Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. It can be regarded as a special kind of DNA replication in vitro.

DNA polymerase I was first discovered in 1955, and the Klenow fragment of Escherichia coli with more experimental value and practicality was discovered by Dr. H. Klenow in the early 1970s. However, because this enzyme is not resistant to high temperature and can be denatured at high temperature, it is not suitable for using high temperature denatured polymerase chain reaction. The enzyme used today (Taq polymerase for short) was isolated from bacteria in hot springs in 1976. Because of its high temperature tolerance, it is an ideal enzyme, but it has been widely used since 1980s. The original prototype concept of PCR is similar gene repair and replication, which was put forward by Dr. Kjell Kleppe in 197 1. He published the first simple and short-lived gene replication experiment (similar to the first two cycles of PCR). The PCR developed today was developed by Dr. Kary B. Mullis in 1983. Dr. Mullis worked in PE company, so PE company has a special position in the field of PCR. Dr Muhlis published the first related paper with Saiki and others in 1985. Since then, the application of PCR has advanced by leaps and bounds, and the quality of related papers can be said to be unmatched by many other research methods. Subsequently, PCR technology was widely used in biological research and clinical application, and became the most important technology in molecular biology research. Muhlis also won the 1993 Nobel Prize in chemistry.

[Edit this paragraph] [PCR principle]

Semi-conservative replication of DNA is an important way of biological evolution and passage. Double-stranded DNA can be denatured and melted into single strand under the action of various enzymes, and copied to the same bimolecular mussel according to the principle of base complementary pairing with the participation of DNA polymerase. It was found in the experiment that DNA can be denatured and melted at high temperature, and can be refolded into double chains when it is cooled. Therefore, the denaturation and renaturation of DNA can be controlled by temperature change, and DNA polymerase and dNTP can replicate specific genes in vitro by adding designed primers.

However, DNA polymerase will be inactivated at high temperature, so it is necessary to add new DNA polymerase every cycle, which is not only cumbersome to operate, but also expensive, which restricts the application and development of PCR technology.

The discovery of thermostable DNA polymerase-Taq enzyme is a milestone in PCR application. The enzyme can tolerate the high temperature above 90℃ without losing its activity, which makes the PCR technology very simple and greatly reduces the cost. PCR technology has been widely used and gradually applied in clinic.

The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence. PCR consists of three basic reaction steps: denaturation-annealing-extension: ① denaturation of template DNA: after the template DNA is heated to about 93℃ for a certain time, the double-stranded DNA of the template DNA or the double-stranded DNA amplified by PCR dissociates into single strands, which can be combined with primers to prepare for the next round of reaction; (2) Annealing (renaturation) of template DNA and primer: after the template DNA is denatured into single strand by heating, the temperature is reduced to about 55℃, and the primer and the complementary sequence of the template DNA single strand are paired and combined; ③ Primer extension: Under the action of TaqDNA polymerase, DNA template-primer conjugate takes dNTP as the reaction raw material and the target sequence as the template. According to the principle of base complementary pairing and semi-conservative replication, a new semi-conservative replication chain complementary to the template DNA chain can be synthesized, and more "semi-conservative replication chains" can be obtained by repeating the three processes of cyclic denaturation-annealing-extension, which can be used as the template for the next cycle. It takes 2 ~ 4 minutes to complete a cycle, and the target gene to be amplified can be amplified by a million times in 2 ~ 3 hours.

[Edit this paragraph ][PCR reaction system and reaction conditions]

1. standard PCR reaction system

10× amplification buffer 10μl

200μl mixture of four dNTP.

Primer10 ~100μ l

Template DNA 0. 1 ~ 2 μ g

Taq DNA polymerase 2.5 microliters

Mg2+ 1.5 mmol/L.

Add double or triple distilled water100μ l.

Five elements of PCR reaction: There are five main substances involved in PCR reaction, namely:

Primers (PCR primers are DNA fragments, and primers for DNA replication in cells are RNA strands), enzymes, dNTP, templates, and buffers (Mg2+ is required).

There are many methods to design primers, which are determined by the purpose of PCR in the experiment. But the basic principle is the same.

There are two main sources of enzymes used in PCR: Taq and Pfu. From two different thermophilic bacteria. Among them, Taq amplification efficiency is high, but there is a mismatch. Pfu has weak amplification efficiency, but it has error correction function. Therefore, in actual use, we must make different choices according to our needs.

The template DNA used for amplification can come from any source, but there are two principles. First, the purity must be high, and second, the concentration should not be too high to avoid inhibition.

The composition of buffer solution is the most complex, generally including four effective components except water: buffer system, generally using HEPES or MOPS buffer system; Potassium ions are generally used for monovalent cations, but ammonium ions can also be used in special cases; The divalent cation, that is, magnesium ion, is determined according to the reaction system and does not need to be adjusted except in special circumstances; Auxiliary components, such as DMSO and glycerol, are mainly used to maintain the activity of enzymes and help DNA contact winding structures.

2.PCR primer design

There are two primers in PCR reaction, namely 5' primer and 3' primer. Primers are designed based on a single DNA strand (usually based on information strand), and the 5'- end primer is the same as the short DNA sequence located upstream of the 5'- end of the fragment to be amplified; The primer at the 3' end is complementary to the short DNA sequence at the 3' end of the fragment to be amplified.

Basic principles of primer design

① Primer length: 15-30bp, generally around 20bp.

② The content of primer base: G+C should be 40-60%. Too little G+C is not conducive to amplification, and too much G+C is easy to produce non-specific bands. ATGC should be randomly distributed to avoid the string arrangement of more than 5 purine or pyrimidine nucleotides.

③ There should be no complementary sequence in the primer.

④ There should be no complementary sequence between the two primers, especially to avoid the complementary overlap at the 3' end.

⑤ The homology between the primer and the sequence of the non-specific amplification region should not exceed 70%, and the 8 bases at the 3' end of the primer should not be completely complementary to the sequence outside the amplification region, otherwise it will easily lead to non-specific amplification.

⑥ The bases at the 3' end of the primer, especially the last base and the penultimate base, should be strictly matched, and it is best to choose G and C. ..

⑦ The 5' end of the primer can be modified. Such as adding restriction sites, introducing mutation sites, labeling with biotin, fluorescent substances and digoxin, and adding other short sequences, including start codons and stop codons.

V. Primer design software

Primer Premier5.0 (automatic search) *

VOligo6 (primer evaluation)

Vector NTI suit

vDNAsis

Womiga

vDNAstar

VPrimer3 (online service)

3. Preparation of templates

The template of PCR can be DNA or RNA.

The template is mainly based on the amplification object of PCR, which can be samples of pathogens such as viruses, bacteria and fungi. It can also be a pathophysiological specimen such as cells, blood and amniotic fluid cells. Forensic specimens include blood spots, sperm spots, hair and so on.

The basic requirements of sample processing are to remove impurities and partially purify the nucleic acid in the sample. Most samples need to be treated with SDS and protease K, and bacteria that are difficult to break can be treated with lysozyme and EDTA. The obtained crude DNA was extracted and purified by phenol and chloroform, and then precipitated by ethanol as a template for PCR reaction.

4. Control of 4.PCR reaction conditions

① ① The buffer of ①①PCR reaction provides suitable pH and some ions.

② The total concentration of magnesium ion should be higher than dNTPs, which is usually1.5 mmol/L..

(3) the substrate concentration dNTP is prepared into an equimolar concentration of 20-200μ mol/L.

④TaqDNA polymerase 2.5U( 100ul)

⑤ Primer concentration is generally 0.1~ 0.5 umol/L..

⑥ reaction temperature and cycle time

Degeneration temperature and time 95℃, 30s.

The annealing temperature and time are about 5℃ lower than the Tm value of the primer, generally at 45 ~ 55℃.

The extension temperature and time are 72℃,1min/kb (within10kb).

Tm value = 4 (g+c)+2 (a+t)

Cycle times: generally 25 ~ 30 times. The number of cycles determines the yield of PCR amplification. The initial concentration of template is low, and the number of cycles can be increased to achieve effective amplification. But the number of cycles cannot be increased indefinitely. Generally, the number of cycles is about 30 times. After more than 30 cycles, the activity of DNA polymerase gradually reaches saturation, and the product quantity no longer increases with the increase of cycles, resulting in the so-called "platform period".

[Edit this paragraph] [PCR step]

The standard PCR process is divided into three steps:

1.DNA denaturation (90℃-96℃): Under the action of heat, the hydrogen bond of the double-stranded DNA template is broken to form single-stranded DNA.

2. Annealing (25℃-65℃): The temperature of the system decreases, and the primer and the DNA template combine to form a local double strand.

3. Extension (70℃-75℃): Under the action of Taq enzyme (about 72℃, the activity is the best), dNTP is used as raw material to extend from the 5' end to the 3' end of the primer to synthesize a DNA strand complementary to the template.

After denaturation, annealing and extension in each cycle, the DNA content doubles. As shown in the figure:

At present, some PCR can be replicated in a short time even if the Taq enzyme activity is not optimal due to the short amplification region, so it can be changed to a two-step method, that is, annealing and extension are carried out at 60℃-65℃ at the same time, so as to reduce the heating and cooling process once and improve the reaction speed.

[Edit this paragraph] [PCR detection]

The PCR reaction amplified a high copy number, and the next detection became the key. Fluorescein (EB) staining gel electrophoresis is the most commonly used detection method. The specificity of electrophoresis detection is not too high, so nonspecific hybridization such as primer dimer is easy to cause misjudgment. But because of its simplicity, it has become the mainstream detection method. In recent years, the detection method represented by fluorescent probe has gradually replaced electrophoresis.

[Edit this paragraph] [PCR reaction characteristics]

Strong specificity

The specific determinants of PCR reaction are:

① Correct combination of specificity of primer and template DNA;

② the principle of base pairing;

③ The authenticity of ③Taq DNA polymerase synthesis reaction;

④ The specificity and conservation of the target gene.

The correct combination of primers and templates is the key. The combination of primer and template and the extension of primer chain follow the principle of base pairing. Because of the fidelity of polymerase synthesis reaction and the high temperature resistance of TaqDNA polymerase, the combination (renaturation) of template and primer in the reaction can be carried out at higher temperature, the specificity of combination is greatly increased, and the amplified target gene fragment can maintain high accuracy. By selecting highly specific and conservative target gene regions, its specificity will be higher.

high sensitivity

The amount of PCR products increases exponentially, and the initial template to be tested can be amplified to the level of μg=-6 according to Pique (pg= 10- 12). A target cell can be detected from 6.5438+0 million cells; In virus detection, the sensitivity of PCR can reach 3 RFU (plaque forming unit); In bacteriology, the lowest detection rate is 3 bacteria.

Simple and quick

Application of thermostable Taq DNA polymerase in PCR reaction. After the reaction solution is added at one time, denaturation-annealing-extension reaction is carried out in DNA amplification solution and water bath pot, and the amplification reaction is generally completed within 2 ~ 4 hours. Amplification products are generally analyzed by electrophoresis, and isotopes are not needed, so there is no radioactive pollution and it is easy to popularize.

The purity of the specimen is low.

There is no need to separate viruses or bacteria from cultured cells, and crude DNA and RNA can be used as amplification templates. It can be directly used for DNA amplification and detection of clinical samples such as blood, body cavity fluid, mouthwash, hair, cells and living tissues.

[Edit this paragraph] [Loop parameter] [PCR]

1, initial denaturation.

The complete denaturation of template DNA and the complete activation of PCR enzyme are very important for the success of PCR. It is suggested that the heating time refer to the reagent description, and the activation time of unmodified Taq enzyme is generally two minutes.

2, the denaturation step in the cycle

Generally, 30 seconds at 95℃ is enough to completely denature various target DNA sequences, and the time of this step can be shortened if possible.

Too long denaturation time impairs enzyme activity, and too short denaturation of target sequence is not complete, which easily leads to amplification failure.

3. Primer annealing

The annealing temperature needs to be determined from many aspects. Generally, according to the Tm value of primers and the amplification length, the annealing temperature is appropriately reduced. Then make an estimate on the basis of this experiment.

Annealing temperature has a great influence on the specificity of PCR.

4. Primer extension

Primer extension is usually carried out at 72℃ (the optimum temperature of Taq enzyme). However, when the amplification length is short and the annealing temperature is high, this step can be omitted.

The extension time depends on the length of the amplified fragment, and it is generally recommended to be above 1000bp, and the derivative of Pfu and its derivatives should be set as 1min/kbp.

5. Number of cycles

Most PCR contains 25-40 cycles, which is easy to produce non-specific amplification.

6. Last extension

After the last cycle, the reaction was maintained at 72℃ for 5- 15 minutes, so that the primers were completely extended and the single-stranded products were annealed into double-stranded.

(Pollution and false positives in PCR.

The pollution in PCR mainly comes from

1, cross-contamination between samples;

2. Legacy of previous PCR products.