Zea31085
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 Posted: Sat 7:15, 26 Mar 2011 Post subject: |
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PCR (Polymerase Chain Reaction), referred to as PCR, is a molecular biology technique used to amplify specific DNA fragments. Can be regarded as a special DNA replication in vitro. [overview] DNA polymerase (DNApolymerase I) was first discovered in 1955, while the more practical with the experimental value and the Klenow fragment of E. Coli is in the early 70s by the Dr. H. Klenow found However, as this enzyme is not high temperature, high temperature can denature, so do not make use of the heat denaturation of the polymerase chain reaction. Enzyme used in the present (the Taq polymerase), is in 1976 from the hot springs bacteria (Thermusaquaticus) isolated. It lies to the characteristics of high temperature, is an ideal enzyme, but it is widely used in the 80's after. The original concept of PCR is similar to the original prototype gene repair replication, it was in 1971 by Dr. Kjell Kleppe proposed. He made the first simple and transient DNA replication (similar to the first two cycles PCR reaction) experiment. And now is the developed PCR in 1983 by the Dr. Kary B. Mullis developed by, Dr. Mullis PE year service company, so PE sector in PCR has a special status. Dr. Mullis and Saiki in 1985, who published his first formal paper related. Thereafter, PCR of the use of rapid, quality of related papers published can be said of many other research methods that come anywhere close. Followed by PCR in biological research and clinical applications can be widely used as the most important molecular biology techniques. Mullis also won the 1993 Nobel Prize in Chemistry. [PCR principle] DNA semi-conservative replication of biological evolution and an important way to passage. Double-stranded DNA in a variety of enzymes may be denatured into single-stranded helicase, in the participation of DNA polymerase, according to the principle of complementary base pairing of two elements copied into the same shell collapse. Found in experiments, DNA can also occur at high temperature denaturation solution chain, when the temperature decreases and then you can become a double-stranded renaturation. Therefore, temperature control by DNA denaturation and renaturation on the design of primers, DNA polymerase, dNTP to complete in vitro replication of specific genes. However, DNA polymerase inactivation in the high temperature will, therefore, each cycle had to add new DNA polymerase, not only the operation cumbersome, and expensive, which restricts the use and development of PCR technology. Found that heat-resistant DNA polymerase - Taq enzyme for PCR applications include landmark, the enzyme can tolerate temperatures above 90 ℃ and yet live, not every cycle enzyme, the PCR technology has become very simple, but also greatly reduce the cost, PCR technology can be a large number of applications, and gradually used in clinical. PCR technology is similar to the basic principles of the natural DNA replication process, and its specificity depends on both ends of the target sequence complementary oligonucleotide primers. PCR by the degeneration - annealing - extension of three basic reaction steps constitute: ① template DNA denaturation: template DNA by heating to around 93 ℃, after a certain time, so that the template DNA to form double-stranded or double-stranded by PCR amplification of DNA solution away, making a single chain so that it primer binding, for the next round of reactions to prepare; ② template DNA, primer annealing (recovery of): template DNA by heat denaturation to single chain, the temperature dropped to 55 ℃ or so, primers and template DNA single strand pairing with complementary sequences; ③ extension primer: DNA template - primer conjugates under the action of the TaqDNA polymerase to dNTP as the reaction raw material, the target sequence as a template by complementary base pairing and a half Copy retained the principle of synthesis of a new chain with the template DNA chain complementary to semiconservative replication, duplication cycle variability - annealing - the process can be extended three more become a template for the next cycle. Required to complete a cycle every 2 to 4 minutes, 2 to 3 hours will be able to expand the target gene to be amplified several million times magnification. [PCR reaction system and reaction conditions] 1. Standard PCR amplification reaction buffer 10 × 10μl
4 种 dNTP mixture of 200μl
primer 10 ~ 100μl
template DNA0.1 ~ 2μg
Taq DNA polymerase 2.5 μl
Mg2 +1.5 mmol / L
with double or triple distilled water 100 μl
PCR Reaction five elements: participation in PCR reaction substances are five, namely: primers (PCR primers for the DNA fragments, cell DNA replication primer for the period of RNA chain), enzyme, dNTP, template and buffer solution (which requires Mg2 +). There are many design primer, and PCR The purpose of the decision in the experiment. However, the same basic principles of PCR enzyme used in two main sources: Taq and Pfu. Macrophages from two different thermophilic bacteria. Which Taq amplification efficiency but prone to mismatch. Pfu amplification efficiency but weak error correction. So the actual time needed to be done using different choices template for amplification with the DNA, can be any source, but there are two principles, the first purity to be higher, and the second is not too high so as not to inhibit the concentration of the composition of most buffer complex than water generally consists of four active ingredients: buffer system, the general use of HEPES or MOPS buffer system; a cation, generally use potassium ions, but in exceptional circumstances can also use the ammonium ion; divalent cations, namely, Mg ion, according to reaction system to determine, except in special circumstances without adjustment; auxiliary components, a common DMSO, glycerol, etc., mainly used to help maintain the enzyme activity and structure of DNA contact with winding 2, PCR primer design PCR reaction with two primers, that is, 5 'primer and 3' primers. Designed primers to a DNA single strand as a benchmark (often the information chain as the base), 5 'primer to be amplified in the 5' end of a short DNA sequence upstream of the same; 3 'primers and be amplified in 3 'end of a short DNA sequences complementary. The basic principle of primer design primers ① length :15-30bp, commonly used for 20bp or so. ② primer bases: G + C content of 40-60% is appropriate, G + C poor little amplification, G + C too prone to non-specific band. ATGC best random distribution, to avoid more than 5 purine or pyrimidine nucleotides arranged in clusters. ③ primers should not appear within the complementary sequence. ④ should not exist between the two primers complementary sequence, in particular to avoid the 3 'end of the complementary overlap. ⑤ non-specific amplification primers and homology region no more than 70% of the primer 3 'end of the 8 consecutive bases outside the amplified region does not have to be completely complementary sequence, otherwise easily lead to non-specific amplification. ⑥ primer 3 'end of the base, in particular the last and penultimate bases, should be strictly requires pairing, the best option is to G and C. ⑦ primer 5 'end can be modified. Additional restriction enzyme sites such as the introduction of mutations, with biotin, fluorescent substances, digoxin tag, add other short sequence, including the initiation codon, stop codon and so on. v primer design software Primer Premier5.0 (automatic search) * vOligo6 (primer evaluation) vVector NTI Suit vDNAsis vOmiga vDNAstar vPrimer3 (online services) 3, the template can be prepared by PCR template DNA, can also be RNA. Derived mainly based on PCR template amplified objects, specimens can be pathogens such as viruses, bacteria, fungi and so on. Can also be a pathological specimens such as biological cells, blood, amniotic fluid cells. Forensic specimens have blood spots, fine spot, hair and so on. The basic requirements of sample preparation is to remove impurities, and partially purified samples of nucleic acid. Most samples need to go through SDS and proteinase K treatment. Hard broken bacteria, lysozyme plus EDTA treatment available. The resulting crude DNA, after phenol and chloroform extraction and purification, ethanol precipitation and then used for PCR reaction template. 4, PCR reaction conditions ① PCR reaction buffer to provide a suitable pH and the concentration of certain ions ② total magnesium concentration of dNTPs should be higher than the commonly used 1.5mmol / L ③ dNTP substrate concentration to molarity preparation, 20 ~ 200umol / L ④ TaqDNA polymerase 2.5U (100ul) ⑤ primer concentration is generally 0.1 ~ 0.5umol / L ⑥ reaction temperature and denaturation temperature and time cycles 95 ℃, 30s annealing temperature and time than primer Tm values of about 5 ℃ general extension of the 45 ~ 55 ℃ temperature and time 72 ℃, 1min/kb (10kb inside) Tm value = 4 (G + C) +2 (A + T) cycles: typically 25 to 30 times. PCR amplification cycles determine the yield. The initial template concentration is low, increase the number of cycles in order to achieve effective amplification volume. But not unlimited number of cycles increased. Number of general circulation for the 30 or so, after more than 30 cycles, DNA polymerase activity gradually reached saturation, the amount of the product is no longer with the increase of number of cycles, there has been so-called [PCR steps] the standard PCR process is divided into three steps: 1.DNA denaturation (90 ℃ -96 ℃): the role of double-stranded DNA template in the heat, the hydrogen bond breaking, the formation of single-stranded DNA2. Annealing (25 ℃ -65 ℃ ): The system temperature decreases, primer and DNA template, forming partial duplexes. 3. Extension (70 ℃ -75 ℃): In the Taq enzyme (at about 72 ℃, the highest activity) under the action of the dNTP as raw material, from the primers 5 '→ 3' end of an extension of the complementary template synthesis DNA chain. After each cycle denaturation, annealing and extension, DNA content is doubled. As shown: Now some PCR amplified region is very short because, even if the Taq enzyme activity can not be the best in a very short period of time the copy is complete, it can be changed to two-step method, namely, annealing and extension at the same time 60 ℃ - between 65 ℃ to reduce a heating and cooling process and improve the response speed. [PCR testing] PCR reaction amplified high copy number, the next step to become a key test. Fluorescence (ethidium bromide, EB) staining gel electrophoresis is the most common means of detection. Electrophoresis assay specificity is not too high, so the primer dimer and other non-specific hybrids can easily lead to miscarriage of justice. But because of its simple and easy to become a mainstream detection method. In recent years, as the representative of the fluorescent probe detection method, and a trend of gradually replacing electrophoresis. [PCR reaction Features] specificity determinants of the specificity of PCR reaction were: ① specific primers and the correct combination of template DNA; ② the principle of base pairing; ③ Taq DNA polymerase, reaction fidelity; ④ specificity of target genes conservative. Primers and templates in which the correct combination is the key. The combination of primers and templates, and primer extension of the chain is to follow the principle of base pairing. Polymerase, reaction and TaqDNA polymerase fidelity high temperature, the reaction in the combination of templates and primers (refolding) can be carried out at higher temperatures, combined with greatly increased the specificity of the amplified target gene fragments also be able to maintain a very high accuracy. And then by selecting the specific and highly conserved target gene region of the higher degree of specificity. High sensitivity PCR product formation is increased exponentially, and can Peak (pg = 10-12) tested the order of the starting template amplified micrograms (μg =- 6) levels. From 1 million cells were detected in a target cell; in virus detection, PCR sensitivity of up to three RFU (plaque forming units); the smallest bacterial detection rate in the three bacteria. Simple, rapid PCR reaction with thermostable Taq DNA polymerase, plus a one-time After the reaction solution, that solution and DNA amplification variability on water bath - annealing - extension reaction, generally 2 to 4 hours to complete expansion reaction. PCR products were analyzed by electrophoresis general, do not use the isotopes, no radioactive contamination, easy to promote. Low purity of samples of viruses or bacteria and does not require separation of cells, DNA and RNA can be used as crude products amplified template. Clinical specimens can be directly used, such as blood, body cavity fluid, washing liquid cough, hair, cells, living tissue and other DNA amplification test. [PCR cycle parameters] 1, pre-denaturation (Initial denaturation). PCR template DNA completely denatured and fully activate the enzyme essential to the success of PCR, the proposed reference reagent manual heating time is generally not modified Taq enzyme activation time is two minutes. 2, cycle denaturation step loop typically 95 ℃, 30 秒 enough to complete a variety of target DNA sequence variability, possible to shorten the procedure time can be too long and damage to enzyme activity variability, short target sequence variability is not complete, easily lead to amplification failure. 3, primer annealing (Primer annealing) the annealing temperature needs to decide in many ways, generally based on the Tm value of primers for reference, according to the length of appropriate amplification down as the annealing temperature. Then make a prediction on the basis of this experiment. The specificity of the PCR annealing temperature has a greater impact. 4, primer extension primer extension at 72 ℃ for general (Taq enzyme optimum temperature). However, amplification of shorter and higher annealing temperature, extension time this step can be omitted with the amplified fragment length, are generally recommended over 1000bp, containing Pfu and its derivatives derived set 1min/kbp. 5, the majority of PCR with 25-40 cycles loop, too easy to produce non-specific amplification. 6, the last extension in the last cycle, the reaction maintained at 72 ℃ for 5-15 minutes. The primer extension completely, and to anneal into double strand single-stranded product. (D) PCR contamination and false positive in the PCR in the pollution comes mainly from 1, cross-contamination between samples; 2, PCR product of the previous left (carry-over) [PCR instrument] pcr pcr temperature cycling instrument development is essential, pcr amplification of each parameter must be accurate. Since perkin elmercetus pcr amplification company's first inception, has dozens of different manufacturers in the domestic and international production and sales of pcr amplification. In a few short years, the amplification of several generations of development, and constantly adopting new technologies, and further toward convenient, practical, high intelligence and automation direction. In order to facilitate understanding and appropriate pcr purchase laboratory equipment, now part of the domestic and pcr amplification are listed below; these instruments mainly aluminum block with a variable temperature, variable temperature water bath and the air temperature way to achieve the purpose of thermal cycling, each has its advantages and disadvantages . 1109-type dna amplification made is with the constant temperature bath robot displacement type. Closer to the manual. ptc-51a / b, small size, high intelligence and automated processes that can cater to nested pcr, convenient and practical. All of the above instruments are generally equipped with microcomputer automatic control of temperature, time and cycles, the purpose of labor saving can be achieved. Pcr in using these instruments as prior to the test tube should be generally measured temperature cycling conditions in order to understand the rise, cooling pipe internal heat conduction when the temperature lag caused by the situation and the actual arrival of the maximum, minimum temperature, the cycle parameters for the modified design. Temperature lag was used in eppendorf tube material, wall thickness and shape (hole match with the aluminum block level) to the effect that temperature aluminum block on the impact of bigger instruments. Equipped with a refrigerator for cooling a semiconductor equipment components, the temperature below room temperature using cold pcr reaction to a small tube, should pay attention to the problem of water condensation, without rendering the instrument into the wet semiconductor components and damage to equipment. Several domestic and foreign production-type dna amplification 1109 Beijing New technologies combined with the military arm ASTRI bath temperature electronic thermostat 40 × 0.5ml 16400 Yuan / 90a / b-type arm of Genetics Chinese Academy of heating 14,000 yuan temperature water bath / Taiwan ptc-51a / b-type air temperature of Military Medical Sciences doubles as a set of electronic transfer type 30 × 0.5ml 12000 Yuan / dna thermal cycler perkin elmercetus (U.S.) compressor refrigeration temperature aluminum block 48 × 0.5ml us $ 20000. - thermocycler # 60 # 00 # 180 b.. braun biotech (Germany) water bath temperature 98 ℃ fourth gear electric, water cooled 60 × 1.5ml 100 × 1.5ml 180 × 1.5ml dm 30000 .- automatic temperature cy-cler single block twin block ericomp inc. (U.S.) aluminum block temperature 25 ~ 100 ℃ electric, water cooled 29 × 1.5ml 58 × 1.5ml us $ 4985 .- us $ 7980 .- grant autogen grant instrumant ltd (U.S.) temperature 0 ~ 99 ℃ water bath, electric, Add cold water to cool or circulator 50 × 1.5ml or 50 × 1.5ml us $ 250. plus us $ 15.-for rack (x2) techne phc-1 phc-2 techne ltd. (English) aluminum block temperature 0 ~ 99 ℃ third gear cooled electric 500w 100w 54 × 0.5ml 54 × 0.5ml 24 × 1.5ml £ 3383. £ 3997. - biooven biothrm corp (U.S.) air temperature -150 ℃ 115v 11a 200's of 4 × 96plate us $ 2990. - trio-thermo-block tb-1 biomertra (Germany) semiconductor temperature 220v 3 × 20 tubes were temperature dm12900. - micro cycler e eppendorf (U.S.) temperature aluminum block 220v/100v 3 × 29 管 us $ 3500. - [PCR common issue] PCR products electrophoresis detection time is generally less than 48h, Some of the best on the same day electrophoresis, more than 48h after the bands irregular or even disappear. False negative, does not appear with the PCR reaction amplified the key to a nucleic acid template prepared ①, ② the quality and specificity of primers, ③ the quality of the enzyme and the use of ethidium bromide, ④ PCR cycling conditions. Should address the above links to find the reasons were analyzed. Template: ① template contains complex proteins, ② template containing Taq enzyme inhibitors, ③ the template does not digest protein than the net, especially histone chromosome, ④ Preparation of template in the extraction of losing too much, or inhalation of phenol. ⑤ template nucleic acid denaturation is not complete. The enzymes and good quality primer, the amplified band does not appear very likely that the digestion of samples, template nucleic acid extraction process goes wrong,[link widoczny dla zalogowanych], and therefore effective and stable to digestion and preparation of treatment liquid, the procedure should not be free to change the fixed . Enzyme inactivation: the need to replace a new enzyme, or enzymes use both old and new, to analyze whether the loss of enzyme activity due to insufficient or false negative result. Should be noted that sometimes forget to add Taq enzyme or ethidium bromide. Primers: primer quality, the concentration of primers, two primers, the concentration is symmetrical, is a failure or PCR amplified bands is not ideal, easy dispersion of the common cause. Some batches of primer synthesis quality problems, a high concentration of the two primers, a low concentration, resulting in low efficiency of the asymmetric amplification, measures as follows: ① a good primer synthesis of the selected units. ② primer concentration not only depends on OD values, but also to pay attention to primer stock solution made by agarose gel electrophoresis, there must be a primer bands appear, and two primers with the brightness should be broadly consistent with, such as a primer bands, a primer-free band, this time may fail to do PCR, and primer synthesis units should be resolved through consultation. Such as a primer of high brightness, a low brightness, the dilution primers to balance the concentration. ③ high concentration of a small amount of primer should Aliquot to prevent freezing and thawing or long-term Refrigerate part, leading to degradation of primer failure deterioration. ④ unreasonable primer design, such as primer length is not enough, the formation of dimers between the primers and so on. Mg2 + concentration: Mg2 + ion concentration on the efficiency of PCR amplification of great influence, the concentration is too high can reduce the specificity of PCR amplification, the concentration of PCR amplification yield is too low, and even the failure of PCR amplification without the amplified bands. Reaction volume changes: usually used for PCR amplification of the volume of 20ul, 30ul, 50ul. Or 100ul, what volume of applications for PCR amplification and detection is based on scientific research and clinical settings with different purposes, such as in a small volume of 20ul, the do a large volume, be sure to mold claims, or else prone to failure. Physical causes: variability of the PCR amplification is very important, such as the low denaturation temperature, denaturation time is short, very likely false negative; annealing temperature is too low can lead to reduced non-specific amplification efficiency of annealing temperature is too specific amplification high impact combination of primers and template PCR amplification efficiency decreases. Sometimes necessary to use a standard thermometer, about amplification or detection of soluble pot of degeneration, annealing and extension temperature, which is one of the reasons of failure PCR. Target sequence variation: If the target sequence mutation or deletion of specific binding of primers and templates, or for a certain period of absence of target sequences of primers and templates to lose complementary sequence, the PCR amplification is not successful. False positive PCR amplification bands appeared with the purpose of target sequence with the same article, sometimes the band more orderly, more bright. Inappropriate primer design: choice of the purpose of amplification and non-amplified sequence of sequence homology, thus making PCR amplification, the PCR amplified products of non-purposeful sequence. Primer target sequence is too short or too short, prone to false positives. To be re-designed primers. Target sequence or amplification products of cross-contamination: this kind of pollution for two reasons: First, the entire genome or large fragments of cross-contamination, leading to false positives. The following methods can be used to resolve false positive: the operation should be carefully gently to prevent the inhalation of sample target sequence or scatter gun centrifuge tube. In addition to enzyme and can not heat the material, all reagents or equipment should be autoclaved. Samples used in centrifuge tubes and into the tips and so should be a one-time use. If necessary, additional samples before the reaction tubes and reagents used ultraviolet radiation to destroy the existence of the nucleic acid. Second, small fragments of nucleic acid in air pollution, these small fragments shorter than the target sequence, but has some homology. Can be spliced to each other, and complementary primers, the amplified PCR products, resulting in the generation of false positive, nested PCR method can be used to reduce or eliminate. Experience non-specific amplification with PCR amplification bands appeared inconsistent with the expected size, large or small, or simultaneously with non-specific amplification with a specific amplification band. The emergence of non-specific bands, the reasons: First, primers are not completely complementary with the target sequence, or the formation of primer dimer aggregate. Second, Mg2 + ion concentration is too high, the annealing temperature is too low, and too much on the number of PCR cycles. Followed by the quality and quantity of the enzyme, the enzyme is often prone to a number of sources of non-specific bands and there is not another source of the enzyme, the enzyme also appears excessive and sometimes non-specific amplification. The measures are: necessary, re-designed primers. Reduce the amount of enzyme or exchange of another source of enzyme. Reduce the amount of primers, the appropriate amount of increase in the template, reduce cycle times. Appropriately increasing the temperature of annealing temperature or by two-point method (93 ℃ denaturation, 65 ℃ annealing and extension of left and right). Smear sheet appears with PCR amplification of towing or sometimes apply tape or carpet tape or sheet-like band. The reason often due to excessive activity or poor quality of the enzyme, dNTP concentration is too high, Mg2 + concentration is too high, too low annealing temperature, cycle times caused by too much. The measures are: reducing the amount of enzyme, or exchange of another source of the enzyme. ② reduced dNTP concentration. Appropriate to reduce the Mg2 + concentration. Increase the amount of template, reduce cycle times. Cloning of PCR product 1) the optimal conditions for cloning PCR products of what? Optimal insert: vector ratio to be determined experimentally. 1:1 (insert: vector) often is the best ratio of 1:8 or 8:1 molar ratio is required. Shall determine the ratio range. Connection connection with 5ul 2X liquid, 50ng plasmid DNA, 1Weiss units T4 ligase, insert a total of 10ul. Heat 1 hour at room temperature, or overnight at 4 ℃. In these 2 temperatures, the lack of T-protruding from the side of the vehicle will be connected, resulting in blue spots. 1 hour at room temperature thermal insulation to meet the requirements of the majority of clones, to improve the connection efficiency, be 4 ℃ overnight. 2) PCR products were purified by gel need? Gel analysis of PCR products, such as only one band, no gel purification. As seen with other miscellaneous, may be the accumulation of a large number of primer dimers. Small number of moles of primer-dimers is also high, which will produce a high percentage of clones with primer-dimers, not an end inserts. Need to do this gel purified before cloning. 3) If no fragment was recovered, but also what needs to be controlled experiment? A) conversion coating is not competent cells. If the colony, indicating that ampicillin failure, or contamination on the type of plasmid with ampicillin resistance, or type of colony produced ampicillin resistance. B) into whole plasmid, calculate the number of colony growth, determination of transformation efficiency. For example, 1ug/ul plasmid 1:100 dilution, 1ul to 100ul competent cell transformation. After dilution to 1000ul with SOC with 100ul ceiling. Cultured overnight, resulting in 1,000 colonies. Conversion rate: the total number of colonies generated / ceiling amount of DNA. Ceiling amount of DNA used in the conversion reaction is divided by the dilution factor. Specifically, transformation with 10ng DNA, diluted with SOC after 1000u containing 10 ng DNA, with 1 / 10 planking, shared 1 ng DNA. Conversion rate: 1000 clone X10 (3 power) ng / ceiling 1 ng DNA ug = 10 (6 th) cfu / ug into pGEM-T using 10 (8 th) cfu / ug competent cell colonies, or if there is no few colonies, the transformation of competent cells is too low. C) using pGEM-T as the positive control, or PCR products, resulting in> 20-40 blue plaques (10 with the specified procedure (8 th) cfu / ug competent cells), indicating that the carrier lost T. May be contaminated DNA ligase enzyme. T4DNA ligase (M1801, M1804, M1794) good quality standards without nuclease contamination from other sources should not be used T4 DNA ligase replaced. D) with the pGEM-T or pGEM-T Easy vector, pGEM-T connections are controlled, high-frequency transformation of competent cells (10 (8 th) cfu / ug), in accordance with the procedures specified in the experiment can be obtained 100 colonies, 60% should be white, such as generating> 20-40 blue spots, no colonies or very few colonies, connection problem. 4) The results were good, but not recovered fragment experiment gone wrong? A) connected with the room temperature heat 1 hour, to meet the majority of clones, for efficiency, need 4 ℃ overnight. B) insert with pollution, the 3 `-T deficiency, or inhibition of connectivity, inhibition of transformation. To do this, insert the positive control and pGEM-T mixture, and then connected. Such as reducing the number of colonies in control, inserts to be purified, or re-preparation. If a large amount of blue spots, inserts pollution nuclease, the pGEM-T or pGEM-T Easy vector 3 `-T is missing. C) into the fragment is not suitable for connection. Purified with gel inserts, due to excessive UV radiation, have occurred. Excessive exposure to UV will produce pyrimidine dimers, is not conducive to connect, DNA must be re-purified. D) with a heat-resistant DNA polymerase repair the amplified product was no end A, which is the pGEM-T or pGEM-T Easy vector cloning needs. Add Taq DNA polymerase and nucleotide can be added at the end A. Details of the investigation pGEM-T pGEM-T Easy Vector Data (TM042). E) highly repetitive sequences may be unstable and produced in the absence of amplification and rearrangement, such as the inserts were found to generate high-frequency deletions and rearrangements, required recombinant E. coli strains defects, such as the SURE cell PCR reaction system and reaction conditions standard PCR reaction system: 10 × PCR buffer, dNTP mixture 10ul 4 种 the 200umol / L of each primer, 10 ~ 100pmol template DNA 0.1 ~ 2ug Taq DNA polymerase 2.5u Mg2 + 1.5mmol / L with double or triple distilled water to 100ul PCR reaction of five elements: participation in PCR reaction substances are mainly there are five or primers, enzyme, dNTP, template and Mg2 + primers: primers PCR-specific reaction of the key, PCR products specific depends on the primers and template DNA complementary level. Theoretically, as long as that of any DNA sequence of a template, we can design according to their complementary oligonucleotide primers do chain, the use of PCR template DNA can be a large number of in vitro amplification. Primers were designed to follow the following principles: ① primer length: 15-30bp, commonly used for 20bp or so. ② primers span: the 200-500bp is appropriate, under certain conditions can be extended up to 10kb fragments. ③ primer bases: G + C content of 40-60% is appropriate, G + C poor little amplification, G + C too prone to non-specific band. ATGC best random distribution, to avoid more than 5 purine or pyrimidine nucleotides arranged in clusters. ④ avoid secondary structures occur within the primer to avoid the complementarity between the two primers, especially the 3 'end of the complementary, or the formation of primer dimers, resulting in non-specific amplified bands. ⑤ primer 3 'end of the base, in particular the last and penultimate bases, matching requirements should be strictly in order to avoid the bases do not match the end result of PCR failure. ⑥ primers have or be able to add appropriate restriction sites, the best target sequence was amplified with the appropriate restriction sites, and this restriction enzyme analysis or molecular cloning is very good. ⑦ specific primers: primer nucleic acid sequence database should be no significant homology with other sequences. The amount of primers: the concentration of each primer 0.1 ~ 1umol or 10 ~ 100pmol, produced the lowest amount of primer needed for good results, high concentrations can cause primer mismatch and non-specific amplification, and the increase between the two primers polymer opportunity. There are two kinds of enzymes and the concentration of Taq DNA polymerase supply, a raw water from the habitat of the natural bacteria in the purified enzyme, the other is the genetically engineered E. coli enzyme synthesis. A typical PCR reaction of catalytic amount of enzyme is about 2.5U (refer to the total reaction volume of 100ul), the concentration is too high can cause non-specific amplification, the concentration is too low to reduce the amount of synthetic products. quality and concentration of dNTP dNTP concentration and the quality and efficiency of PCR amplification is closely related to, dNTP granular powder, such as the volatility of loss of biological activity preserved improperly. dNTP solution acidic when paired with high concentration, to 1M NaOH or 1M Tris. HCL buffer to adjust the PH to 7.0 to 7.5, a small-packing, -20 ℃ stored frozen. Freezing and thawing will dNTP degradation. In the PCR reaction, dNTP should be 50 ~ 200umol / L, with particular attention to four kinds of dNTP concentrations were equal (equimolar preparation), such as the concentration is different from any of several other (the high or low), will cause a mismatch. Too low will reduce the concentration of PCR product yield. dNTP combination with Mg2 +, the free Mg2 + concentration decreased. Template (target gene) the amount of DNA template and purification of nucleic acid level, is the key to success or failure of PCR, one of the traditional DNA purification methods commonly used to SDS and proteinase K digestion of samples. The main function of SDS: dissolve membrane lipids and proteins, which damage cell membranes and membrane proteins dissolved and dissociated cell nuclear protein, SDS can precipitate with protein binding; proteinase K to digest protein hydrolysis, in particular, histone and DNA binding, and then the organic solvents phenol and chloroform extraction out of proteins and other cell components, nucleic acids precipitated with ethanol or isopropanol. DNA can be extracted as a template for PCR reaction. General clinical testing samples, fast and easy way can be dissolved cells, lysis of pathogens, digestion and removal of chromosomal proteins to target genes of free, directly used for PCR amplification. Extraction of RNA template or the general use of proteinase K guanidine isothiocyanate method, to prevent RNase degradation of RNA. Mg2 + Mg2 + concentration and specificity of PCR amplification yield a significant impact, in general, PCR reactions, a variety of dNTP concentration 200umol / L time, Mg2 + concentration of 1.5 ~ 2.0mmol / L is appropriate. Mg2 + concentration is too high, the reaction specificity of reduced non-specific amplification occurs, the concentration is too low will reduce the activity of Taq DNA polymerase, the reaction products decreased. PCR reaction and the conditions of PCR reaction conditions were temperature, time and cycle times. Temperature and time settings: Based on the principle of three-step PCR is set up degeneration - annealing - extension of three temperature points. Reaction in the standard method using three temperature points, double-stranded DNA denaturation in 90 ~ 95 ℃, and then rapidly cooled to 40 ~ 60 ℃, annealing and primer binding to the target sequence, and then quickly heated to 70 ~ 75 ℃, the Taq DNA polymerase under the effect of the primer along the template chain extension. For shorter target gene (the time length of 100 ~ 300bp) can be the second temperature point method, in addition to the denaturation temperature, the annealing and extension temperature can be combined, generally at 94 ℃, 65 ℃ annealing and extension of left and right (this temperature Taq DNA enzyme catalytic activity is still high.) ① denaturation temperature and time: modified low temperature melting lead to PCR failure is not entirely the most important reason. Under normal circumstances, 93 ℃ ~ 94 ℃ min enough to make the template DNA denaturation, if less than 93 ℃ extension of time is required, but the temperature is not too high, because high temperatures affect the activity of the enzyme. If this step can make a template or PCR products of target genes completely denatured, it will lead to PCR failure. ② annealing (recovery of) temperature and time: Specific PCR annealing temperature is the more important factor. Rapid cooling after the denaturation temperature to 40 ℃ ~ 60 ℃, can occur with primers and templates. Because the template DNA is much more complex than primer, primer and template combination of collision between the opportunity is much higher than the collision between the complementary strands of the template. Annealing temperature and time, depending on the length of primer, base composition and concentration, as well as the length of the target base sequence. For the 20 nucleotides, G + C content of about 50% of the primers, 55 ℃ for the selection of the optimal annealing temperature starting point is better. Primer annealing temperature by the following formula to help choose the right temperature: Tm value (melting temperature) = 4 (G + C) +2 (A + T) annealing temperature = Tm value - (5 ~ 10 ℃) in the Tm value of the permissible range, select a higher annealing temperature can greatly reduce the template between primers and non-specific binding and improve the specificity of PCR reaction. Refolding time is generally 30 ~ 60sec, enough to make between the primers and templates are fully integrated. ③ extension temperature and time: Taq DNA polymerase of the biological activity: 70 ~ 80 ℃ 150 nucleotides / S / enzyme molecule 70 ℃ 60 nucleotides / S / enzyme molecule 55 ℃ 24 nucleotides / S / enzyme molecules When higher than 90 ℃, DNA synthesis is almost not possible. Extension of PCR reaction temperature is generally chosen between 70 ~ 75 ℃,[link widoczny dla zalogowanych], the temperature commonly used 72 ℃, high temperature is not conducive to the extension primer and template combination. PCR extension reaction time, can be amplified fragment length, are generally less than 1Kb DNA fragment is sufficient extension time 1min. 3 ~ 4kb of the target sequences to be 3 ~ 4min; amplified 10Kb be extended to 15min. Extends into the lead for too long with the emergence of non-specific amplification. Low concentration of template for amplification, an extension of time to be slightly longer. PCR Laboratory and Method 1, the laboratory needs of the layout of PCR required four laboratories, namely, a reagent storage and preparation areas, two sample preparation area, three amplification reaction mixture preparation and amplified region, 4 PCR product analysis area, the layout shown in Figure 1. Figure 1 PCR Laboratory of four regional settings if you use automatic analyzer, may be appropriate to merge the regions, we recommend that the amplified region and product analysis into a total of three PCR detection area, the layout shown in Figure 2. Figure 2, three PCR laboratory set up by the regional requirements of the Ministry of Health, into the various work areas must strictly follow the order in one direction, that is only from the reagent storage and preparation areas? Sample preparation zone amplification reaction mixture preparation and amplification areas? PCR product analysis area to avoid cross-contamination. The work area must install the appropriate equipment to ensure that the exhaust air flow at a single direction. Work area walls, floors, office supplies, disinfectant must be selected corrosion resistant materials. Work area must have a clear marker, different working areas to avoid the equipment, goods mix. 2, set the standard of the work area must be equipped with exhaust fans, air conditioning, corrosion-resistant surface and work table, wardrobe, shoe, special office supplies, special clothes and work shoes, mobile UV lamps to ensure the safety and health needs. According to the Ministry of Health, 3, micro pipette (covering 1-1000μl) 4, Mobile ultraviolet light (near the work surface) 5, consumables: disposable gloves, disposable absorbent paper, high pressure processing and processing injector tube tip (with filter ) 6, 7, special work clothes and work shoes, special office supplies (b) of the sample preparation area refrigerator 1,2-8 ℃, -20 ℃ or -80 ℃ refrigerator 2, 3 high-speed refrigerated centrifuge desktop, allowing mixing device 4, water bath or heating block 5, the micro pipette (covering 1-1000μl) 6, removable ultraviolet light (near the work surface) 7, Clean Benches 8, consumables: disposable gloves, disposable absorbent paper, high pressure processing Canadian injector tube tip (with filter) 9, 10 special work clothes and work shoes, office supplies for dealing with specific macromolecules DNA, should have the ultrasonic water bath apparatus. (C) gene amplification and product analysis area 1, automatic quantitative nucleic acid amplification (including computers, printers) 2, micro pipette (covering 1-1000μl) 3, removable ultraviolet light (near the work surface) 4 , consumables: disposable gloves, disposable absorbent paper, high pressure processing and processing injector tube tip (with filter) 5, 6, special work clothes and work shoes, office supplies, dedicated work area must be clear of the mark, to avoid different working areas of equipment, goods mix. In different work areas should use a different color or a significant difference between the uniform signs to facilitate identification. When workers leave the work area shall not be out of district-specific clothes. 3, laboratory quality control PCR laboratory staff must participate in the provinces by the Ministry of Health or the Clinical Laboratory Centre, the clinical gene amplification organized training courses and certificates. PCR laboratory passed the acceptance, the laboratory must be at least should have held two or more PCR laboratory must establish a strict laboratory management system, establish standard operating procedures (SOP), established series of quality management documents, to ensure that laboratories meet the daily running of the Ministry of Health's requirements to ensure accurate test results, to ensure health and safety laboratory, to ensure long-term stable operation of the laboratory.
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