PCR sequencing experiment
PCR sequencing experiment
Since the introduction of dideoxynucleotide triphosphate (ddNTP) as a chain terminator by Sanger et al. (1975), the technology of DNA sequencing has developed rapidly. Currently, there are two main methods used for PCR sequencing: direct sequencing and circular sequencing.
Principle
The basic principle of direct sequencing is that the double-stranded DNA product from PCR amplification is denatured to form a single strand, and the sequencing primer is annealed to a complementary sequence on one of the template strands. The annealed primer is catalytically extended by DNA polymerase by 20 to 80 nucleotides under low-progressivity reaction conditions (e.g., low temperature and low dNTP concentration); because the reaction system is doped with radiolabeled dNTP, the newly synthesized DNA strand contains multiple radiolabels, which facilitates the generation of high radioactivity. The labeled DNA strand is catalyzed by DNA polymerase to extend under highly progressive reaction conditions, and the chain extension reaction is terminated by doping ddNTP into the reaction system. The reaction product is separated by electrophoresis and autoradiography, and then the sequence can be read.
The basic principle of the cycle sequencing method is to utilize the efficient autocycling ability of the thermal cycler to obtain amplification of the chain terminated sequence products in a linear manner, thus generating a highly visible sequence gradient. The PCR-amplified DNA is first denatured to form a single-stranded form, and a labeled primer ( 32P, biotin, or fluorescent) is annealed to a complementary sequence on one of the strands. The annealed primer undergoes a chain extension-termination reaction catalyzed by heat-resistant DNA polymerase. The resulting partial double-stranded product formed by the template strand and the extended terminated strand is denatured again in the next sequencing cycle, releasing the template strand to serve as a template for another round of the initiation reaction, while accumulating the strand termination product generated in each cycle. This cycling step is repeated 20 ~ 40 times so that the chain termination products are amplified in a linear fashion.
Operation method
cycle sequencing method
Principle
The basic principle of the cycle sequencing method is to utilize the efficient autocycling capability of the thermal cycler to obtain amplification of chain-terminated sequence products in a linear fashion, resulting in a highly visible sequence gradient. The PCR-amplified DNA is first denatured to form a single-stranded form, and a labeled primer (32P, biotin, or fluorescent) is annealed to a complementary sequence on one of the strands. The annealed primer undergoes a chain extension-termination reaction catalyzed by heat-resistant DNA polymerase. The resulting partial double-stranded product formed by the template strand and the extended terminated strand is denatured again in the next sequencing cycle, releasing the template strand to serve as a template for another round of the initiation reaction, while accumulating the strand termination product generated in each cycle. This cycling step is repeated 20 ~ 40 times so that the chain termination products are amplified in a linear fashion.
Materials and Instruments
Equipment: Move The basic process of the cycle sequencing method can be divided into the following steps: A Labeling primers. Take 10~15 pmol of sequencing primer, 50 μCi of [ γ-32P ] ATP, 5 μL of 10X kinase buffer (provided by the manufacturer), add water to 50 μL of the reaction system, mix well, and then preheat at 37 ℃ for 5 minutes. Incubate for 30 min at 37 ℃ with 10 U of T4 polynucleotide kinase diluted at 1 μL, then add 10 U of T4 polynucleotide kinase and incubate for 30 min at 37 ℃. The unadulterated [ γ-32P ]ATP was removed by gel filtration column. The labeled primers can be stored at -70 °C for more than 2 weeks. B. Preparation of dNTP/ddNTP mixture Add 2 μL of one dNTP/ddNTP mixture to each of the four microcentrifuge tubes. C. Prepare the reaction mixture: Take the appropriate DNA template, 1~2 pmol of labeled sequencing primer, 3 μL of 10X Sequencing Reaction Buffer, 5 U of Taq DNA Polymerase, add water to a total volume of 20 μL, and mix well. The quality of the sequence gradient can be improved by adding some reagents such as DMSO, Triton X-100, Tween-20 or NP-40 to the mixture and mixing thoroughly. D Cycling Reactions 4 μL of the reaction mixture was added to 4 tubes of dNTP/ddNTP mixture and placed on a thermal cycler at 94 °C for cycling. Each cycle consists of denaturation at 94 °C for 1 min, annealing at 40-60 °C for 30 s, and chain extension-termination at 72 °C for 30 s. The number of cycles is 20-40. E Terminate the reaction system. At the end of the cycle, add 4 μL of termination solution (95% formamide, 20 mmol/L ED-TA, 0.05% desert phenol blue, 0.05% xylene blue FF) to each tube and mix well. The samples can be stored at -70 °C for 2~7 d. Heat the samples at 80 °C for 3 min before electrophoresis. F. Electrophoretic Separation and Sequence Interpretation 2 ~ 3 μL of sample per lane was used for electrophoresis. Sequence interpretation is performed by autoradiography; 32P-labeled samples should be exposed overnight, and 32P-labeled samples should be exposed for 2 to 3 days. Caveat 1 In the labeling primer process, in order to obtain a high specific activity of the labeling primer, the primer, kinase and [y-] are used in the reaction system.32P] ATP in the reaction system should be maintained at an optimal level.2 It is critical to determine the optimal dNTP/ddNTP ratio, which should be used to produce a low background, highly visible sequence gradient.3 To increase the autoradiographic intensity of sequence gradients close to the primers (in the range of 1-100 bases), Mn2+to the sequencing reaction system to increase the efficiency of the DNA polymerase for ddTNP incorporation. To increase the autoradiographic intensity of the sequence ladder away from the primer (in the range of 200-400 bases), it is necessary to increase the amount of dNTP in the chain-extension-termination reaction mix, depending on the polymerase used. For more product details, please visit Aladdin Scientific website.
① PCR instrument;
② Electrophoresis instrument, etc.
Reagents:
① Template: PCR amplified DNA can be used as template, or low concentration of dNTP (10-20 μmol/L) and primers (1 mmol/L) can be used for PCR amplification of the target DNA, and then use the PCR product as template directly. concentration of DNA template: 0.01 mmol/L.
DNA template concentration: 0.01 mmol/L. Plasmid DNA templates were extracted using the Plasmid Purification Kit. For high copy number plasmids, sequencing can be performed directly from the colony. Typical amount of plasmid for sequencing reaction: 50 ~ 200 mol.
Other templates (M13, Mucilage and DNA) are prepared by conventional molecular biology methods. 10 ~ 100 mol of M13 and human DNA as templates. 50 ~ 200 mol of Mucilage DNA as templates.
② Sequencing primers. Isotope-labeled sequencing primers: ［γ-32
32
P]ATP or [γ-] ATP
33
The 5' end of the primer is isotopically labeled with ［γ-32P］ATP or ［γ-33P］ATP and T4 polyribonucleotide kinase. Concentration of primer: 0.1 - 0.2 mmol/L.
Non-isotopically labeled sequencing primers: fluorescently labeled with 4 dideoxyribonucleotides, allowing fluorescence-based dideoxy sequencing reactions.
③ DNA polymerase. It should be a heat-resistant DNA polymerase without 3'f5' exonuclease activity. For example, Taq DNA polymerase.
④ Sequencing reaction buffer is specific to the polymerase used.
⑤ Radiolabeled dNTP Depending on the polymerase used.