1 Definition
PCR is a molecular biology experimental method for the in vitro enzymatic synthesis of specific DNA fragments, mainly consisting of a repeated thermal cycle of three steps: high temperature denaturation, low temperature annealing and moderate temperature extension: that is, the template DNA is first denatured to a single strand at high temperature, two primers anneal to a complementary sequence on each of the two template DNA strands under DNA polymerase and appropriate temperature, followed by DNA polymerase The annealed primers are then extended with four deoxynucleotide triphosphates (dNTPs) as substrates catalyzed by DNA polymerase. This is repeated so that the DNA fragment located between the two known sequences is amplified geometrically.
2 Historical review of polymerase chain reaction
2.1 The earliest conception of in vitro amplification of nucleic acids
It was proposed by Khorana and colleagues in 1971 that “tRNA genes could be cloned by denaturing DNA, hybridizing it with suitable primers, extending the primers with DNA polymerase, and giving constant attention to the process”. However, the early idea of Khorana et al. was forgotten because of the difficulty of sequencing and synthesizing oligonucleotide primers at that time, and the discovery of DNA restriction endonucleases by Smith et al. at that time (1970), which made it possible to clone genes in vitro.
3.2 Invention of polymerase chain reaction
It was not until 1985 that Mullis et al. invented the groundbreaking Polymerase Chain Reaction (PCR) at the Human Genetics Research Laboratory of PE-Cetus, Inc. in the United States, making the coveted unlimited amplification of nucleic acid fragments in vitro a reality. The principle is similar to the in vivo replication of DNA, except that it provides a suitable condition for the in vitro synthesis of DNA in a test tube. It started with the use of the E. coli DNA polymerase Klenow fragment to amplify specific fragments of the human genome. Since the enzyme is not heat resistant, the Klenow enzyme has to be refilled after each heating of denatured DNA. This process is time-consuming, laborious and error-prone when working with multiple specimens. The application of heat-resistant DNA polymerase made it easier to automate the polymerase chain reaction reaction, and PE-Cetus introduced the first polymerase chain reaction thermal cycler, making automation of this technology a reality.
3 Development of Polymerase Chain Reaction Related Technologies
The development of the polymerase chain reaction and its related technologies has been phenomenal. The first and second international symposia on polymerase chain reaction technology were held in the United States and the United Kingdom in 1988 and 1990, respectively. The first symposium focused on the application of polymerase chain reaction and the optimization of the technology itself; the second symposium focused on the Human Genome Project and the latest advances in polymerase chain reaction. This reflects the importance that biologists attach to the polymerase chain reaction.
(Table) Polymerase chain reaction related technologies
Name Main use of simplex primer amplification method
Amplification of unknown gene fragments
Nested polymerase chain reaction
Improving sensitivity and specificity of polymerase chain reaction and analyzing mutations
Complex polymerase chain reaction
Simultaneous detection of multiple mutations or pathogens
Reverse polymerase chain reaction
Amplify unknown sequences on both sides of a known sequence, causing mutations in the product
Single specific primer polymerase chain reaction
Amplifies unknown genomic DNA
Single-sided primer polymerase chain reaction
Amplification of unknown cDNA by known sequences
Anchored polymerase chain reaction
Analysis of sequences with different ends
Efficient Polymerase Chain Reaction
Reduces primer dimerization and improves polymerase chain reaction specificity
Curing polymerase chain reaction
Facilitates product separation
Membrane-bound polymerase chain reaction
Removal of contaminating impurities or polymerase chain reaction product residues
Expression cassette polymerase chain reaction
Produces DNA fragments for synthetic or mutant proteins
Ligation-mediated polymerase chain reaction
DNA methylation analysis, mutation, cloning, etc.
RACE-Polymerase Chain Reaction
Amplification of cDNA ends
Quantitative polymerase chain reaction
Quantification of mRNA or chromosomal genes
In situ polymerase chain reaction
Study of the proportion of cells expressing a gene, etc.
Hypothetical polymerase chain reaction
Identification of bacteria or genetic effects
Generic primer polymerase chain reaction
Amplification of related genes or detection of related pathogens
Messenger amplification for phenotypic typing (mapping)
Simultaneous analysis of mRNA from a small number of cells
4 Other amplification techniques
In parallel with the development of polymerase chain reaction and its related techniques, new amplification techniques have been created. These techniques, each with its own advantages and disadvantages, complement the polymerase chain reaction, and some can be combined to form a large family of in vitro nucleic acid amplification techniques. We believe that with the development of molecular biology technology, new members of this family will emerge.
Other in vitro nucleic acid amplification techniques (Table) Technical applications Transcription-dependent amplification system (TAS) detection HIV ligase chain reaction (LCR) detection point mutation autonomous sequence replication (3SR) system to study RNA, clinical applications, forensic medicine and other chain substitution amplification (SDA) detection, identification gene Qβ replicase system to increase the sensitivity of probe detection circular probe reaction to increase the sensitivity of probe detection.
4.1 Ligase Chain Reaction (A)
Ligase chain reaction (LCR), a new technique for in vitro amplification and detection of DNA, is mainly used for the study of point mutations and amplification of target genes. It was invented and patented by Backman in 1997 to detect point mutations in target gene sequences.
The basic principle of LCR is to use DNA ligase. The LCR amplifies a large number of target genes by specifically ligating double-stranded DNA fragments in a three-step cycle of denaturation-annealing-joining.
The amplification efficiency of LCR is comparable to that of polymerase chain reaction, and only two temperature cycles are used for LCR with heat-resistant ligase: denaturation at 94°C min and denaturation and ligation at 65°C for about 30 cycles. The detection of its products is also more convenient and sensitive. At present, this method is mainly used for the study and detection of point mutations, detection of microbial pathogens and targeted mutagenesis, etc. It can also be used for the diagnosis of single-base genetic disease polymorphisms and products of single-base genetic diseases, the identification of microbial species types, and the study of point mutations in oncogenes.
4.2 Nucleic acid sequence-dependent amplification (A)
Nucleic acid sequence-based amplification (NASBA), also known as self-sustained sequence replication (3SR) or regenerative sequence replication technique, was first reported by Guatelli et al. in 1990. NASBA is mainly used for RNA amplification, detection and sequencing.
The basic method is: add primers and specimens to the amplification reaction solution, open the secondary structure of RNA molecule at 65℃ for 1 min, then cool down to 37℃ and add reverse transcriptase, T7RNA polymerase and RNase H, and react at 37℃ for 1~1.5 h. The product can be seen under UV by agarose electrophoresis and bromoethidium staining.
NASBA is characterized by easy operation, no special apparatus and no temperature cycling. The whole reaction process is controlled by three enzymes, with less cycles and high fidelity, and its amplification efficiency is higher than that of polymerase chain reaction, with good specificity.
4.3 Transcription-dependent amplification system (A)
Transcript-based amplification sytem (TAS), which was reported by Kwen et al. in 1989, is mainly used for amplifying RNA.
The main feature of TAS is the high amplification efficiency because its RNA copy number increases exponentially by 10, reaching 2×106 copies of the target sequence in only 6 cycles. another feature is its high specificity because TAS can only perform 6 temperature cycles with low mismixing rate, coupled with hybridization with dextran beads sandwich.
Although this method has high specificity and sensitivity, its cycling process is complex and requires repeated addition of reverse transcriptase and T7RNA polymutase, which needs further study.
4.4 Qβ replicase reaction (A)
Kacian et al. first reported in 1972 that Qβ replicase (Q-beta replicase) catalyzes the self-replication function of RNA template, which can amplify its natural template MDV-1 RNA to 109 at room temperature for 30 min. In 1986, Chu et al. reported that with a biotagged target sequence-specific probe that can hybridize to affinity-linked MDV-1 RNA In 1986, Chu et al. reported that a biotagged target sequence-specific probe could be used to hybridize to affinity-linked MDV-1 RNA, which was eluted from unbound MDV-1, followed by the addition of Qβ replicase to amplify replicate MDV-1 copies, and then detected by ethidium bromide staining or hybridized with a homologous second probe.
Qβ replicase is an RNA-directed RNA polymerase with 3 features: ① It can initiate RNA synthesis without oligonucleotide primer guidance. (ii) It can specifically recognize the unique RNA folding structure in RNA genes due to intramolecular base pairing. (iii) Insertion of a short nucleic acid sequence into the unfolded structural region of MDV-1 RNA, the natural template of Q β replicase, does not affect the replication of the enzyme. Thus, if a nucleic acid probe is inserted in this region, its sequence may be amplified by Qβ replicase as well.
In 1988, Lizardi et al. inserted the target gene sequence into the MDV-1 plasmid and transcribed the MDV-1 RNA probe catalyzed by T7 RNA polymerase, which could hybridize with the target sequence, then washed the non-hybridized probe and added Qβ replicase to amplify the probe, and the amplified probe could be used as a template to amplify exponentially. The product is detected by the two methods described above. Now the technique has developed sandwich hybridization, molecular switch and target-dependent replication.
5 Examples of applications of polymerase chain reaction techniques.
Research: gene cloning; DNA sequencing; analysis of mutations; gene recombination and fusion; identification and regulation of protein conjugation DNA sequences; mapping of transposon insertion sites; detection of gene modifications; construction of synthetic genes; construction of cloning or expression vectors; detection of endonuclease polymorphism of a gene
Diagnosis: bacteria (spirochetes, mycoplasma, chlamydia, mycobacterium, rickettsia, diphtheria, pathogenic Escherichia coli, dysentery, Aeromonas hydrophila and Clostridium difficile, etc.); viruses (HTLV, HIV, HBV, HPVS, EV, CMV, EBV, HSV, measles virus, rotavirus, B19 of microvirus); parasites (malaria, etc.); human genetic diseases ( Lesh-Nyhan syndrome, geodystrophy, hemophilia, BMD, DMD, cystic fibrosis, etc.)
Immunology: HLA splitting; characterization of T-cell receptor or antibody diversification; genetic mapping of autoimmune diseases; lymphokine quantification
Human genome engineering: generation of DNA signatures with scattered repetitive sequences; construction of genetic maps (detection of DNA, polymorphisms or sperm mapping); construction of physical maps; sequencing, expression mapping
Forensics: crime scene specimen analysis; HLA-DQ
Oncology: pancreatic cancer, colon cancer, lung cancer, thyroid cancer, melanoma, hematological malignancies
Tissue and population biology: genetic clustering studies; animal conservation studies; ecology; environmental science; experimental genetics.
Paleontology: analysis of archaeological and museum specimens
Zoology: diagnosis of zoonotic diseases, etc.
Botany: detection of plant pathogens, etc.