Chapter 1: PCR Technology
1.1 Principles of PCR
PCR (Polymerase Chain Reaction), or polymerase chain reaction, refers to the DNA polymerase catalyzed by the parent strand of DNA as a template, with a specific primer for the extension of the starting point, the addition of dNTP, Mg2+ and extension factors, amplification enhancement factors. Through denaturation, annealing and extension steps, the in vitro replication of the daughter strand is complementary to the parent strand template DNA, which can rapidly and specifically amplify any target DNA in vitro.
1.2 Classification of DNA polymerases
DNA pol is an important enzyme for cellular replication of DNA. According to thermal stability, synthesis ability, speed, fidelity and specificity, it can be classified as Taq DNA polymerase, high-fidelity DNA polymerase, hot-start DNA polymerase, direct-expansion DNA polymerase, long-fragmented DNA polymerase enzyme, fast DNA polymerase, multiple DNA polymerase and so on.
The most common of these is Taq DNA polymerase, which has polymerase activity at the 5'→3' end and exonuclease activity at the 5'→3' end, but no 3'→5' end correction activity. The most important feature of Taq is its good heat resistance, which can withstand the thermal denaturation step of PCR, making it unnecessary to add more enzyme halfway through the process. However, Taq has low fidelity because it has no proofreading activity. Its fidelity is mainly achieved by the concentration and ratio of magnesium ions and dNTPs.
High-fidelity DNA polymerase contains a polymerization center and a cleavage center, the polymerization center has a 5'→3' DNA polymerase activity, which can catalyze the synthesis of DNA along the direction of 5'→3'; the cleavage center has a 3'→5' exonuclease activity, which can carry out the repair of base mismatches. Therefore, in addition to the characteristics of Taq DNA polymerase, high-fidelity DNA polymerase also has corrective activity, responsible for excising the mismatched nucleotides, thus ensuring the accuracy of the product.
The diagram above shows how DNA polymerase works [1].
Direct PCR (Direct PCR) is a reaction that uses unpurified samples for PCR amplification and animal or plant tissues for direct amplification. Currently, Yeasen Direct PCR kits can be used for PCR amplification of raw samples such as plants, animal tissues, blood, etc. without the need for nucleic acid purification, which greatly simplifies the process of PCR experiments.
The above figure shows the Direct PCR technique.
A. Direct method: take a small amount of sample and add it directly to the PCR Master Mix for PCR identification;
B. Lysis method: after the sample is taken, add it to the lysis solution to release the genome, take a small amount of the lysis supernatant and add it to the PCR Master Mix for PCR identification.
1.3 Frequently Asked Questions and Solutions
|
Trouble |
Reason |
Solution |
|
Without amplified bands |
Electrophoresis system problems |
Troubleshoot nucleic acid dye, gel concentration, and electrophoresis buffer. |
|
Inaccurate denaturation temperature |
Is the indicated temperature of the PCR instrument consistent with the actual temperature? If the temperature is too high, the enzyme is rapidly in the first few cycles; if it is too low, the template denaturation is not complete. |
|
|
Contamination of protease and nuclease in the reaction system |
The reaction system should be heated at 95°C for 5-10 minutes before the addition of Taq enzyme. |
|
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Primer error |
Check primer specificity or redesign primers using BLAST. |
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Template contains impurities |
In particular, formaldehyde-fixed and paraffin-embedded tissues often contain formic acid, causing DNA depurination and affecting PCR results. |
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Primer deterioration and failure |
Confirm that the synthetic primers are correct , purified , or inactivated due to improper storage conditions. |
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Less amount of PCR product |
Inappropriate annealing temperature |
A gradient PCR reaction was designed to optimize the annealing temperature with a gradient of 2 degrees. |
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Presence of inhibitors in the DNA template |
Make sure the DNA template is clean. |
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Prolonged denaturation |
Prolonged denaturation leads to inactivation of DNA polymerase. |
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Extension too short |
The extension time is too short. Set the extension time on the principle of 1kb/min. |
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Amount of DNA template too low |
Increase the amount of DNA template. |
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Insufficient quantity of primers |
Increase the primer content in the system. |
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Insufficient number of PCR cycles |
Increase the number of reaction cycles. |
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Multiple bands |
Poor primer specificity |
Primer design software such as BLAST and Primer were utilized to check primer specificity or redesign primers. |
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Excessive number of loops |
Increase the amount of template appropriately , and reduce the number of cycles. |
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Exogenous DNA Contamination |
Ensure clean operation. |
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Excessive amount of templates |
The amount of plasmid DNA should be <50ng , while genomic DNA should be <200ng. |
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Too much primer |
Reduce the amount of primers in the reaction system. |
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The reaction solution is not well mixed |
Make sure the reaction buffer is completely melted and thoroughly mixed. |
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Mg High concentration |
Adjust the concentration of Mg used appropriately. |
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High enzyme dosage or poor enzyme quality |
Reduce the amount of enzyme or replace it with another source. |
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Low annealing temperature, long annealing and extension time |
Increase the annealing temperature to reduce the denaturation and extension time, or design a gradient PCR reaction to optimize the annealing temperature. |
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Problems with the PCR mix itself |
If it is PCR premix, it may be the quality of the reagent itself, it is recommended to change the batch or other brands. |
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Wrong Size |
Contamination |
Clean the bench with new reagents and tips. |
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Incorrect use of templates or primers |
Replace primers and templates. |
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Genotype |
Sequence analysis and BLAST studies were performed on the studied genes. |
Chapter 2: Nucleic Acid Electrophoresis
2.1 Principles of nucleic acid electrophoresis
The movement of charged particles under the action of an electric field towards an electrode opposite to their electrical properties is called Electrophoresis. Using the charged particles in the electric field move at different speeds and achieve separation of the technology is called electrophoresis. Nucleic acid electrophoresis is an important tool for nucleic acid research and is an integral part of techniques such as nucleic acid probes, nucleic acid amplification and sequence analysis. Nucleic acid electrophoresis is usually carried out in agarose or polyacrylamide gels. Different concentrations of agarose and polyacrylamide can form gels with different molecular sieve mesh sizes, which can be used to separate nucleic acid fragments of different molecular weights.
2.2 Agarose gel electrophoresis
Agarose is a linear polymer extracted from seaweed. Agarose is heated in a buffer solution and melted into a clear, transparent sol, which is then poured into a gelatin mold and solidifies to form a solid matrix known as a gel, the density of which depends on the concentration of agarose.
Agarose gel electrophoresis is a kind of electrophoresis method using agarose as support medium, and the main difference between the analytical principle and other support electrophoresis is that it has the dual role of "molecular sieve" and "electrophoresis". The gel is placed in the electric field, under the action of the electric field, the charged nucleic acids migrate to the positive pole through the mesh of the gel. The rate of migration is affected by the size of nucleic acid molecules, agarose concentration, applied voltage, electric field, electrophoresis buffer, and the amount of embedded dye. After appropriate time of electrophoresis under different conditions, nucleic acid fragments with different sizes and conformations will be in different positions on the gel, thus achieving the purpose of separation. The separation of agarose gel has a wide range, commonly used in DNA cutting gel recovery, DNA isolation and used to support whether the DNA is recombinant, plasmid, etc. Whether the plasmid is cut or not, different concentrations of agarose gel can be separated from the length of 200bp to 50kb of DNA fragments.
2.3 Experimental Methods
Making glue
Take 1% concentration as an example: weigh 1 g agarose, pour it into a 250 ml conical flask, add 100 ml 0.5×TBE electrophoresis buffer and mix it well, boil it in a microwave oven and then air dry it to about 60℃ , add an appropriate amount of nucleic acid dye and shake it well and then pour it into a clean gel plate that has already been prepared, take a comb with both hands to insert into gel solution vertically, and wait for gel to be cooled down naturally until it is completely solidified (25-30min).
Remove the glue making comb
Carefully transfer the gel to the electrophoresis tank (either with the gel tray or just the gel), place the sample well side in the negative pole, and add 0.5× TBE electrophoresis buffer until the gel is about 1 mm below.
Add sample
Add 10× loading buffer (loading buffer) to the DNA samples, mix well, and then slowly add the sample mixture to the submerged gel wells with a pipette gun, adding 5-10 μL of sample per well (not more than 40 μL). Generally, the first well should be filled with DNA marker, the second with positive control (defined DNA), and the third with negative control (reagent or water), and the order of the samples should be recorded.
Electrophoresis
Cover the electrophoresis tank, connect the wires, turn on the power, and set the electrophoresis voltage, current and time parameters. Generally, the voltage should not exceed 5 v/cm (the length refers to the distance between positive and negative poles of the electrophoresis tank), and the electrophoresis time is generally 15-30min.
End of electrophoresis
Remove the gel (without gel tray) and image it under the UV imaging system to get a clear electrophoretic image, then edit the electrophoretic image with the image editing software and label it with the sample information, date and operator.
2.4 Frequently Asked Questions and Solutions
|
Trouble |
Reason |
Solution |
|
Missing target band |
Small DNA runs out of gel |
Shorten the electrophoresis time, reduce the voltage , increase the gel concentration. |
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DNA bands of similar molecular weight size are not easily distinguished |
Increase the electrophoresis time to match the optimal gel concentration. |
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The target fragment is too large for conventional electrophoresis. |
Analyzed on pulsed gel electrophoresis. |
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No Purpose Clip |
The PCR process was faulty and did not amplify the target product. |
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Blurred DNA bands |
Stale electrophoresis buffer |
When electrophoresis buffer is used for many times, the ionic strength will be reduced, the PH value will rise slowly, and the flushing ability will be weakened, thus affecting the electrophoresis effect, so it is recommended to change the electrophoresis buffer frequently. |
|
DNA degradation |
Avoid nuclease contamination. |
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The electrophoresis conditions used are not suitable for electrophoresis |
electrophoresis voltage should not exceed 20V/cm and the temperature should be <30°C; the temperature of electrophoresis of huge DNA strands should be <15°C; check whether the electrophoresis buffer used has sufficient buffering capacity. |
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Excessive DNA sampling |
Reduce the amount of DNA up-sampled in the gel. |
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DNA samples are too salty |
Excess salt was removed by ethanol precipitation prior to electrophoresis. |
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protein contamination |
Phenol extraction prior to electrophoresis removes proteins. |
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Sample concentration too high |
The concentration of the upper sample should be less than 500ng/well, the concentration is too high will affect the speed of electrophoresis, running, resulting in dragging and blurring . |
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Weak or no bands |
Insufficient sample size of DNA |
Increasing the amount of DNA uptake |
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DNA degradation |
Avoiding nuclease contamination of DNA |
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Inappropriate light source for nucleic acid dyes |
Select the appropriate wavelength light source according to the instructions for the nucleic acid dye. |
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Bands non-separated |
lack of time |
Increase electrophoresis time |
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Incorrect gel concentration |
The concentration of glue is too high, resulting in too much resistance to separation. |
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Concentration of salt ions in the sample is too high |
High concentration of salt ions increases electrophoretic resistance and prevents separation of bands, e.g., digested samples containing endonuclease buffer. |
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Non-specific bands |
RNA contamination |
Re-preparation of samples |
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Contamination between samples |
Replacement of the tip during filling; avoiding spilling of samples with volumes >40 μl. |
2.5 Polyacrylamide gel electrophoresis
Polyacrylamide gels are formed by the chemical reaction between acrylamide monomer, chain polymerization catalysts N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulphate, and cross-linking agent N,N'-methylenebisacrylamide. The acrylamide monomer polymerizes in the presence of a catalyst to form long chains, which are cross-linked by a cross-linking agent to form a gel, the pore size of which is determined by the chain length and the degree of cross-linking. The pore size is determined by the chain length and the degree of cross-linking. The chain length depends on the concentration of acrylamide, and the degree of cross-linking of the polymer can be changed by adjusting the ratio of acrylamide to cross-linking agent.
Polyacrylamide gel electrophoresis can be used to separate samples based on differences in charge, molecular size, and shape of the electrophoresed samples. It combines molecular sieving and electrostatic , and has a higher resolving power than agarose gel electrophoresis. DNA fragments differing by only one nucleotide can be separated.
Polyacrylamide gel electrophoresis is used to analyze and prepare DNA fragments less than 1 kb in length. Depending on the size of the nucleic acid fragments to be isolated, gels of different can be prepared.
The effective separation ranges for different concentrations of acrylamide and DNA are shown in the table below:
|
Conc. (%) |
Effective separation range (bp) |
|
3.5 |
100 to 2000 |
|
5 |
80 to 500 |
|
8 |
60 to 400 |
|
12 |
40 to 200 |
|
15 |
25 to 150 |
|
20 |
10 to 100 |
2.4 Guidelines for the selection of related products
Conventional PCR |
||||
| Specification | 10167ES | 10102ES | 10103ES | 10108ES |
| Amplification Length | ≤10-15 kb | ≤5 kb | ≤5 kb | ≤4 kb |
| Extension Time | 1-10 sec/kb | 30 sec/kb | 30 sec/kb | 30 sec/kb |
| Product End Structure | 3’-dA | 3’-dA | 3’-dA | 3’-dA |
| Annealing Temperature | 60℃ | Tm-(2~5)℃ | Tm-(2~5)℃ | Tm-(2~5)℃ |
| GC Compatibility Range | 30-70% | 40-70% | 40-70% | 30-70% |
| 5'-3' Exonuclease Activity | Present | Present | Present | Present |
| Colony PCR | Suitable | Suitable | Suitable | Suitable |
| Gene Identification | Suitable | Suitable | Suitable | Suitable |
| Multiplex PCR | Not Suitable | Not Suitable | Not Suitable | 3-4 plex PCR |
| Electrophoresis Indicator | Purple-red | Blue | Colorless | Blue |
| Hot Start | Hot Start | Not Hot Start | Not Hot Start | Hot Start |
| Pre-mix/Kit | Pre-mix | Pre-mix | Pre-mix | Pre-mix |
Direct PCR |
||||
| Specification | 10185ES | 10188ES | 10187ES | |
| Product Type | Mouse Direct Amplification | Blood Direct Amplification | Plant Direct Amplification | |
| Amplification Length | ≤1 kb | ≤8 kb | ≤1 kb | |
| Extension Time | 30 s/kb | 3-5 s/kb for ≤2 kb, | 60 s/kb | |
| 10 s/kb for ≤8 kb | ||||
| Sample lysis time | 15 min | 0-3 min | 0-10 min | |
| Product End Structure | Blunt End | Blunt End | Blunt End | |
| Annealing Temperature | Tm-(2~5)℃ | Tm-(1~2)℃ | Tm-(2~5)℃ | |
| GC Compatibility Range | 30-70% | 30-75% | 40-65% | |
| 5'-3' Exonuclease Activity | √ | √ | √ | |
| Gene Identification | √ | √ | √ | |
| Multiplex PCR | 3-4 plex | |||
| Direct Sample Amplification | √ | √ | √ | |
| Electrophoresis Indicator | Blue | Colorless | Blue | |
| Hot Start | √ | √ | √ | |
| Pre-mix/Kit | Kit (with Mix) | Kit (with Mix + Buffer) | Kit (with Mix) | |
| Suitable Organisms | Mouse, Rat | Human, Mouse, Goat, Chicken, Pig, etc. | Rice, Corn, Tobacco, Rapeseed, Wheat, Soybean, etc. | |
| Suitable Tissue/Material | Tail, Ear, Toe (with muscle), and other organs | Fresh blood with EDTA, Heparin, Citrate, etc., refrigerated (frozen) blood, commercial dry blood spots | Young leaves, old leaves, seedlings, young stems | |
| Sample input | Tissue: 5-10 mg; Tail: 1-5 mm | Whole blood: 0.5%-20%, dry blood spot: 1 mm² | Leaf: 1-10 mm, Seed: 1-3 mm | |
High-Fidelity PCR |
||||
| Specification | 10164ES | 10153ES | 10154ES | |
| Amplification Length | ≤10 kb gDNA, ≤10 kb cDNA, ≤16 kb λDNA | ≤10 kb gDNA, ≤10 kb cDNA, ≤13 kb λDNA | ≤10 kb gDNA, ≤10 kb cDNA, ≤13 kb λDNA | |
| Extension Time | 5 sec/kb | 30 sec/kb | 30 sec/kb | |
| Fidelity (Taq) | 83× | 83× | 83× | |
| Product End Structure | Blunt End | Blunt End | Blunt End | |
| Annealing Temperature | 60℃ | 68℃ | 68℃ | |
| GC Compatibility Range | 20-80% | 30-60% | 30-60% | |
| 5’-3’ Exonuclease Activity | Absent | Absent | Absent | |
| Electrophoresis Indicator | Blue | Colorless | Blue | |
| Single Enzyme/Pre-mix | Pre-mix | Single Enzyme | Pre-mix | |
| Tolerance | / | / | Blood, Mouse Tissue Lysate | |
Nucleic Acid Electrophoresis |
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| Product Category | Cat NO. | Product Name | Application | |
| Agarose | 10208ES | Agarose | Routine nucleic acid electrophoresis | |
| 10221ES | High Sieving Agarose (PCR Grade) | Suitable for separating DNA fragments of 20 bp-800 bp, comparable to polyacrylamide gel | ||
| 10226ES | Agarose Tablets (0.5 g/tablet) | Convenient for routine agarose applications | ||
| Nucleic Acid Dye | 10202ES | YeaRed Nucleic Acid Gel Stain (10,000× in Water) | Water-soluble, with spectral properties similar to EB, excitable at 300 nm UV light | |
| DNA Marker | 10510ES | GoldBand 1 kb DNA Ladder | 250-12000 bp (13 bands) | |
| 10515ES | GoldBand 50 bp DNA Ladder | 50-1000 bp (14 bands) | ||
| 10507ES | GoldBand 100bp DNA Ladder | 100-1500 bp (12 bands) | ||
| 10516ES | GoldBand 100 bp plus DNA Ladder | 100-3000 bp (14 bands) | ||
| 10517ES | GoldBand 200 bp DNA Ladder | 200-5000 bp (12 bands) | ||
| 10518ES | GoldBand 500 bp DNA Ladder | 500-5000 bp (8 bands) | ||
| 10501ES | GoldBand DL2000 DNA Marker | 100-2000 bp (6 bands) | ||
| 10504ES | GoldBand DL5000 DNA Marker | 100-5000 bp (9 bands) | ||
| 10505ES | GoldBand DL10,000 DNA Marker | 100-10000 bp (10 bands) | ||
| 10511ES | GoldBand Full-Scale DNA Ladder | 100-12000 bp (20 bands) | ||
| 10512ES | GoldBand DL15000 DNA Marker | 250-15000 bp (7 bands) |
2.5 Publications using Nucleic Acid Electrophoresis Products (Partial)
[1] Luo J, Yang Q, Zhang X, et al. TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile. Cell. 2022;185(6):980-994. e15. doi:10.1016/j.cell.2022.02.010 (IF:41.584)
[2] Huang N, Chen H, Gong H, et al. SeHed, a novel gene expression system with stress-evoked hydrogen perox ide elimination property and anti-aging e ffect. signal Transduction and Targeted Therapy. 2022, 7, 235. doi: 10.1038/s41392-022-01047-2. (IF: 38.1)
[3] Zhang C, Zhou B, Gu F, et al. Micropeptide PACMP inhibition elicits synthetic lethal effects by decreasing CtIP and poly(ADP-ribosyl) ation. mol Cell. 2022;82(7):1297-1312.e8. doi:10.1016/j.molcel.2022.01.020 (IF:17.970)
[4] Zhu M, DaiX. Growth suppression by altered (p)ppGpp levels results from non-optimal resource allocation in Escherichia coli. Nucleic Acids Res. 2019;47(9):4684-4693. doi:10.1093/nar/gkz211 (IF:11.147)
[5] Rizwan HM,Zhimin L, HarsonowatiW, et al. Identification of Fungal Pathogens to Control Postharvest Passion Fruit (Passiflora edulis) Decays and Multi-Omics Comparative Pathway Analysis Reveals Purple Is More Resista nt to Pathogens than a Yellow Cultivar. j Fungi (Basel). 2021;7(10):879. Published 2021 Oct 19. doi:10.3390/jof 7100879 (IF:5.816)
[6] LiT, Zhou B, Luo Z, et al. Structural Characterization of a Neutralizing Nanobody With Broad Activity Against SARS-CoV-2 Variants. Front Microbiol. 2022;13:875840. Published 2022 Jun2. doi:10.3389/fmicb.2022.875840 (IF:5.640)
[7] Wang T, Ren D, Guo H, et al. CgSCD1 Is Essential for Melanin Biosynthesis and Pathogenicity of Colletotrichum gloeosporioides. pathogens. 2020;9(2) :141. published 2020 Feb 20. doi:10.3390/pathogens9020141(IF:3.018) :141. Published 2020 Feb 20. doi:10.3390/pathogens9020141 (IF:3.018)
[8] Lv Y, LiX, Zhang H, Zou F, Shen B. CircRNA expression profiles in deltamethrin-susceptible and -resistant
pipiens pallens (Diptera: Culicidae). Comp Biochem Physiol B Biochem Mol Biol. 2022;261:110750. doi:10.1016 /j.cbpb.2022.110750 (IF:2.231)
[9] Hu M, DaiX. Growth suppression by altered (p) ppGpp levels results from non-optimal resource allocation in Escherichia coli[J]. Nucleic acids research, 2019, 47(9): 4684-4693.(IF11.6)
[10] Guo L, Yang W, Huang Q, et al. Selenocysteine-specific mass spectrometry reveals tissue-distinct selenopro teomes and candidate selenoproteins[J ]. Cell chemical biology, 2018, 25(11): 1380-1388. e4. (IF6.762)
2.6 Publications using PCR Products (Partial)
[1] Wang Y, Fu Z, LiX, et al. Cytoplasmic DNA sensing by KU complex in aged CD4+ T cell potentiates T cell activa tion and aging-related autoimmune inflammation. Immunity. 2021;54(4):632-647.e9. doi:10.1016/j.immu
ni.2021.02.003(IF:31.745)
[2] Yang X, Gao F, Zhang W, et al. "Star" miR-34a and CXCR4 antagonist based nanoplex for binary cooperative migration treatment against metastatic breast cancer. J Control Release. 2020;326:615-627. doi:10.1016/j. jconrel.2020.07.029(IF:7.727)
[3] QiaoY, Du J, Ge R, et al. A Sample and Detection Microneedle Patch for Psoriasis MicroRNA Biomarker
Analysis in Interstitial Fluid. Anal Chem. 2022;94(14):5538-5545. doi:10.1021/acs.analchem.1c04401(IF:6.986)
[4] Lin Q,Ye X, Huang Z, et al. Graphene Oxide-Based Suppression of Nonspecificity in Loop-Mediated Isother malAmplification Enabling the Sensitive Detection of Cyclooxygenase-2 mRNA in Colorectal Cancer. Anal Chem. 2019;91(24):15694-15702. doi:10.1021/acs.analchem.9b03861(IF:6.350)
[5] Lin Q, Huang Z,Ye X, et al. Lab in a tube: Isolation, extraction, and isothermal amplification detection of exosomal long noncoding RNA of gastric cancer. Talanta. 2021;225:122090.
doi:10.1016/j.tta.2021.122090(IF:6.057)
[6] Liu C, Zou G, et al. 5-Formyluracil as a Multifunctional Building Block in Biosensor Designs[J]. Angew Chem Int Ed Engl. 2018 Jul 26;57(31):9689-9693.（IF 11.992）
[7] Wang M, Zhang S, Zheng G, et al. Gain-of-Function Mutation of Card14 Leads to Spontaneous Psoriasis-like Skin Inflammation through Enhanced Keratinocyte Response to IL-17A. Immunity. 2018;49(1):66-79.e5.
doi:10.1016/j.immuni.2018.05.012(IF:19.734)
[8] Zhang Y, Ding H, Wang X, et al. MK2 promotes Tfcp2l1 degradation via β-TrCP ubiquitin ligase to regulate mouse embryonic stem cell self-renewal. Cell Rep. 2021;37(5):109949. doi:10.1016/j.celrep.2021.109949 (IF:9.423)
[9] Lin Q,Ye X, Yang B, et al. Real-time fluorescence loop-mediated isothermal amplification assay for rapid and sensitive detection of Streptococcus gallolyticus subsp. gallolyticus associated with colorectal cancer[J].
Analytical and bioanalytical chemistry, 2019, 411(26): 6877-6887.（IF6.35）
[10] Lin Q,Ye X, Huang Z, et al. Graphene Oxide-Based Suppression of Nonspecificity in Loop-Mediated Isother malAmplification Enabling Sensitive Detection of Cyclooxygenase-2 mRNA in Colorectal Cancer[J]. Analyti cal chemistry, 2019.（IF6.35）
Citiation:
[1] Loeb LA, Jr R. DNA polymerases and human disease[J]. Nature Reviews Genetics.