PCR considerations
Electrophoretic detection time of PCR products
Generally, it is within 48 hours, and some are best detected on the same day. After more than 48 hours, the band irregularity disappears.
False negative, no amplification bands
The key steps of the PCR reaction are 1 template nucleic acid preparation, 2 primer quality and specificity, 3 enzyme quality and 4 PCR cycle conditions. The reason for finding the problem should also be analyzed and researched on the above links.
Template: 1 template contains heteroprotein, 2 template contains Taq enzyme inhibitor, protein in 3 template is not digested, especially histone in chromosome, 4 is excessively lost when extracting template, or inhaled phenol. 5 template nucleic acid degeneration is not complete. When the quality of the enzyme and the primer is good, there is no amplification band, and it is very likely that the sample is digested, and the template nucleic acid extraction process is out of order. Therefore, an effective and stable digestion treatment solution should be prepared, and the procedure should be fixed and should not be changed at will. .
Enzyme inactivation: new enzymes need to be replaced, or both old and new enzymes should be used simultaneously to analyze whether false negatives are caused by loss or insufficient enzyme activity. It should be noted that sometimes Taq enzyme or ethidium bromide is forgotten.
Primers: Primer quality, primer concentration, and the concentration of the two primers are symmetrical, which is a common cause of PCR failure or unsatisfactory expansion of the band. Some batches have problems with the quality of primer synthesis. Two primers have a high concentration and a low concentration, resulting in low-efficiency asymmetric amplification. The countermeasures are: 1 Select a good primer synthesis unit. 2 The concentration of the primer should not only look at the OD value, but also pay attention to the primer solution for agarose gel electrophoresis. There must be a primer band, and the brightness of the two primer bands should be roughly the same, such as a primer with a band and a primer without Strips, PCR may fail at this time, and should be resolved in consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance the concentration when diluting the primer. 3 Primers should be stored in high concentration and small amount to prevent multiple freeze-thaw cycles or long-term storage of refrigerators, resulting in failure of primer degradation. 4 Primer design is not reasonable, such as the length of the primer is not enough, the formation of dimers between the primers.
Mg2+ concentration: Mg2+ ion concentration has a great influence on PCR amplification efficiency. Too high concentration can reduce the specificity of PCR amplification. If the concentration is too low, it will affect the PCR amplification yield and even the PCR amplification will not be amplified.
Change in reaction volume: The volume used for PCR amplification is usually 20 ul, 30 ul, 50 ul. Or 100ul, the application of large volume for PCR amplification, is set according to the different purposes of scientific research and clinical testing. After making a small volume such as 20ul, and then making a large volume, it is necessary to mold the condition, otherwise it is easy to fail.
Physical reasons: Denaturation is very important for PCR amplification, such as low denaturation temperature, short denaturation time, and highly likely false negatives; annealing temperature is too low, which can cause non-specific amplification and reduce specific amplification efficiency. High-intensity primers bind to the template to reduce PCR amplification efficiency. It is sometimes necessary to use a standard thermometer to detect denaturation, annealing and extension temperatures in the instrument or water bath, which is one of the reasons for PCR failure.
Target sequence variation: If the target sequence is mutated or deleted, it affects the specific binding of the primer to the template, or the primer and the template lose complementary sequence due to a certain deletion of the target sequence, and the PCR amplification is not successful.
False positive
The PCR amplification bands appearing are consistent with the target target sequence bands, and sometimes the bands are more tidy and the brightness is higher.
Primer design is not suitable: the selected amplified sequence has homology with the non-target amplified sequence, so when PCR amplification is performed, the amplified PCR product is a non-target sequence. The target sequence is too short or the primer is too short to be prone to false positives. Primers need to be redesigned.
Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: one is cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be resolved by the following methods: Care should be taken to prevent the target sequence from being drawn into the sample gun or spilled out of the centrifuge tube. All reagents or equipment should be autoclaved except for enzymes and substances that cannot withstand high temperatures. The centrifuge tube and sample tip used should be used at one time. If necessary, the reaction tubes and reagents are irradiated with ultraviolet light to destroy the nucleic acid present before the specimen is added. The second is the contamination of small fragments of nucleic acids in the air. These small fragments are shorter than the target sequences, but have some homology. The splicing can be spliced ​​to each other, and after complementing the primers, the PCR product can be amplified, resulting in the generation of false positives, which can be alleviated or eliminated by nested PCR.
Non-specific amplification band
The bands appearing after PCR amplification are inconsistent with the expected size, either large or small, or both specific amplification bands and non-specific amplification bands. The occurrence of non-specific bands is due to the fact that the primers are not completely complementary to the target sequence or the primers polymerize to form a dimer. Second, the Mg2+ ion concentration is too high, the annealing temperature is too low, and the number of PCR cycles is too high. The second is the quality and quantity of the enzyme. Sometimes the enzymes of some sources are prone to non-specific bands and the enzymes of another source do not. Excessive amounts of enzymes sometimes lead to non-specific amplification. The countermeasures are: redesigning the primers if necessary. Reduce the amount of enzyme or exchange enzymes from another source. Reduce the amount of primers, increase the amount of template, and reduce the number of cycles. Appropriately increase the annealing temperature or use two temperature point method (denaturation at 93 ° C, annealing and extension at around 65 ° C).
a sheet-like tow or smear
PCR stripping sometimes occurs with a smear strip or a strip or carpet strip. The reason is often due to the excessive amount of enzyme or the poor quality of the enzyme, the dNTP concentration is too high, the Mg2+ concentration is too high, the annealing temperature is too low, and the number of cycles is too high. The countermeasures are: reducing the amount of enzyme, or transposing an enzyme from another source. 2 reduce the concentration of dNTP. Appropriately reduce the Mg2+ concentration. Increase the amount of templates and reduce the number of cycles.
Cloning PCR products 1) What are the optimal conditions for cloning PCR products?
Optimal insert: The vector is determined experimentally. 1:1 (insert: carrier) is often the best ratio, and the molar ratio is 1:8 or 8:1. The ratio range should be determined. The ligation was carried out with 5 ul of 2X ligation solution, 50 ng of plasmid DNA, 1 Weiss unit of T4 ligase, and a total of 10 ul of the insert. Incubate for 1 hour at room temperature or overnight at 4 °C. At these two temperatures, the carrier lacking the T-bulk will self-ligate and produce a blue spot. Incubation at room temperature for 1 hour can meet most of the cloning requirements. To improve the efficiency of the connection, it is necessary to overnight at 4 °C.
2) Does the PCR product need to be purified by gel?
For example, the gel analysis of the amplified product has only one band and does not require gel purification. If other bands are visible, it may be a dimer that accumulates a large number of primers. The small number of primer dimers is also high in moles, which results in a high proportion of clones with primer dimers, rather than the insert of interest. For this purpose, gel purification is required prior to cloning.
3) What kind of control experiment is needed if the target fragment is not recovered?
A) Coating untransformed competent cells.
If there are colonies, it indicates that the ampicillin has failed, or the plasmid with the ampicillin-resistant type is contaminated, or the colony that produces the ampicillin-resistant type.
B) Transformation of the intact plasmid, calculation of the number of colony growth, and determination of transformation efficiency.
For example, 1 ug/ul plasmid was diluted 1:100 and 1 ul was used for 100 ul of competent cell transformation. After diluting to 1000 ul with SOC, plate with 100 ul. Incubate overnight to produce 1000 colonies. Conversion rate: Total number of colonies produced / total amount of plated DNA.
The total amount of plating DNA is the amount used for the conversion reaction divided by the dilution factor. Specifically, 10 ng of DNA was used for transformation, and 10 ng of DNA was diluted with SOC to 1000 u, and plated with 1/10 to share 1 ng of DNA. Conversion rate:
1000 clone X10 (3th power) ng / planking 1 ng DNA ug=10 (6th power) cfu/ ug
Transformation of pGEM-T application 10 (8th power) cfu/ ug competent cells
If there are no colonies or few colonies, the conversion rate of competent cells is too low.
C) If a positive control of pGEM-T, or a PCR product, is used to generate >20-40 plaques (using a specified step 10 (8th power) cfu/ug competent cells), indicating that the vector has lost T. It may be that the ligase contaminates the nuclease. T4 DNA ligase (M1801, M1804, M1794) has good quality standards without nuclease contamination and is not replaced with other sources of T4 DNA ligase.
D) Using pGEM-T or pGEM-T Easy vector, connect pGEM-T positive control, transform high frequency competent cells (10 (8th power) cfu / ug), according to the specified experimental procedure, can get 100 colonies, 60% of them should be white spots, such as producing >20-40 blue spots, no colonies or few colonies, and there is a problem with the connection.
4) The results of the control experiment are good, but the target fragment is not recovered. What problems have been tested?
A) The connection is incubated for 1 hour at room temperature to satisfy most clones. To increase efficiency, 4 °C is required overnight.
B) The insert is contaminated, causing the 3'-T to be deleted, or inhibiting the junction, inhibiting transformation. To do this, the insert was mixed with the pGEM-T positive control and ligated. If the number of colonies in the control is reduced, the insert is purified or re-prepared. If a large amount of blue spots are produced, the insert is contaminated with nuclease, and the pGEM-T or pGEM-T Easy vector 3'-T is deleted.
C) The insert is not suitable for connection. Gel-purified inserts sometimes occur due to excessive UV exposure. UV over-irradiation produces pyrimidine dimers that are not conducive to ligation and the DNA must be repurified.
D) The amplified product of the thermostable DNA polymerase with repair function has no A at the end, and the latter is required for pGEM-T or pGEM-T Easy vector cloning. Add Taq DNA polymerase and nucleotides to add A to the end. For details, check the pGEM-T pGEM-T Easy carrier technical data (TM042).
E) Highly repetitive sequences may be unstable, causing deletions and rearrangements in amplification. If the insert is found to be highly frequent in deletion and rearrangement, recombinant defective E. coli strains such as SURE cells are required.
PCR reaction system and reaction conditions
Standard PCR reaction system:
10× amplification buffer 10ul
4 kinds of dNTP mixture each 200umol/L
Primers 10 to 100 pmol each
Template DNA 0.1~2ug
Taq DNA Polymerase 2.5u
Mg2+ 1.5mmol/L
Add double or triple distilled water to 100ul
Five elements of the PCR reaction: There are five main substances involved in the PCR reaction, namely primers, enzymes, dNTPs, templates, and Mg2+.
Primers: Primers are the key to PCR-specific reactions, and the specificity of the PCR product depends on the extent to which the primer is complementary to the template DNA. In theory, as long as any template DNA sequence is known, the complementary oligonucleotide strands can be used as primers, and the template DNA can be amplified in vitro by PCR.
Primers should be designed to follow the following principles:
1 Primer length: 15-30bp, usually about 20bp.
2 Primer amplification span: 200-500 bp is appropriate, and fragments up to 10 kb can be amplified under specific conditions.
3 primer base: G + C content is 40-60% is appropriate, G + C too little amplification effect is not good, G + C too much is prone to non-specific bands. ATGC is preferably randomly distributed to avoid the string arrangement of more than 5 purine or pyrimidine nucleotides.
4 Avoid secondary structure inside the primer to avoid complementation between the two primers, especially the 3' end, otherwise a primer dimer will be formed, resulting in a non-specific amplification band.
The bases at the 3' end of the 5 primers, especially the last and second to last bases, should be strictly matched to avoid PCR failure due to terminal base mismatch.
The 6 primers may or may be added with a suitable restriction site, and the target sequence to be amplified preferably has a suitable restriction site, which is advantageous for digestion analysis or molecular cloning.
7 Primer specificity: Primers should have no significant homology to other sequences in the nucleic acid sequence database. Primer amount: The concentration of each primer is 0.1~1umol or 10-100pmol. It is better to produce the desired result with the lowest primer amount. The high primer concentration will cause mismatch and non-specific amplification, and increase the formation of primers. The opportunity for the polymer.
Enzymes and their concentrations are currently supplied by two Taq DNA polymerases, one is a natural enzyme purified from the genus Bacillus, and the other is a genetically engineered enzyme synthesized by Escherichia coli. Catalyzing a typical PCR reaction requires about 2.5 U of enzyme (meaning that the total reaction volume is 100 ul). If the concentration is too high, it can cause non-specific amplification. If the concentration is too low, the amount of synthetic product will decrease.
The quality of dNTP is closely related to the concentration and concentration of dNTP and the efficiency of PCR amplification. The dNTP powder is granular, and loses its biological activity if it is improperly preserved. The dNTP solution is acidic and should be formulated to a high concentration with 1M NaOH or 1M Tris. The pH of the HCL buffer is adjusted to 7.0-7.5, and the small amount is dispensed and stored at -20 °C. Multiple freeze-thaw cycles will degrade dNTP. In the PCR reaction, the dNTP should be 50 ~ 200umol / L, especially pay attention to the concentration of the four dNTPs are equal (equal molar preparation), such as when any one of the concentrations is different from the other (higher or lower), It will cause a mismatch. Too low a concentration will reduce the yield of PCR products. dNTP can bind to Mg2+ to reduce the concentration of free Mg2+.
The amount and purification degree of the template (target gene) nucleic acid template nucleic acid is one of the key steps in the success or failure of PCR. Traditional DNA purification methods usually use SDS and proteinase K to digest the specimen. The main functions of SDS are: lysing lipids and proteins on the cell membrane, thus dissolving membrane proteins to destroy cell membranes and dissociating nuclear proteins in cells. SDS can also bind to proteins and precipitate; proteinase K can hydrolyze and digest proteins, especially The histone bound to the DNA is extracted with the organic solvent phenol and chloroform to extract the protein and other cellular components, and the nucleic acid is precipitated with ethanol or isopropanol. The extracted nucleic acid can be used as a template for the PCR reaction. For general clinical test specimens, the cells can be lysed by a quick and simple method, the pathogen is lysed, the protein of the chromosome is digested and the target gene is released, and it is directly used for PCR amplification. RNA template extraction is generally performed using guanidinium isothiocyanate or proteinase K to prevent RNase from degrading RNA.
The Mg2+ concentration of Mg2+ has a significant effect on the specificity and yield of PCR amplification. In the general PCR reaction, when the concentration of various dNTPs is 200umol/L, the concentration of Mg2+ is preferably 1.5-2.0mmol/L. When the concentration of Mg2+ is too high, the specificity of the reaction is lowered, and non-specific amplification occurs. If the concentration is too low, the activity of Taq DNA polymerase is lowered, and the reaction product is reduced.
Selection of PCR reaction conditions
The PCR reaction conditions are temperature, time, and number of cycles.
Temperature and time setting: Three temperature points of denaturation-annealing-extension are set based on the three steps of the PCR principle. In the standard reaction, the three-temperature method is used, the double-stranded DNA is denatured at 90-95 ° C, and then rapidly cooled to 40-60 ° C. The primer is annealed and bound to the target sequence, and then rapidly heated to 70-75 ° C in Taq DNA. The primer strand is extended along the template by the action of the polymerase. For shorter target genes (100-300 bp in length), a two-temperature method can be used. Except for the denaturation temperature, the annealing and extension temperatures can be combined into one. Generally, the denaturation at 94 °C is used, and the annealing and extension are performed at about 65 °C. Taq DNase still has high catalytic activity).
1 Denaturation temperature and time: The denaturation temperature is low, and the incomplete melting is the most important cause of PCR failure. In general, 93 ° C ~ 94 ° Cmin is enough to denature the template DNA, if it is lower than 93 ° C, it needs to extend the time, but the temperature can not be too high, because the high temperature environment has an effect on the activity of the enzyme. If this step does not completely denature the target gene template or PCR product, PCR will fail.
2 annealing (refolding) temperature and time: annealing temperature is an important factor affecting PCR specificity. After the denaturation, the temperature is rapidly cooled to 40 ° C ~ 60 ° C, which can be combined with the template. Since the template DNA is much more complex than the primer, the chance of collisional binding between the primer and the template is much higher than the collision between the complementary strands of the template. The annealing temperature and time depend on the length of the primer, the base composition and its concentration, and the length of the target sequence. For 20 nucleotides, a primer with a G+C content of about 50%, 55 ° C is preferred as the starting point for selecting the optimum annealing temperature. The renaturation temperature of the primers can be used to help select the appropriate temperature by the following formula:
Tm value (melting temperature) = 4 (G + C) + 2 (A + T)
Refolding temperature = Tm value - (5 ~ 10 ° C)
Selecting a higher renaturation temperature within the allowable range of Tm values ​​can greatly reduce non-specific binding between the primer and the template, and increase the specificity of the PCR reaction. The renaturation time is generally 30 to 60 sec, which is sufficient to completely bind the primer to the template.
3 extension temperature and time: biological activity of Taq DNA polymerase:
70～80°C 150 nucleotides/S/enzyme molecule
70 ° C 60 nucleotide / S / enzyme molecule
55 ° C 24 nucleotide / S / enzyme molecule
Above 90 ° C, DNA synthesis is almost impossible.
The extension temperature of the PCR reaction is generally selected between 70 and 75 ° C, and the usual temperature is 72 ° C. The excessive extension temperature is not conducive to the combination of the primer and the template. The time of the PCR extension reaction may depend on the length of the fragment to be amplified, and generally a DNA fragment within 1 Kb, and an extension time of 1 min is sufficient. The target sequence of 3 to 4 kb takes 3 to 4 minutes; the amplification of 10 Kb needs to be extended to 15 minutes. Excessive elongation can lead to the appearance of non-specific amplification bands. For the amplification of low concentration template, the extension time is slightly longer.
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