Polymerase Chain Reaction (PCR)

Summary

PCR technology can be used to (1) test whether infectious diseases are in a recessive or subclinical state; (2) effectively detect mutations in oncogenes and accurately detect the expression of oncogenes; and (3) clinically applied to detect gene mutations in thalassemia.

Operation method

PCR technology

Principle

The basic principle is similar to the natural process of DNA replication, and its specificity depends on the oligonucleotide primers complementary to the ends of the target sequence.PCR consists of three basic reaction steps: ① denaturation of the template DNA: the template DNA is heated to 94 ℃ for a certain period of time, so that the template DNA double-stranded or double-stranded DNA formed by the PCR amplification dissociates, so as to become a single-stranded, in order for it to bind to the primer for the next round of reaction. so that it can combine with primers and prepare for the next round of reaction;so that it combines with the primer to prepare for the next round of reactions; ② template DNA and primer annealing (denaturation): template DNA by heating denaturation into a single-stranded, the temperature is reduced to 55 ℃ or so, the primer and the template DNA single-stranded complementary sequences paired binding; ③ primer extension: DNA template - primer conjugate in the Taq enzyme under the action of the target sequence as a template with dNTP as the raw material for the reaction, according to the base pairing and half-conserved replication. Under the action of Taq enzyme, with dNTP as the reaction material and the target sequence as the template, a new semi-conserved replicated strand complementary to the template DNA strand is synthesized according to the principle of base pairing and semi-conserved replication. By repeating the cycle of denaturation-annealing-extension, more "semi-conserved replicated strands" can be obtained, and this new strand can become the template for the next cycle. It takes 2-4 minutes to complete each cycle, and the target gene can be amplified millions of times in 2-3 hours.

Materials and Instruments

Template DNA
dNTP Taq DNA polymerase Distilled water PCR buffer Primers Magnesium chloride
PCR instrument Pipette gun PCR plate Thin-walled tubes Centrifuge tubes Tube cassettes

Move

I. Standard PCR reaction system

10×amplification buffer 10 ul 4 dNTP mixtures 200 umol/L each Primers 10-100 pmol each Template DNA 0.1～2 ug Taq DNA polymerase 2.5 u ^Mg2+ 1.5 mmol/L Add double or triple distilled water to 100 ul PCR primer design

There are two primers in the PCR reaction, i.e. the 5′-end primer and the 3′-primer. The primers are designed based on a single strand of DNA (often based on the information strand), the 5′-end primer is identical to a small segment of DNA sequence located at the 5′-end of the fragment to be amplified; the 3′-end primer is complementary to a small segment of DNA sequence located at the 3′-end of the fragment to be amplified.

1. Basic principles of primer design

(1) Primer length: 15-30 bp, commonly used is about 20 bp.

(2) Primer base: G+C content of 40-60% is appropriate, G+C is too little amplification effect is not good, too much G+C is easy to appear non-specific bands. ATGC is best distributed randomly, to avoid more than 5 purine or pyrimidine nucleotides arranged in bunches.
(3) Complementary sequences should not appear within the primers. (4) There should be no complementary sequences between two primers, especially avoiding complementary overlap at the 3 ′ end. (5) The homology between the primers and the sequences of the non-specific amplification region should not exceed 70%, and the 8 consecutive bases at the 3 ′ end of the primers should not have fully complementary sequences outside of the region to be amplified, or else it will easily lead to non-specific amplification. (6) The bases at the 3' end of the primer, especially the last and penultimate bases, should be strictly required to be paired, and the best choice is G and C.
(7) The 5 ′ end of the primer can be modified. Such as additional restriction enzyme sites, introduction of mutation sites, labeling with biotin, fluorescent substances, digoxin, addition of other short sequences, including start codon, stop codon and so on.
2. Primer design software

Primer Premier5.0 (automatic search), Oligo6 (primer evaluation), Vector NTI Suit, DNAsis, Omiga, DNAstar, Primer3 (online service).

III. Template preparation

(1) The template for PCR can be DNA or RNA.

(2) The template is mainly based on the PCR amplification object, can be a pathogen specimen such as viruses, bacteria, fungi and so on. It can also be pathophysiological specimens such as cells, blood, amniotic fluid cells and so on. Forensic specimens such as blood spots, sperm spots, hair, etc..

(3) The basic requirement of specimen processing is to remove impurities and partially purify the nucleic acids in the specimen. Most of the samples need to be treated with SDS and proteinase K. Bacteria that are difficult to fragment can be treated with lysozyme plus EDTA. The crude DNA obtained is purified by phenol and chloroform extraction, and then precipitated with ethanol and used as a template for PCR reaction.

Control of PCR reaction conditions

1. The buffer for PCR reaction should provide suitable pH with certain ions. 2.

2. The total concentration of magnesium ions should be higher than that of dNTPs, commonly used 1.5 mmol/L. The buffer for PCR reaction provides suitable pH with certain ions.
3. substrate concentration dNTP is prepared in equimolar concentration, 20 to 200 umol/L.
4. taqDNA polymerase 2.5 U (100 ul).
5. primer Concentration is generally 0.1 to 0.5 umol/L.
6. reaction temperature
(1) Denaturation temperature and time 95 °C for 30 s.
(2) Annealing temperature and time About 5 ℃ below the primer Tm value, usually at 45-55 ℃.
(3) Extension temperature and time 72 ℃, 1 min/kb (within 10 kb).
(4) Tm value = 4(G+C) +2(A+T) 7. Number of cycles : Generally 25 ~ 30 times. The number of cycles determines the yield of PCR amplification. If the initial concentration of the template is low, the number of cycles can be increased to achieve efficient amplification. However, the number of cycles cannot be increased indefinitely. Generally, the number of cycles is about 30. After the number of cycles exceeds 30, the DNA polymerase activity gradually reaches saturation, and the amount of product no longer increases with the increase in the number of cycles, resulting in the so-called "plateau period".
V. PCR cycle parameters

1. Pre-denaturation (Initial denaturation)

The complete denaturation of the template DNA and the complete activation of the PCR enzyme are crucial to the success of PCR, and it is recommended that the heating time should be referred to the instructions of the reagents, and the activation time of the unmodified Taq enzyme should be two minutes. 2.

2. Denaturation steps in the cycle

Generally, 95 ℃, 30 seconds is enough to denature various target DNA sequences in the cycle, if possible to shorten the time of this step, denaturation time is too long to damage the enzyme activity, too short target sequence denaturation is not complete, easy to cause amplification failure.

3. Primer annealing

The annealing temperature needs to be decided from various aspects, generally based on the Tm value of the primer as a reference, according to the length of the amplification of the appropriate downward adjustment as the annealing temperature. Then make a prediction based on this experiment.

The annealing temperature has a greater impact on the specificity of PCR.

4. Primer extension

Primer extension is generally carried out at 72℃ (the optimal temperature for 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 fragments, and is generally recommended to be above 1000 bp, and 1 min/kbp for derivatization containing Pfu and its derivatives.

5. Number of cycles

Most PCRs contain 25-40 cycles, too many cycles may cause non-specific amplification.

6. Final extension

After the last cycle, the reaction is maintained at 72°C for 5-15 minutes. The primers are extended completely and the single-stranded product is annealed to double-stranded.

Steps of PCR

1. DNA denaturation

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

2. Annealing

(25℃-65℃): The temperature of the system decreases, the primer combines with the DNA template to form a partial double strand.

3. extension

(70℃-75℃): under the action of Taq enzyme (at about 72℃, the best activity), with dNTP as raw material, from the 5′ end of the primer → 3′ end extension, synthesize DNA strand complementary to the template.

VII. PCR Detection

PCR reaction amplified a high copy number, the next step of detection becomes critical. Fluorescein (ethidium bromide, EB) stained gel electrophoresis is the most commonly used detection means. The specificity of the electrophoresis assay is not very high, so non-specific hybrids such as primer dimers can easily cause misclassification. However, because of its simplicity and ease of use, it has become the mainstream detection method. In recent years, the fluorescent probe as a representative of the detection method, there is a trend to gradually replace the electrophoretic method.

Caveat

1. Due to the high sensitivity of PCR reactions, special care should be taken to prevent DNA contamination. Reciprocal contamination of samples may produce false-positive results, especially when PCR is applied to the determination of clinical pathogenic bacterial infections.

2. if the PCR instrument is a shared, non-password protected PCR instrument, always check that the program on the PCR instrument is correct.

3. do not use excessive amounts of reagents "less is usually better (more specific)".

4. reagents should be purchased and used in small portions, so that in case of contamination, the contaminated reagents can be discarded immediately without causing major losses; reagents in small portions can also help to minimize the number of repeated freezing and thawing (e.g., dNTPs are sensitive to repeated freezing and thawing).

5. After adding all reagents, blow with the tip of the gun several times or vortex or flick the walls of the tube to ensure adequate mixing.

6. use a negative control to check for the occurrence of contamination.

7. use positive controls (samples that amplify well).

8. use a DNA molecular weight standard for electrophoresis (to indicate if PCR fails or bands run off the gel or the photo system fails).

9. use additives in moderation when amplifying very long, GC-rich templates or templates prone to secondary structure (glycerol, formamide, NMP reduce denaturation and annealing temperatures by a few degrees; glycerol also acts as a stabilizer of the polymerase activity; DMSO reduces the occurrence of secondary structure and improves specificity of the reaction by destabilizing the binding of non-specific primers). (increasing the specificity of the reaction).

10. Do not use a refrigerator with automatic defrost to store enzymes (avoid repeated freezing and thawing), use a new shotgun enzyme each time you pick up the enzyme, and return the enzyme to the refrigerator immediately after use. The enzyme should be added after the buffer has been added. Adding the enzyme directly to water may result in denaturation and inactivation of the enzyme.

11. The electrophoretic detection time of PCR products is generally within 48 hours, and some of them are better to be detected by electrophoresis on the same day, because the band shape is irregular or even disappears after more than 48 hours.

Common Problems

I. Key aspects of PCR reaction

1. Preparation of template nucleic acid.

2. Quality and specificity of primers.

3. Quality of enzyme.

4. PCR cycling conditions.

Common problems and countermeasures of PCR

1. False-negative, no amplification bands appear

(1) Template reasons

The template contains heterogeneous proteins.

The template contains Taq enzyme inhibitors.

③ Proteins in the template have not been eliminated, especially histones in the chromosomes.

(4) Too much is lost during the extraction and preparation of the template, or phenol is inhaled.

⑤ Incomplete denaturation of nucleic acids in the template. If the enzyme and primer are of good quality and no amplification band appears, it is very likely that there is something wrong with the digestion of the specimen and the extraction of template nucleic acid. Therefore, it is necessary to prepare an effective and stable digestion solution, and the procedure should be fixed and should not be changed arbitrarily.

(2) Enzyme inactivation

① Replace the enzyme with a new one, or use both the old and new enzymes to analyze whether the enzyme activity has been lost or is insufficient to cause false negatives.

② Forget to add Taq enzyme or ethidium bromide.

(3) Primers

① Quality of primers.

② Concentration of the primer.

③ Whether the concentrations of the two primers are symmetrical.

Solve the countermeasures:

① Select a good primer synthesis unit.

② primer concentration should not only look at the OD value, but also focus on the primer solution for agarose gel electrophoresis, there must be primer bands, and the brightness of the two primer bands should be generally consistent, such as a primer band, a primer without a band, at this time to do the PCR is likely to fail, and should be negotiated with the primer synthesis unit to solve the problem. If one primer has high brightness and one has low brightness, the concentration should be balanced when diluting the primers.

③ The primers should be stored in high concentration and small amount to prevent multiple freezing and thawing or long term storage in the refrigerator, which will lead to degradation and failure of the primers.

④ The primer design is unreasonable, such as the primer length is not enough, and the dimer is formed between the primers.

(4) ^Mg2+ concentration

① The concentration is too high to reduce the specificity of PCR amplification.

② concentration is too low to affect the PCR amplification yield or even make PCR amplification failure and not amplified bands.

(5) Change of reaction volume

① Usually used for PCR amplification volume of 20 ul, 30 ul, 50 ul or 100 ul, how much volume should be used for PCR amplification, according to the different purposes of scientific research and clinical testing and set; ② After doing a small volume such as 20 ul, and then do a large volume of PCR amplification, the reaction volume should be changed to a larger volume.

② After doing a small volume such as 20 ul, when doing a large volume, must be molded conditions, otherwise it is easy to fail.

(6) Physical reasons

① Denaturation is quite important for PCR amplification. If the denaturation temperature is low and the denaturation time is short, false negatives are very likely to occur.

② annealing temperature is too low, can lead to non-specific amplification and reduce the efficiency of specific amplification; annealing temperature is too high to affect the combination of primers and templates and reduce the efficiency of PCR amplification.

③ Problems with the denaturation, annealing and extension temperatures in the amplifier or water soluble pot.

(7) Target sequence variation

① target sequence mutation or deletion, affecting the specific binding of primers and templates.

② The loss of a segment of the target sequence makes the primer and the template lose the complementary sequence.

2. False positives, the PCR amplification bands appear consistent with the target sequence bands, and sometimes the bands are more neat and brighter.

(1) Inappropriate primer design

① The selected amplification sequence is homologous to the non-target amplification sequence.

② The target sequence is too short or the primer is too short.

(2) Cross-contamination of target sequences or amplification products

① The whole genome or a large segment of the cross-contamination.

Solution: Be careful and gentle during operation to prevent the target sequence from being sucked into the spiking gun or spilled out of the centrifuge tube. All reagents or equipment should be autoclaved, except for enzymes and substances that cannot withstand high temperature. Centrifuge tubes and sample feeder tips should be disposable. If necessary, the reaction tubes and reagents are irradiated with ultraviolet light to destroy nucleic acids present before adding specimens.

② Airborne contamination with small fragments of nucleic acids that are shorter than the target sequence but have some homology. They can be spliced to each other and complementary to the primers, they can amplify the PCR product and lead to false positives.

Solution: Can be mitigated or eliminated by nested PCR.

3. Appearance of non-specific amplification: The bands appearing after PCR amplification are not consistent with the expected size, either large or small, or both specific amplification bands and non-specific amplification bands appear at the same time.

(1) The primers are not fully complementary to the target sequence or the primers polymerize to form a dimer.

(2) ^Mg2+ ion concentration is too high.

(3) Annealing temperature too low.

(4) Too many PCR cycles.

(5) The quality and quantity of enzymes; some sources of enzymes tend to produce nonspecific bands while others do not, and too much enzyme can sometimes result in nonspecific amplification.

Countermeasures include: redesigning the primers if necessary. Reduce the amount of enzyme or switch to another source of enzyme. Reduce the amount of primer, increase the amount of template, and reduce the number of cycles. Increase the annealing temperature or use the two-temperature point method (denaturation at 93°C, annealing and extension at about 65°C).

4. Patchy or smeared bands appear

(1) Cause

① Excessive amount of enzyme.

② Poor quality of enzyme.

③ The dNTP concentration is too high.

④ Excessive concentration of ^Mg2+.

⑤ The annealing temperature is too low.

⑥ Caused by too many cycles.

(2) Countermeasures

① Reduce the amount of enzyme.

② Switch to another source of enzyme.

③ Reduce the concentration of dNTP.

④ Reduce the concentration of ^Mg2+ appropriately.

⑤ Increase the amount of template.

⑥ Reduce the number of cycles.

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