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SGD Help: Web Primer

The Web Primer tool facilitates the design of primers for use in sequencing or PCR. Sequencing primers will be evenly spaced along the DNA. PCR primers will be at the ends of the DNA selected in a region of DNA the length of which you define.

Contents

  1. Using Web Primer
  2. Primers for PCR
  3. Primers for Sequencing

Using Web Primer

  • Step 1: Enter a DNA Source
    Enter either a locus name or an actual DNA sequence; either will then act as the source of DNA from which the primers are chosen. For the Locus field, any standard ORF name (i.e. YBL004W) or gene name (i.e. BUD4, TUB2) is allowed. If an entry in this field is ambiguous (matching more than one entry in the database) a list of matching entries is created and you can choose one. An example of this is YJL12, which matches ORFs YJL120W through YJL129C. If these ORFs have gene names associated with them this name is present as well (i.e. YJL125C=GCD14). Entries which match MORE than 25 ORFs will not be processed and you are requested to refine the entry. The DNA sequence may include numbers but should NOT include letter characters (other than the DNA sequence).
  • Step 2: Choose the experiment type for the primer
    1. a. PCR primers
      Some of the properties of primers that can affect the outcome of PCR include: the GC / AT ratio, length, melting temperature, and the extent of annealing between primers. The location of a primer also heavily influences its usefulness. All of these parameters can be customized by the user and are further described in the Primers for PCR section below.
    2. b. Sequencing primers
      Sequencing primers are also highly customizable. Potential valid primers are evenly spaced along the DNA of interest starting at each 5' end. The user can specify the area of DNA to be sequenced, how many strands to order sequencing primers for, which strand to order primers for, the approximate distance between primers, the length and percent GC content of the primers, and the maximum self annealing of the primers. See the Primers for Sequencing section below for more discussion.
  • Step 3: Click the "Submit" button
    After submitting your request, an intermediate page will appear allowing you to customize the parameters of the primers (see below). Set the parameters and click the "Submit" button. If the parameters are not satisfactory and primers cannot be designed, follow the instructions in the resulting error message and re-set the parameters.

Primers for PCR

  • Location of Primers (with respect to a chosen Locus) - When the DNA source is a Locus, the location of the primers will be determined relative to the start and stop codons of the gene. The default option will find "forward" primers of given length(s) that wholly reside somewhere within the first 35 basepairs upstream of the coding sequence, and likewise will find "reverse" primers that reside within the 35 basepairs immediately following the coding sequence. You can alter the endpoints of either of these "primer selection regions" by changing the number in the "Distance from Start" and "Distance from Stop" fields (note that entering negative numbers will cause the primer selection region to be located inside the coding region of the gene). The user may also define exact 5' endpoints of the primers by selecting the button marked "YES" on the line that asks about exact endpoints. Thus while the default option will allow amplification of a region whose endpoints are somewhere within 35 basepairs upstream and 35 bp downstream of the gene, choosing an exact endpoint will cause each primer to share the same 5' end.
  • Location of primers (with respect to DNA entered) - Primer location is most influenced by selection of a gene. Possible primers are determined by their relationship to this gene. The user may choose where in relation to the start and stop codons the primers are located. The default option will find primers in the first and last 35 basepairs of the DNA sequence entered. You may define exact endpoints to start and stop the primer; thus the default option will allow amplification of a region whose endpoints are in the first and last 35 basepairs, while choosing exact endpoints will cause all primers evaluated to start and end with exactly the same sequence.
  • Primer composition - Primers that contain a skewed AT/GC ratio can fail to give high specificity, or be poorly behaved in other ways. You can enter minimum, optimal, and maximum values for the percentage of basepairs which are either G or C.
  • Primer melting temperature - Melting temperature heavily influences the results of PCR. This tool calculates the Tm of an oligonucleotide by the nearest neighbor method. (For more information, see Borer et al., 1974, Rychlik et al., 1990, or Breslauer et al., 1986.) Minimum, optimum, and maximum values may be set by the user.
  • Primer Annealing - Primers also tend to dimerize and anneal to themselves, which can present significant problems in using PCR. One method to account for this problem was developed by Hillier and Green, 1991 and calculates a local alignment score, either anywhere within the primer or anchored from the 3'-end. For a visual representation of this problem, see the help from Primer3 on max complementarity and max 3' complementarity. Maximum allowable alignment scores for annealing between primers can be set by the user.
  • Ranking the pairs of primers - Primers are assigned a value based on their features and based on the user defined preferences. The best pair of primers is defined to be the pair of primers with the lowest score. This score is calculated in the following way: +1 per 10% difference from the optimal GC percentage, +1 per degree Celsius difference from the optimal Tm, +1 per 5 units of annealing at the end of primers, +1 per 10 units of annealing in the middle of primers, and +1 per 2 basepairs difference from the optimal length.

Primers for Sequencing

  • Location (with respect to Gene chosen) - The segment of DNA that is analyzed to find sequencing primers is determined by the values entered in the Distance boxes as well as the gene entered. For example, if you wanted to sequence YAR024C and approximately 1000 basepairs on either side of the gene then you would enter 1000 in each of the boxes. The segment of DNA chosen is then chopped up into pieces. The length of these pieces is represented by the spacing variable. If you enter 400, these pieces would be 400 basepairs in length. One primer will be chosen from each of these regions. You are allowed to specify how big a region (the "length of DNA in which to search for valid primers" field) in which to accept a primer. If you use the default 35 basepairs, then the FIRST 35 basepairs of each of the pieces of DNA created by the "distance between.." algorithm is analyzed for valid primers. The lengths of valid primers are user definable and can be set by the various "length" fields.
  • Location (with respect to DNA entered) - The segment of DNA entered is first chopped up into pieces. The length of these pieces is represented by the spacing variable. If you enter 400, for example, these pieces will be 400 basepairs in length. One primer will be chosen from each of these regions. You can specify how big a region (the "length of DNA in which to search for valid primers" field) in which to accept a primer. If you use the default 35 basepairs, then the FIRST 35 basepairs of each of the pieces of DNA created by the "distance between.." algorithm is analyzed for valid primers. The lengths of valid primers are user definable and can be set by the various "length" fields.
  • Primer composition - Primers that contain a skewed AT/GC ratio can fail to give high specificity, or behave poorly in other ways. You can enter minimum, optimal, and maximum values for the percentage of basepairs which are either G or C.
  • Primer Annealing - Primers tend to dimerize and anneal to themselves. One method for accounting for this problem was developed by Hillier and Green, 1991. You can set maximum values for annealing between primers.
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