first commercial DNA sequencer PRISM 370A in 1986, replacing the radionuclide labeling and autoradiography detection systems with fluorescent signal reception and computer signal analysis. The company's first capillary electrophoresis sequencer PRISM 310, introduced in 1995, has greatly increased the throughput of sequencing. Sanger sequencing is the most classic generation of sequencing technology and remains the most commonly used method for obtaining nucleic acid sequences.
(B) second-generation sequencing
1. Pyro-sequencing
Unlike the post-synthesis sequencing concept used by the Sanger sequencing method, Ronaghi proposed in 1996 and 1998 the method of sequencing by pyrosynthesis in solid-phase and liquid-phase carriers-pyrophosphate sequencing. The basic principle is to use the pyrophosphate group released when the primer strand is extended to excite fluorescence, and to judge the number of bases matched by the peak height. Due to the concept of real-time fluorescence monitoring, pyrosequencing enables quantification of base load ratios at specific sites, and is therefore widely used for SNP site detection, allele (mutation) frequency determination, and bacterial and viral typing. . Due to the different principles of fluorescence reporting, the sensitivity of detection for sequence variation has increased from 20% of Sanger sequencing to 5%. However, due to the high cost of instrument procurement and single detection of this technology, large-scale clinical use has not yet been obtained.
2. High-throughput second-generation sequencing
The current high-throughput second-generation sequencing platforms include Roche454, IlluminaSolexa, ABISOLiD, and LifeIon Torrent, all of which are constructed by DNA fragmentation, library and vector cross-linking, and synthesis on the carrier surface. The sequencing reaction allows the parallel flow of the highest 96-well plate in the first-generation sequencing to be expanded to a million-parallel parallel reaction on the carrier, completing high-throughput detection of massive data. The technology can be used for true omics detection of genomes, transcriptomes, etc., in guiding disease molecular targeted therapy, mapping pharmacogenomic maps to guide individualized drugs, pathogenic microbial genome identification of infectious diseases, and through fetal DNA information in maternal Prenatal diagnosis and other aspects have achieved gratifying results. However, because the technology requires fragmentation of DNA, the sequencing reaction has a short read length (such as Solexa and SOLiD systems with a single read length of only 50 bp), which requires large-scale splicing of the data, so the molecular diagnostics master biological information. The knowledge has put forward higher requirements to facilitate the analysis of sequencing data in the later stage.
(3) Third generation sequencing
The core concept of the 3rd generation sequencing technology is sequencing of side synthesis targets targeting single molecules. The technology's operating platform currently includes Heliscope from Helicos, SMRT from Pacific Biosciences, and nanopore technology from Oxford Nanopore Technologies. The technology further reduces the cost, and can perform single molecule detection on the mixed genetic material, so the identification of SNP and CNV is more effective. However, there is still a long way to go before the commercialization of the product and its final application in clinical applications.
3. Molecular diagnostic techniques based on molecular conformation
(1) Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP)
From 1970 to 1980, Fischer et al. and Orita et al. proposed the method of separating and identifying the variation sequence by denaturation and non-denaturing PAGE by using the variation of nucleic acid sequence to the difference of double-strand denaturing conditions and single-strand space folding, namely DGGE and SSCP. Both of the above techniques detect the change in electrophoresis rate under specific conditions by the difference in spatial conformation of the mutated nucleic acid molecule. Because nucleic acid molecule conformations are sequence specific and very sensitive to sequence changes, often a one base change can also be identified. However, since both DGGE and SSCP must be subjected to PCR and open lid electrophoresis, it is not commonly found in clinical testing.
(2) Denaturing high-performance liquid chromatography (dHPLC)
In 1997, Oefner and Underhill established a technique for the separation of variant sequences using heteroduplex degeneration and chromatographic elution, called dHPLC, which automatically detects single base substitutions and insertions or deletions of small fragment nucleotides. For a double-stranded mixture of nucleic acids with a certain proportion of mutated sequences, after the denaturation and renaturation process, two double-stranded chains will appear in the system: one is a homoduplex, and the wild sense strand-wild antisense strand or variant justice A double-stranded nucleic acid consisting of a strand-variant antisense strand; the other is a heteroduplex, ie, one single strand in the double strand is wild-type and the other one is variant. Due to the different melting characteristics of heterologous double-stranded DNA with partial base mismatch and homologous double-stranded DNA, the heteroduplex is more susceptible to denaturation due to the presence of mismatched regions under the same partial denaturing conditions, and is retained by the column. The time is shorter than the homoduplex, so it is eluted first, and thus appears as a bimodal or multimodal elution curve in the chromatogram. Because the technique uses a chromatographic technique with higher analytical sensitivity for detection, a <5% load variant sequence can be quickly detected. However, it should be noted that since the technique mainly detects the sequence variation by heteroduplex, it does not distinguish between the wild type and the variant homozygote.
(3) High-resolution melting analysis (HRMA)
In 2003, Wittwer et al. revolutionized the use of supersaturated fluorescent dyes for the first time to perform fluorescent passive labeling of PCR products, and then identified individual base changes by simple product melting analysis. The principle of this technique is also identified by sequence variation by heteroduplex. After PCR amplification of the sample to be tested, if there is a sequence variation heterozygote, a heteroduplex is formed, and the melting temperature thereof is greatly decreased. At this time, since the double chain is completely filled with the saturated dye, the change in the melting temperature of the product can be determined by the difference in the melting curve. For mutant homozygotes, HRMA can also use its higher resolution to identify the difference in thermostability between single site A:T double bond pairing and G:C triple pairing of PCR products, but for class II and III SNPs. The homozygous variation cannot be effectively distinguished.
How to use the DNA conformation to speculate the sequence, so as to avoid costly sequence determination or cumbersome hybridization reaction has always been a hot issue in molecular biology research and application. At present, the convenience of indirect detection of sequence variation using conformational changes has been unanimously confirmed, especially HRMA can complete the amplification detection reaction of a single closed tube of the variant sequence. However, it should be noted that since the molecular detection means based on conformational change cannot strictly guarantee the specificity of detection by probe hybridization or nucleic acid sequence determination, it is only suitable for large-scale primary screening, and true diagnosis still needs to be diagnosed. Verification of hybridization or sequencing.
4. Quantitative PCR (qPCR)
Compared with other molecular diagnostic detection techniques, qPCR has two advantages, namely, nucleic acid amplification and detection are carried out by fluorescent signals in the same closed system, which eliminates the contamination of amplification products caused by open-cap treatment after PCR; Dynamic monitoring of fluorescent signals allows quantification of low copy templates. Due to the above technical advantages, qPCR has become the most accepted technology in clinical gene amplification laboratories. Identification and gene quantification of various pathogenic microorganisms such as viruses and bacteria, genetic polymorphism typing, and gene mutation screening. A large number of applications have been applied in various clinical practices such as monitoring of gene expression levels. However, with the rapid development of qPCR technology, the quality management issues related to this technology are becoming more and more prominent. How to eliminate the detection variation caused by various biological variables, reduce or inhibit the various interference factors in experimental operation and methodology is qPCR The problem facing technology.
( 1 ) Real-time PCR (real- time PCR)
Double-strand incorporation
In 1992, Higuchi et al. measured the fluorescence intensity of each nucleic acid after thermal cycling by incorporating ethidium bromide into the PCR reaction solution, and proposed a nucleic acid amplification curve using fluorescence intensity and thermal cycle number to quantitatively determine the reaction system. The reaction kinetics (real-time PCR) model of the initial template opens up a method for nucleic acid quantification by detecting fluorescent signals in real time. The nucleic acid dye can be inserted into the DNA double strand, and the fluorescence is released only when the double strand is embedded. The fluorescence intensity of the reaction tube is detected after each amplification cycle, and the S-shaped nucleic acid amplification curve of the fluorescence intensity-thermal cycle number is plotted to The projection of the intersection of the fluorescence threshold and the amplification curve on the number axis of the amplification cycle is used as a cycle threshold (Ct), and Ct has a negative exponential relationship with the number of initial templates contained in the reaction system, and the initial template amount is inferred. Morrison [22] subsequently proposed a method for quantification of low-copy templates in a reaction system using the highly sensitive double-stranded dye SYBR Green I. This method is simple to operate, but since the nucleic acid amplification is initiated using only the sequence of the amplification primer, the product specificity cannot be sufficiently ensured. Although product specificity can be tested by melting curve after real-time PCR, its specificity is significantly lower than that of detection using fluorescent probes, so double-stranded incorporation is not recognized in clinical practice.
2. Taqman probe
Due to the low specificity of the double-stranded incorporation method, the concept of 5' nuclease activity and fluorescence resonance energy transfer (FRET) probe of the Taq enzyme previously discovered by Heid [23] in 1996 was proposed. A method of performing qPCR using a Taqman probe. The essence of the TaqMan probe is a FRET oligonucleotide probe, which is labeled with a fluorescent reporter group at the 5' end of the probe, a fluorescent quenching group at the 3' end, and a 5'3' exonuclease activity using the Taq enzyme. The oligonucleotide probe that binds to the target sequence is hydrolyzed during the PCR to free the fluorophore and release the fluorescent signal. Thus, a probe capable of hybridizing to a target sequence releases fluorescence during amplification, and is quantified by the principle of real-time PCR. Due to its high specificity and successful commercialization, Taqman probe has become the most widely used qPCR method in clinical practice. It has various viral gene quantitative detection, genotyping and tumor-related gene expression detection. An irreplaceable position.
3. Molecular beacons
Also in 1996, Tyagi et al. proposed a method for qPCR using molecular beacons (moleuclarbeacons), which are oligonucleotide probes labeled with a fluorescent reporter group and a quencher group at the 5' and 3' ends, respectively. The two ends have complementary high GC sequences, and have a hairpin structure in the qPCR reaction solution, and the fluorescent group and the quenching group undergo fluorescence resonance energy transfer (FRET) to maintain a resting state. When the PCR reaction starts, the stem-loop structure opens under denaturing high temperature conditions, releasing fluorescence; during the annealing process, the target sequence-specific probe remains linear with the template, and the probe that cannot hybridize with the template is reticular to the stem ring. Structure and fluorescence quenching, by detecting the intensity of the fluorescent signal during annealing in the qPCR system, the initial template concentration in the system can be specifically detected by the real-time PCR principle. Compared with the Taqman probe, the molecular beacon uses a hairpin structure to spatially bind the fluorophore to the quenching group, which greatly reduces the fluorescence background of the detection, and its detection specificity is higher than that of the Taqman probe, which is more suitable for etc. Genotyping of the locus.
4. Two-hybrid probe
In 1997, Wittwer et al. published a method for qPCR using two adjacent oligonucleotide probes labeled with a fluorescent donor group and a fluorescent acceptor group, respectively. The excitation spectra of the donor group and the acceptor group labeled by the two-hybrid probe have a certain overlap, and the hybridization positions of the two probes to the target nucleic acid should be adjacent to each other. Only when the two probes hybridize to the target gene at the same time, the donor and the acceptor genes are close to each other, thereby generating energy transfer through FRET, and exciting the fluorescent signal, and the intensity of the fluorescent signal is proportional to the DNA content of the target sequence in the reaction system. Due to the use of two probes for target sequence hybridization, the specificity of this method is greatly improved compared to the traditional single probe detection system.
( 2 ) Digital PCR
The concept of decentralized detection of a single qPCR reaction using a microfluidic array emerged as early as the 1990s. Based on this concept, Vgelstein and Kinzter published a digital PCR method in 1999 to quantify trace amounts of K-RAS mutations in the stool of colon cancer patients. Compared with the traditional qPCR method, the core of digital PCR is to liquefy the qPCR reaction into the microspheres, and then disperse the chylo liquid into the micro-reaction wells of the chip to ensure that only ≤1 nucleic acid template is present in each reaction well. After PCR, the fluorescence signal of each micro-reaction well is detected, and the reaction well of the target nucleic acid template releases the fluorescent signal, and the reaction well without the target template has no fluorescence signal, thereby deducing the nucleic acid to be tested in the original solution. concentration. Therefore, digital PCR is a qPCR reaction that detects an absolute quantification of the fluorescence signal at the end of the reaction, rather than real-time PCR for nucleic acid quantification using the template Ct value.
The micro-drop digital PCR developed by Quantalife (acquired by BIO-RAD in 2011) is the first commercial digital PCR detection system, which has been widely used in micro-pathogenic microbial gene detection, low-load genetic sequence identification, Multiple clinical frontier areas such as gene copy number variation and single cell gene expression detection. Compared with traditional qPCR, this technology has super high sensitivity and precision, making it a new star in the field of qPCR.
V. Outlook for the next five years
The rapid development of molecular diagnostics in the past half century is inseparable from the rapid advancement of molecular biology technology. In summary, in the past 50 years, molecular diagnostic technology has achieved three major transformations and three enhancements: that is, reporting signal detection from radionuclide labeling to fluorescent labeling, and the operation method is from manual operation to fully automated conversion, detection and analysis. The flux is transformed from a single marker to a high-throughput multi-omics combination; the detection sensitivity, precision, and specificity are rapidly improved.
In the next five years, China's molecular diagnostics industry will usher in two aspects of progress. As the health regulatory authorities continue to deepen their understanding of the importance of molecular diagnostics and the entry of more highly educated and highly qualified personnel, molecular diagnostics will revolutionize the concept, and high-throughput technologies will enter the clinical arena. Practical applications. With the further development of technology, traditional methods for identifying specific genetic abnormalities and pathogenic microbial infections will also make great progress in the analysis performance and ease of operation. For the traditional human and time cost detection method, there will be a polarization situation, that is, the classical gold standard such as Southern will be retained; and the sensitivity and specificity such as ASO-RDB can not meet the actual clinical needs. Quickly replaced by new technologies. In the end, molecular diagnosis will also change the current situation of only the detection of pathogenic microorganisms and the diagnosis of some genetic diseases, forming a scene of rapid development of oncology, genetics, microbiology, and pharmacogenomics.
Source: Laboratory Medicine Network
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