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The advent of the Polymerase Chain Reaction (PCR) brought about the ability to rapidly make many copies of a segment of DNA. The PCR reaction depends on short pieces of DNA, called primers, to bind to the denatured DNA strands and act as a template for replication. Primers are usually 17-24 nucleotides in length and must be specific to the target DNA. Listed below are some rules and helpful tools that scientists use in designing primers. Finally, there are instructions on how to perform a nucleotide BLAST to check the specificity of each primer. Read through the information and then use these instructions to complete the Primer Design Exercise. Primer Length The optimum length of a primer depends upon its (A+T) content, and the T m. Apart from the Tm, a prime consideration is that the primers should be complex enough so that the likelihood of annealing to sequences other than the chosen target is very low. For example, there is a ¼ chance of finding an A, G, C or T in any given DNA sequence; there is a 1/16 chance of finding any dinucleotide sequence (eg. AG); a 1/256 chance of finding a given 4-base sequence. So, a sixteen base sequence will statistically be present only once in every 4,294,967,296 bases (or 4 billion): this is about the size of the human or maize genome, and 1000x greater than the genome size of E. coli. Thus, the association of a greater-than-17-base oligonucleotide with its target sequence is an extremely sequence-specific process. Consequently, 17-mer or longer primers are routinely used for amplification from genomic DNA of animals and plants. Elongation Temperature and Time This is normally 70 - 72oC, for 0.5 - 3 min. Taq actually has a specific activity at 37 oC which is very close to that of the Klenow fragment of E. coli DNA polymerase I, which accounts for the apparent paradox which results when one tries to understand how primers which anneal at an optimum temperature can then be elongated at a considerably higher temperature - the answer is that elongation occurs from the moment of annealing , even if this is transient, which results in considerably greater stability. At around 70oC the activity is optimal, and primer extension occurs at up to 100 bases/sec . About 1 min is sufficient for reliable amplification of 2kb sequences (Innis and Gelfand, 1990). Longer products require longer times: 3 min is a good bet for 3kb and longer products. Longer times may also be helpful in later cycles when product concentration exceeds enzyme concentration (>1nM), and when dNTP and / or primer depletion may become limiting. A simple set of rules for primer sequence design is as follows (adapted from Innis and Gelfand, 1990): 1. primers should be 17-28 bases in length; 2. base composition should be 50-60% (G+C);
The BLAST tool is maintained by the National Center for Biotechnology Information (NCBI) and can be found at the following link: www. base pair). runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing). 8. Tms between 55-80oC are preferred. Calculating Melting Temperatures of primers and target DNA 1. Tm. Enter the query sequence by typing the primer sequence into the text box. run a computer search against the vector and insert DNA sequences to verify that the primer and especially the 8-10 bases of its 3' end are unique. primers should end (3') in a G or C. primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided.ncbi. One way to determine the uniqueness of the primer sequence is to compare the primer to all other sequence data using a bioinformatics tool that is a Basic Local Alignment Search Tool. DNA calculator This website will allow you to fill in your primer sequence to determine the length.asp Determining the uniqueness of the primer sequence: Specificity to the target gene is one of the most important properties of a primer.gov/BLAST/ . “The Wallace Rule” Tm (in ºC) = 2(A+T) + 4(C+G) (A+T) = sum of A and T residues in the primer (C+G) = sum of C and G residues in the primer 2. 1. and molecular weight for your primers and if there will be any secondary structures or possibilities of primer dimers. 2 .nlm. Go to the above website.3. and should be avoided. There are many types of BLASTs. as otherwise primer dimers will be synthesised preferentially to any other product. otherwise known as BLAST. If possible. 5.nih. 7. 3'-ends of primers should not be complementary (ie. 4.sigma-genosys. In the “Choose Search Set” box select “nucleotide collection (nr/nt)” from the Database category. 2. but we will only be dealing with the nucleotide BLAST (blastn) to determine the specificity of the primers. Under the heading “Basic BLAST” click on the link titled “nucleotide BLAST”. 6. or CG or GC: this prevents "breathing" of ends and increases efficiency of priming. http://www.com/calc/DNACalc. 3.
gelatin or BSA to 100ug/ml.1uM each primer. 6. 8. and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X100 (0. 5.they are also generally proprietary. Modern formulations may differ considerably. If more than 5 matches are found. the primers should amplify only your gene of interest.0. Higher than 50mM KCl or NaCl inhibits Taq. “Program Selection”. The first figure on the results page provides an overview of the database sequences that are aligned to the query sequence. The lower the E value. primers 0. The Expectation value (E) is a number that describes the number of matches a particular sequence would get by random chance. 3 .3. Each matched sequence contains a link to view the entire sequence. This page will update until the results are available. which is your primer sequence in this case. select “blastn” as the type of algorithm. but some is necessary to facilitate primer annealing.5mM or higher MgCl2. a score and an E value. up to 50mM KCl. Reaction Buffer Recommended buffers generally contain: • • • • • • 10-50mM Tris-HCl pH 8. the name of the sequence. 9. in this case we are looking for Bacteria (P. Next type in the organism of interest. however .200uM each dNTP. A new page will appear entitled. As long as there are not any matches in the species you are working in. the best way to determine how well the sequences align is to look at the alignment and the length of homology between the primer sequence and the hits from the genome database. Following the figure is a list of sequences that are significantly similar to the query sequence.10% v/v) (Innis and Gelfand. putida) or Viruses (Cauliflower Mosaic Virus). 50 . 1. Click on the blue BLAST Icon near the bottom of the page.4.05 . “Job Title”. the less likely the match would happen by chance and thus making the similarity of the query sequence to another sequence more significant.2 . Scroll down and see how the base pairs align with the sequence matches of the same species. 1990). such as primers. 7. When working with short sequences. In the next box. the primer is not specific enough.
0. so allowances should be made for dNTPs. take a small sample (1ul) of the amplified mix and re-amplify 20-30x in a new reaction mix rather than extending the run to more cycles: in some cases where template concentration is limiting. product specificity.2uM is sufficient for homologous primers. Tm of template. so [Mg 2+] should be 0. dNTPs are the most concentrated. all of which chelate and sequester the cation. as these can differ markedly in their requirements. Taq requires free Mg2+. dNTPs .1.5 2.[Mg2+] affects primer annealing. primers and template. competition for primer binding by re-annealing of concentrated (10nM) product (Innis and Gelfand. others are dependent on it. Some enzymes do not need added protein. Primer concentrations should not go above 1uM unless there is a high degree of degeneracy. 4 . enzyme). of these. Nucleotide concentration need not be above 50uM each: long products may require more.3 . competition for reactants by non-specific products. even under the same conditions of concentrations and cycling times/temperatures. is the attenuation in the exponential rate of product accumulation in late stages of a PCR. A titration should be performed with varying [Mg2+] with all new template-primer combinations. 1990). this can give good product where extension of cycling to 40x or more does not. Some enzymes work markedly better in the presence of detergent. reactant depletion (primers. when product reaches 0. end-product inhibition (pyrophosphate formation). If desired product is not made in 30 cycles. latter for long products). This may be caused by degradation of reactants (dNTPs.0 nM. however. The plateau effect.5mM greater than [dNTP].former a problem with short products. Cycle Number The number of amplification cycles necessary to produce a band visible on a gel depends largely on the starting concentration of the target DNA. product and primer-template associations. probably because it prevents the natural tendency of the enzyme to aggregate. enzyme activity and fidelity.
pp. 5 .Innis MA and Gelfand DH (1990). Academic Press. Optimization of PCRs. 3-12 in: PCR Protocols (Innis. eds. Sninsky and White. Gelfand. New York.).
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