Original Address: PCR primer dna fingerprinting design Author: Ivy

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Original Address: PCR primer dna fingerprinting design Author: Ivy
1. The specific amplification (i.e., amplification of sequences with known), the primer annealing temperature is preferably a little higher, above 65 degrees, 70 degrees 2. To avoid the 3 'end is rich in GC. Primers were designed to ensure when containing dna fingerprinting 3 A or T in the last five nucleosides. 2. The design of the 5 'end and an intermediate region for G or C primers. dna fingerprinting This increases the stability and the stability of the primer with the sequence of the primer hybridizes. dna fingerprinting 2. Typical primers 18 to 24 nucleosides long. Primers need be long enough to ensure that the sequence is unique, and the possibility of non-target sequence is present in the lower sequence site. However, the length of the primer is greater than 24 nucleotides does not mean that greater specificity. 2. The primer 3 'end may not have recurring bases within 5 bases, the 3' end is preferably T end. 3. The primer 3 'end do not have mismatches 4. Length: the length of the general primer complementary to region of 16-25bp, 4 (G ten C) primers complementary regions of ten 2 (A ten T) do not need to be greater than 70 , generally not less than 50 5.G ten c content: preferably between 45% an 55%, but sometimes by template restrictions, can not idealized. However, there are several options, you can put ten c G content close to 50% as one of the parameters 6. random distribution bases: Avoid consecutive four or more single base. Especially not at its 3 'end occurred more than three consecutive G or C, otherwise it would primer G + C-rich sequence dna fingerprinting region mis-priming. 7. primer itself: not contain very long strong self-complementary sequences, especially the primer 3 'end of the folded back to form hairpin not generally over three bases, otherwise it will affect the binding between the primer and primer 8. template: two There should be a long strong complementary or homologous nucleotides between the primer, especially should avoid the 3 'end of the complementary overlap. Specific primers length principle; usually by primers specific length and annealing temperature control. If the PCR annealing temperature is set within a range close to Tm values of primers (primer / template duplex melting temperature) of a few degrees, 18 to 24 base oligonucleotide is sequence specific good a. The longer the primer, the less amplified template annealing is triggered. To optimize the PCR reaction, using ensure dissolution temperature of not less than the shortest of the primer dna fingerprinting 54 obtain best efficiency and specificity. In general, it is best to seek safety in the specificity of the allowable range. Each additional nucleotides, primers specific four times; Thus, for most applications the minimum length of the primer is 18 nucleotides. The synthetic oligonucleotide primer design chains (18-24 mer) for a variety of experimental conditions is nonetheless wise. primer secondary structures including primer dimers itself, hairpin, between primer dimer. These factors will affect the binding of the primers and the template thus affecting the efficiency of the primers. For the 3 'end formation of dimers of primers, which should be controlled ΔG than -5.0kcal / mol or less than three consecutive complementary bases, primer-dimer because dna fingerprinting such situations there is further possible to form a more stable structure resistance, an intermediate primer or the 5 'end of the requirements may be relaxed. Hairpin primer itself forms, but also to the 3 'end or near the 3' end of the primer - Template Binding greater; factors that influence dna fingerprinting the stability of a hairpin structure in addition to the bond energy of complementary dna fingerprinting base pairing, and stem-loop structure also has a great relationship. Should try to avoid the 3 'end of a primer hairpin structure. primer GC content and Tm values of PCR primers GC content should be kept reasonable. Tm values of oligonucleotide containing 50% G + C of about 20 bases in the range of 56 ~ 62 , which provide sufficient heat for effective annealing. A pair of GC content and Tm values of primers should be coordinated. Poor coordination primer pair efficiency and specificity are poor, because of the reduced Tm values result in the loss of specificity. dna fingerprinting In this case the primer Tm value higher probability of error caused by its greater. The use of high annealing temperature, Tm value lower primer may be completely functional. dna fingerprinting When selecting a new pair of primers was synthesized from a number within the range of good specific oligonucleotide sequence, GC content and Tm coordination of such values is critical. Generally, the Tm value of a pair of primers differ as much as possible of not more than 2 to 3 degrees Celsius, dna fingerprinting while primer Tm values of the reactants and products do not differ much, is preferably within the range of 20 C. additional sequences and annealing temperature of the primer sequence, if additional information to be added to the primer, such as T7RNA polymerase binding site, restriction sites or GC plus hairpin primer extension may be used. Generally, the primers 5 'end sequence does not affect unrelated annealing of primers specific sequence. Sometimes, a large number of primers and template dna fingerprinting added unpaired bases, can be 4-5 amplification cycles under the conditions of a lower annealing temperature; then assumed primer 5 'sequence has been added to the template, calculated the annealing temperature of the rest of the cycle the next. An important consideration when adding restriction enzyme sites in the primers are most effective cutting restriction enzymes require the 5 'end dna fingerprinting of their recognition sequence have 2-3 extra bases non-specific, this will increase the non-primer the length of the template-specific sequence. Another disadvantage is the long primer sequences dna fingerprinting affect the accurate calculation of the dissolution temperature, which is determined for the PCR reaction dna fingerprinting annealing temperature and time is required. For less than 20 base primer, Tm value can be (G + C) +2 (A + T) calculated Tm = 4. For longer primers, dna fingerprinting Tm values to consider kinetic parameters, obtained from the "nearest neighbor bits" dna fingerprinting is calculated, the existing PCR primer design software Most have adopted this approach. primer dna fingerprinting 3 'end nucleotide primer 3' dna fingerprinting end and the template nucleotide perfectly matched for obtaining good results is very important, and the primer 3 'end dna fingerprinting of the final 5-6 nucleotides mismatches as possible less. If the 3 'end of the mismatch excessive reaction by lowering the annealing temperature to compensate for this mismatch will not have any effect, and the reaction is almost doomed to fail. The primer 3 'end of another problem is to prevent a pair of primers homologous within. Special attention should not be complementary to the primer, especially at the 3 'end. Complementarity between the primers will result in unwanted primer duplex, PCR product thus obtained is actually an amplification primer itself. This will produce a state of competition between PCR primer duplexes and natural dna fingerprinting product templates, thus affecting the amplification success. The primer 3 'end by the stability of the primer 3' end base composition, typically in consideration of the end of the five bases ΔG. The size of this value have a greater influence on the amplification, a negative value is large, the 3 'end of the high stability, higher amplification efficiency, as well as easier to ectopic initiator. It should be noted that the primer 3 'end should be avoided T. Experiments show that, with the end of the primer T and even T, G or C mismatches still effective extension. Length and position PCR product within dna fingerprinting rake sequence all computer programs are available for selection of PCR product length. In general, PCR product length influence on amplification efficiency. Under certain applications, PCR product length depends partly on the template material. Specific length of the expected product often depends on the application needs. If the aim is to establish clinical laboratory method for the determination of specific DNA fragments, 120 ~ 300bp small DNA amplification products dna fingerprinting may be the best. The product should have good specificity and a high generation efficiency, and sufficient information can be used to capture the probe containing hybridization experiments. The product of this length dna fingerprinting can be achieved by using a two-step amplification cycle methods, thereby reducing dna fingerprinting the amplification time. Other PCR methods have different optimum product length. For example, by quantitative RNA-PCR detection of gene expression, the product should be large enough so as to constitute a competitive template, so that the product and the competitor on the gel can easily tell the difference. These products are generally in the range of 250 ~ 750bp. supplement if looking PCR primers in the cDNA sequence, requiring special attention by two points: First, try the primer and product is maintained within the coding region of the mRNA, since it is to generate a unique sequence of a protein, unlike the 3 'end non-coding region Unlike many other mRNA homology; second, trying to put a primer on different exons in order to make the PCR product specific dna fingerprinting for the RNA products generated from DNA contamination in the relative size difference. If the aim is to clone a PCR gene or cDNA of specific dna fingerprinting sequences, the size of the product dna fingerprinting is preselected depending on the application. Here, the computer program can provide information about a desired area of the flanking primers are selected. In the choice of primers used to amplify DNA from different species, and should avoid the 5 'and 3' end untranslated region of the mRNA sequence, as they may not have any homology. 3. design degenerate primers designed dna fingerprinting degenerate dna fingerprinting primers, be sure to check the simple amino acid amplification of the target region of the genetic code and the selected degrees. Obviously, we expect to select the lowest degenerate amino acids, to improve the specificity of purpose. full attention to the species for codon preference, select the species frequently used codons to reduce degenerate primers and sex. should endeavor to avoid the 3 'end of the degenerate and, for most amino acid residues, means the primer 3' end not located in the third codon. some ambiguity in the position of using deoxyinosine (dI) instead of degenerate bases. 4. Sequencing primer design course design dna fingerprinting sequencing primers generally by sequencing company dna fingerprinting to complete, if required to design their own words; so except in accordance with the above mentioned primer design common standards, but also need to pay attention to two things: sequencing primer specificity Standard control should be more stringent, ie more priority to specific design. Because in a sequencing reaction, if the primer annealed to the template at a desired position dna fingerprinting and the non-induced chain extension, dna fingerprinting the result will be a lot of interference on the results dna fingerprinting can not be read even cause. sequencing primer Tm values appropriate higher. Now most of the sequencing reactions were selected for sequencing grade heat-resistant DNA polymerase catalytic, and using PCR thermal cycling program. Tm value of the sequencing primer selected slightly higher, helps smooth reaction zone measured across the secondary structure of the template, but also helps to reduce non-specific reaction. 5. Design Design probe probe, according to the different uses have their design features, here is carried out on the general principles discussed: probe length is generally between 20-50 nucleotides long synthetic high cost, and error prone polymerases synthesize long hybridization time. Short the specificity decreased. Note that G and C content of 40-60% in efforts to control, while a base successively repeated no more than 4, in order to avoid non-specific dna fingerprinting hybridization to produce. probe sequence itself can not form dimers, nor the "hairpin" structure exists on this point would require much more stringent than the ordinary primer design. If the probe to the target is a mixture of a plurality of target genes, it is necessary to control the probe and the unrelated gene similarity between 70% or less.
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