Format for 78 columns, fixed point font, tabs every 8 characters. (10pt-> tabs 0.65 in, left margin 0.5in, right margin +6.5in; 12pt -> 0.78in) 123456 123456 123456 123456 123456 123456 123456 123456 123456 123456 12345678901234567890123456789012345678901234567890123456789012345678901234567890 PRIMER: A Computer Program for Automatically Selecting PCR Primers Version 0.5, May 1991 by Stephen E. Lincoln, Mark J. Daly, and Eric S. Lander MIT Center for Genome Research and Whitehead Institute for Biomedical Research Nine Cambridge Center Cambridge, Massachusetts 02142 Send Inquiries to "Primer c/o The Lander Lab" at the above address. Email to "primer@genome.wi.edu" (Internet) or "lander@mitwibr" (Bitnet) FAX 617-258-6505 PRIMER is copyright 1991 by The Whitehead Institute for Biomedical Research, all rights are reserved. PRIMER may be freely used and distributed, but only under specific conditions as outlined in the license included in this document. Any other use is a violation of applicable laws and regulations. PRIMER Version 0.5 Manual Contents: (1) Introduction (2) How PRIMER views DNA sequences (3) How PRIMER chooses PCR primers (4) How PRIMER chooses PCR primer pairs (5) A sample automatic analysis with PRIMER (6) Manually testing specific primers with PRIMER (7) PRIMER Sequence File Format (8) PRIMER Repeat File Format (9) License Agreement (10) Registration Form (1) INTRODUCTION PRIMER is a computer program for selecting PCR primer pairs to amplify specific regions of sequenced DNA. While PRIMER was specifically designed to select primers from cloned and sequenced genomic DNAs, many of the tests it performs are useful for selecting oligos for other types of experiments as well, including other PCR applications, oligo hybridization and dideoxy sequencing. For any given sequence, PRIMER applies a variety of analyses which determine: (1) which "target" subsequence should be amplified (2) which of the possible primers flanking the target will be compatible with specific PCR reaction conditions, such as annealing temperature. (PRIMER computes annealing temperature based on recent experimental measurements of base-stacking energies, rather than simply counting A-T and G-C basepairs. We find that it improved our success rate.) (3) whether the primers (or the target sequence itself) have a significant degree of complementarity to known repeat elements (such as Alu), possibly resulting in nonspecific annealing and amplification. (4) whether the primers have a significant degree of complementarity to themselves or to each other. (5) whether the PCR product itself has the desired size and GC content. Once configured for specific criteria, PRIMER can apply these tests to many input sequences in an automated manner. PRIMER also allows you to interactively test specific primers you have selected by hand (with or without the computer's advice) against these criteria. This manual (i) explains how PRIMER approaches the task of selecting PCR primers; (ii) illustrate its use; and (iii) describes the format of the data files. Also included with the PRIMER program is a README file, which includes information regarding systems compatibility, installation procedures, licensing, redistribution, and other issues. PRIMER's license requires that you use and redistribute PRIMER only in accordance with these guidelines. (2) HOW PRIMER VIEWS DNA SEQUENCES Given a DNA sequence, PRIMER adopts the following naming conventions: Reverse Primer 3' <--------GGAA-- 5' Template: Plus Strand |||| 5' -----ATCG---------------------============--------------CCTT------------ 3' |||| Target |||| 3' -----TAGC---------------------============--------------GGAA------------ 5' |||| Minus Strand 5' --ATCG--------> 3' Forward Primer Template sequences may be written in either a single stranded or double stranded text format. For single stranded sequence (always written 5' to 3'), PRIMER calls the sequence given in the data file the "plus strand", with the inferred reverse complement the "minus strand". When reading double stranded sequence, PRIMER calls the first (or top) sequence the "plus strand", with the other being the "minus strand". (Further details of input sequence format are available in the "FILE FORMATS" section of this manual). Base numbers always refer to the plus strand. The 5' base of the plus strand is always assigned to be base number 1, with base number N being the 3' terminus (where N is the length of the sequence). Thus, base number 1 is also the 3' end of the minus strand, and base N the 5' end. The PCR "target" is defined to be the minimal region of the sequence which must be included in a PCR product. In the input file, the target may be specified explicitly (as a particular range of bases) or implicitly (as the region containing some repeated motif). If the user does not care which region is chosen, no target need be specified. If a target is specified, PRIMER allows the user to specify whether primers are allowed to bind within the target or not, that is: Case 1: Primers are NOT allowed within target. <-------- + --------------=====================------------ Target - --------------=====================------------ --------> |---------------- PCR Product ------------| Case 2: Primers ARE allowed within target. <-------- + --------------=====================------------ Target - --------------=====================------------ --------> |------- PCR Product ------| PRIMER calls a "forward primer" an oligo which reads (5' to 3') as the plus strand does. A forward primer is thus chosen to the left of the target (that is, when reading the plus strand), and in the actual PCR reaction, the forward primer will bind to the minus strand of the template DNA. Similarly, a "reverse" primer is chosen to the right of the target, which must read as the minus strand does, so that it binds to the plus strand during the PCR reaction. It is sometimes convenient to provide PRIMER with a larger sequence than you actually wish the consider usable for PCR. For example, you may which to have PRIMER analyze raw DNA sequence data "straight off the gel", in which case PRIMER might need to be careful not to select PCR primers within the sequencing vector's DNA, but rather within the insert. For this reason, PRIMER restricts its analyses to a "region of interest", a subsequence in which all primers must fall (Obviously, the "target" must be strictly contained in the "region of interest.") The region of interest may be specified explicitly or implictly (e.g., the region between specified restiction sites, including the sites themselves). This is illustrated below: <-------CTTA 5' ----------GAATTC----------------============------------GAATTC----- 3' Eco RI Target Eco RI 3' ----------CTTAAG----------------============------------CTTAAG----- 5' ----------> |-------------- Region of Interest ----------------| (3) HOW PRIMER CHOOSES PCR PRIMERS Given the target and region of interest, PRIMER tries to narrow down the regions in which to search for primers. First, if the user has specified a target and a PCR product size range, PRIMER can immediately restrict its search to regions of the sequence at an appropriate distance from the target. Next, PRIMER tries to determine if the sequence has any regions which display significant similarity to specified repeat elements (e.g., Alu repeats in humans). PRIMER avoids primers that would bind to such regions. For example: <---------- 5' --------------+++++++++++++++-------============----------------------- 3' Alu-Like Region Target 3' --------------+++++++++++++++-------============----------------------- 5' ---------> If PRIMER primer cannot identify adequate repeat-free regions, it may be unable to select primers at all and will generate a warning message. After narrowing down possible regions, PRIMER enumerates all forward and reverse primers of the desired length(s), and exhaustively tests each against the user-specified criteria (e.g., annealing temperature). PRIMER allows one to specify a fixed or variable primer length. With variable primer lengths, PRIMER will try to lengthen or shorten any PCR primer in order to adjust its melting temperature into the desired range. For example: If the desired temperature range is 53-58 degrees, and the desired primer length is 18-24 bases, PRIMER will find that: Primer Length Tm ACTTACTTCTTAAGGTCC 18 42.7 ACTTACTTCTTAAGGTCCA 19 46.3 ACTTACTTCTTAAGGTCCAC 20 47.7 ACTTACTTCTTAAGGTCCACA 21 50.9 ACTTACTTCTTAAGGTCCACAC 22 52.0 ACTTACTTCTTAAGGTCCACACC 23 55.7 ACTTACTTCTTAAGGTCCACACCC 24 59.0 Primers which pass the temperature criteria are then tested for "self- complementarity". Two tests are performed in this analysis: one to detect any subsequence self-complementarity along the primer, and one to detect complementarity specifically at the primer's 3' terminus (which might allow false priming). Primers are also tested for whether they cover a region of the sequence with an ambiguous base (e.g. an "N"), a case which may be completely disallowed by the user. The user may also require that the 3' end of the primer be "GC clamped", that is consist of only G's and C's. Finally, primers which pass all of these tests are themselves double-checked for homology to the repeat sequences, using a somewhat more sensitive, although slower, test than employed initially. (4) HOW PRIMER CHOOSES PCR PRIMER PAIRS Having generated a list of allowable forward and reverse primers, PRIMER then enters its final analysis step - finding "optimal" forward and reverse primer pairs to recommend (this last step is optional, or may be performed manually). PRIMER will try to recommend a single pair for any requested PCR product size range. If multiple ranges are allowed, PRIMER will recommend a separate pair for each range. PRIMER applies similar criteria to those above to determine if each pair is acceptable. The primers are tested for pairwise complementarity (that is, for possible primer-heterodimer formation) as well as compatibility of annealing temperatures. When presented with an especially long list of possible pairs, PRIMER limits its search to those pairs with the most similar annealing temperatures. (5) A SAMPLE RUN AUTOMATIC AUTOMATIC ANALYSIS WITH PRIMER PRIMER performs all of its data input and output using a number of ordinary text files, as listed below: File PRIMER will Name Read/Write Contents ---- ---------- -------- xxx.seq read A DNA sequence (or sequences) for PRIMER analysis yyy.cri read/write The criteria to be used for choosing primers zzz.rep read Genomic repeat sequences to look for xxx.pcr write Automatic PRIMER analysis of sequence xxx xxx.for write List of forward primers examined for sequence xxx xxx.rev write List of reverse primers examined for sequence xxx xxx.tst write Results of manual PRIMER analysis of sequence xxx In all cases, you should be able to read, write, create, and print these files by using standard software packages (e.g., word processors) on your computer. You will need to create a sequence (".seq") file by hand in the format described in this document, although you may have a sequence analysis system which can generate a compatible file. Often, you can quickly paste the sequence from any standard sequence analysis package's output into a PRIMER input file. You can create a repeat (".rep") file the same way, although we have included samples which may be useful to you as is. The criteria file (".cri") is most easily created by having PRIMER interactively prompt you through the process, as shown below. PRIMER assumes that all data files reside in the same directory (or folder) that was open when the program was invoked. Here we illustrate a dialog with PRIMER where we have analyzes a *** base sequence, named "seq1" and stored in the file "sample.seq". The criteria for this analysis have previously been entered into the file "standard.cri", and the repeats we use to consider are the mouse sequences L1, B1, and B2, stored in the file "mouse.rep". Our criteria for single primers are as follows: * We require primers to be 18-22 bases long. * We require Tm from 53-58 degrees, with 55 degrees considered best. * Primers must have 30-70% GC content (recall that this is a different test than that for Tm). * Primers may not span ambiguous sequence (they may not contain N's) * Primer's may not have more than 12 bases of self-complementarity, or more than 8 bases if the matching region is 3' terminal. * The single 3' base of the primer should be a G or C. * PCR product length should be 100-150 bases or 200-250 bases. As you see, in many cases these are the default PRIMER criteria, indicated by the program in brackets, and which we accept simply by hitting the return key. As you will see, PRIMER finds a number of acceptable forward and reverse primers, and recommends two pairs, one each in the size range 100-150 and 200-250 bases. ********************************************************************** * Welcome to: * * * * PRIMER * * (Version 0.5) * * * * Copyright 1991, Whitehead Institute for Biomedical Research * ********************************************************************** File containing template sequence(s): sample.seq One sequence available in file "sample.seq": seq1 (422 bases) Would you like: 1) automatic analysis of this sequence or 2) manual testing of primers for this sequence? 1 PRIMER output file [seq1.pcr] File containing PRIMER criteria [hit RETURN to enter new criteria now]: OPTIMAL primer length [20]: MINIMUM primer length [18]: MAXIMUM primer length [22]: OPTIMAL primer melting temperature [60.0]: 55.0 MINIMUM acceptable primer melting temperature [57.0]: 53.0 MAXIMUM acceptable primer melting temperature [63.0]: 58.0 MINIMUM acceptable primer GC% [20]: MAXIMUM acceptable primer GC% [80]: Salt concentration (mM) [50.0]: DNA concentration (nM) [50.0]: MAXIMUM number of unknown bases (Ns) allowed in a primer [0]: 1 MAXIMUM acceptable primer self-complementarity (number of bases) [12]: MAXIMUM acceptable 3' end primer self-complementarity (number of bases) [8]: GC clamp how many 3' bases [0]: 1 Restriction sites which flank region of interest [ ]: Product length ranges [ 100-150 150-250 250-400 ]: File to save these criteria in [hit RETURN for none]: File containing repeat sequences [hit RETURN for none]: mouse.rep Repeat sequence #1: B1 (132 bases) Max score: 12 Max 3' score: 10 Repeat sequence #2: B2 (178 bases) Max score: 12 Max 3' score: 10 Repeat sequence #3: L1 (7713 bases) Max score: 16 Max 3' score: 14 Saving primer lists to files seq1.for and seq1.rev Analyzing sequence #1 (seq1) Searching for repeat #1 (B1)...not found. Searching for repeat #2 (B2)...not found. Searching for repeat #3 (L1)...not found. Examining 142 forward primers...38 forward primers accepted. Examining 192 reverse primers...53 reverse primers selected Testing pairs... Primers selected! To see PRIMER's results, we now look in the output file "seq1.pcr", shown below: ************************************************************************ * Output from: * * * * PRIMER * * (Version 0.5) * * * * Copyright 1991, Whitehead Institute for Biomedical Research * ************************************************************************ Analyzing sequence 1 (seq1) '*' = target, 'X' = restriction site, '?' = N, number = high repeat homology +10 +20 +30 +40 TNNAGCTCGG TACCCGGGGA TCCTAATAGA ATCTCAGAAT CTGAGAATAA 1 -??------- ---------- ---------- ---------- ---------- ANNTCGAGCC ATGGGCCCCT AGGATTATCT TAGAGTCTTA GACTCTTATT +10 +20 +30 +40 CAGGNTCCAT GAAATGANTG TCAAAGAAAA AGATTCAGAG AAGAGAAAGC 51 ----?----- -------?-- ---------- ---------- ---------- GTCCNAGGTA CTTTACTNAC AGTTTCTTTT TCTAAGTCTC TTCTCTTTCG +10 +20 +30 +40 GGGTCAGCTG AGTAATAAAC ACCATGCAAA AATGAAACAA ACAAACCCCT 101 ---------- ---------- ---------- ---------- ---------- CCCAGTCGAC TCATTATTTG TGGTACGTTT TTACTTTGTT TGTTTGGGGA +10 +20 +30 +40 TGCCCCCTCA ACACACACAC ACACACACAC ACACACACAC ACACACACAC 151 ---------- -********* ********** ********** ********** ACGGGGGAGT TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG +10 +20 +30 +40 ACACACACAC ACTTACTTCT TAAGGTCCAC ACCCGCAGCA GCAGCAGCAT 201 ********** *--------- ---------- ---------- ---------- TGTGTGTGTG TGAATGAAGA ATTCCAGGTG TGGGCGTCGT CGTCGTCGTA +10 +20 +30 +40 CACCTGGGGC TGTGTTAGAG ATATGGGCTA TAGTCTAGCT CCNGTCTCTG 251 ---------- ---------- ---------- ---------- --?------- GTGGACCCCG ACACAATCTC TATACCCGAT ATCAGATCGA GGNCAGAGAC +10 +20 +30 +40 AGGCTGAACT GTACACTCTT TCTTTTACCT GGNAACTGTA TTTCCCTGTG 301 ---------- ---------- ---------- --?------- ---------- TCCGACTTGA CATGTGAGAA AGAAAATGGA CCNTTGACAT AAAGGGACAC +10 +20 +30 +40 ATTTCACGGG NCTTTCTTAG GCATGCACGC CAGTTTTCTN ATCTTGCAAT 351 ---------- ?--------- ---------- ---------? ---------- TAAAGTGCCC NGAAAGAATC CGTACGTGCG GTCAAAAGAN TAGAACGTTA +10 +20 +30 +40 TTGGTTAAGG GTAGTAATCC TT 401 ---------- ---------- -- AACCAATTCC CATCATTAGG AA Acceptable regions for a primer: base 1 to 161 base 212 to 422 Examining 142 forward primers...38 forward primers accepted. Examining 192 reverse primers...53 reverse primers accepted. Testing pairs... Product size range 100-150... forward primer 127->147 : CAAAAATGAAACAAACAAACC Tm = 54.9 reverse primer 272->254 : ATCTCTAACACAGCCCCAG Tm = 55.2 PCR product length: 146, GC = 50% Product size range 150-250... forward primer 107->127 : GCTGAGTAATAAACACCATGC Tm = 55.0 reverse primer 278->259 : GCCCATATCTCTAACACAGC Tm = 54.9 PCR product length: 172, GC = 49% Product size range 250-400... forward primer 107->127 : GCTGAGTAATAAACACCATGC Tm = 55.0 reverse primer 401->381 : AATTGCAAGATNAGAAAACTG Tm = 55.0 PCR product length: 295, GC = 47% (6) MANUALLY TESTING SPECIFIC PRIMER PAIRS WITH PRIMER If PRIMER's automatic analysis of a sequence has been completely successful, the output ".pcr" file will fully describe the oligo pairs which PRIMER recommends you use; you need not examine any other information. In some cases however, you may wish to read to the ".for" and ".rev" files describing all of the candidate forward and reverse primers. If you wish, you can then invoke PRIMER's "manual" analysis mode to test any primer pairs you wish. In addition, you can use PRIMER's manual mode to determine whether ANY primer pair you have selected meets your specified criteria, even in cases where the template sequence is not available. As an example, we first look in the "seq1.for" file generated by the previous analysis: salt conc. = 50.00 mM DNA conc. = 50.00 nM |Self |--B1-- --B2-- --L1--| ? base primer 5'->3' Tm GC% |any 3' |any 3' any 3' any 3'| > 113 TAATAAACACCATGCAAAAATG 55.5 27% | 4 3 | 6 4 7 5 12 12 | ok > 114 AATAAACACCATGCAAAAATG 55.3 29% | 4 3 | 6 4 7 5 12 12 | ok > 115 ATAAACACCATGCAAAAATG 53.7 30% | 4 3 | 6 4 7 5 11 11 | ok > 116 TAAACACCATGCAAAAATGA 55.1 30% | 4 2 | 6 3 7 6 11 10 | > 117 AAACACCATGCAAAAATGAA 56.6 30% | 4 2 | 6 3 7 5 11 10 | > 118 AACACCATGCAAAAATGAAA 56.6 30% | 4 1 | 6 4 7 6 11 11 | > 119 ACACCATGCAAAAATGAAAC 56.0 35% | 4 1 | 6 5 7 5 10 10 | ok > 120 CACCATGCAAAAATGAAAC 54.9 37% | 4 1 | 6 5 7 5 10 9 | ok > 121 ACCATGCAAAAATGAAACAA 56.6 30% | 4 1 | 5 3 6 6 10 10 | > 122 CCATGCAAAAATGAAACAAA 57.1 30% | 4 1 | 5 3 7 7 10 10 | > 123 CATGCAAAAATGAAACAAAC 54.3 30% | 4 1 | 5 4 7 6 11 11 | ok > 124 ATGCAAAAATGAAACAAACA 54.3 25% | 4 2 | 5 3 7 6 11 11 | > 125 TGCAAAAATGAAACAAACAA 55.4 25% | 4 1 | 5 4 7 7 12 12 | > 126 GCAAAAATGAAACAAACAAA 54.0 25% | 2 1 | 5 5 8 8 12 11 | > 127 CAAAAATGAAACAAACAAACC 54.9 29% | 2 1 | 7 7 8 8 12 10 | ok > 128 AAAAATGAAACAAACAAACCC 55.8 29% | 2 1 | 8 8 8 7 11 11 | ok > 129 AAAATGAAACAAACAAACCC 54.2 30% | 2 1 | 8 8 8 7 11 11 | ok > 130 AAATGAAACAAACAAACCCC 56.4 35% | 2 1 | 8 7 8 6 11 10 | ok > 131 AATGAAACAAACAAACCCCT 56.1 35% | 2 1 | 8 7 8 7 10 10 | > 132 ATGAAACAAACAAACCCCTT 56.1 35% | 2 2 | 8 7 7 6 11 11 | > 133 TGAAACAAACAAACCCCTT 55.5 37% | 2 2 | 8 7 7 6 11 11 | > 134 GAAACAAACAAACCCCTTG 55.5 42% | 3 3 | 8 6 7 5 11 10 | ok > 135 AAACAAACAAACCCCTTGC 57.6 42% | 3 2 | 8 6 7 5 11 11 | ok > 136 AACAAACAAACCCCTTGC 56.0 44% | 3 2 | 8 6 6 5 11 11 | ok > 137 ACAAACAAACCCCTTGCC 58.4 50% > 138 CAAACAAACCCCTTGCCC 61.3 56% > 139 AAACAAACCCCTTGCCCC 62.0 56% > 140 AACAAACCCCTTGCCCCC 64.2 61% > 141 ACAAACCCCTTGCCCCCT 63.9 61% > 142 CAAACCCCTTGCCCCCTC 65.0 67% In this list, PRIMER has displayed its analysis results for all of the listed primers, including: * 5' base and sequence of the primer * Melting temperature (Tm) and GC percentage * Degree of self-complementarity, both for any subsequence and for a 3' terminal subsequence, specified as # of bases of match. PRIMER's matching algorithm allows mismatches and gaps, with reduced score. * Degree of homology to repeat sequences, specified as above. * Whether the primer has passed the criteria, indicated by an 'ok' in the right column. Note that PRIMER does not perform homology tests if the annealing temperature is unacceptable. From this list, we can choose a forward primer, for example one spanning 20 bases starting with base 10 in the template: 5' GTACCCGGGGATCCTAATAG 3' From the seq1.rev file we select a reverse primer, spanning bases 273-254: 5' TATCTCTAACACAGCCCCAG 3' To manually test these primers, we interactively invoke PRIMER, loading in the sequence and specifying the two primers. PRIMER first performs the "single primer" analysis, described previously, and outputs the same data it would place into the ".for" and ".rev" files. PRIMER then performs a pairwise analysis, also just as before. In this case we will specify primers by their location in the template. However, PRIMER allows us to specify both forward and reverse primers with: * actual base composition (which must exactly match the appropriate strand of the template). * the 5' base of the primer and its length, separated by spaces * the bases spanned by the primer, as we did here. In addition, PRIMER allows you to place particular primer specifications in the input sequence file, which it will test in pairwise combinations as shown here. This feature is useful when developing either multiple or multiplex PCR assays. ********************************************************************** * Welcome to: * * * * PRIMER * * (Version 0.5) * * * * Copyright 1991, Whitehead Institute for Biomedical Research * ********************************************************************** File containing template sequence(s): sample.seq ==== One sequence available in file "sample.seq": seq1 (422 bases) ==== Would you like: 1) automatic analysis of this sequence or 2) manual testing of primers for this sequence? 2 Where would you like output sent ["seq1.pcr"] File containing PRIMER criteria [hit RETURN to enter new criteria now]: sample.cri Would you like to edit these criteria (y/n): n File containing repeat sequences [hit RETURN for none]: mouse.rep Repeat sequence #1: B1 (132 bases) Max score: 12 Max 3' score: 10 Repeat sequence #2: B2 (178 bases) Max score: 12 Max 3' score: 10 Repeat sequence #3: L1 (7713 bases) Max score: 16 Max 3' score: 14 Manual primer examination - enter 0 to quit Enter primers as actual sequence (e.g., AAGTCGTCATACGTCCAT), base range (e.g., 120-140), or starting base and length (e.g., 220 20) FORWARD primer: 10 20 REVERSE primer: 273-254 |Self |--B1-- --B2-- --L1--| ? base primer 5'->3' Tm GC% |any 3' |any 3' any 3' any 3'| > 10 GTACCCGGGGATCCTAATAG 57.4 55% | 6 5 | 5 4 5 4 10 9 | ok < 273 TATCTCTAACACAGCCCCAG 55.4 50% | 2 2 | 7 5 8 8 10 9 | ok PCR product length: 264, GC = 46%, pairwise match (any 3' 3'): 4 2 3 FORWARD primer: 0 (7) PRIMER INPUT SEQUENCE FILE FORMAT The input sequence file must be prepared properly for analysis by PRIMER. In addition to specifying the template sequence itself, this file also provides PRIMER with the name of the sequence and other bits of information which describe the target, region of interest, specify the actions of the program. The first line of information for each sequence is the sequence name which follows the "*sequence: " identifier. After this, several other lines of information may be given. Often, you can quickly paste the sequence from any standard sequence analysis package's output into a PRIMER input file. However, we may be able to modify PRIMER to read other sequence formats, but we need help in deciding which are important. If you have a favorite sequence format (so long as it is a text file of some sort), send an example to us by floppy disk or Email, and we may be able to modify the program to read it. Even better, make the change to the PRIMER code yourself and send us the changes to incorporate! The format is as shown in the following examples. First, a simple example of a file with one sequence and nothing else specified: *sequence: seq1 TNNAGCTCGG TACCCGGGGA TCCTAATAGA ATCTCAGAAT CTGAGAATAA CAGGNTCCAT GAAATGANTG TCAAAGAAAA AGATTCAGAG AAGAGAAAGC GGGTCAGCTG AGTAATAAAC ACCATGCAAA AATGAAACAA ACAAACCCCT TGCCCCCTCA ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACTTACTTCT TAAGGTCCAC ACCCGCAGCA GCAGCAGCAT CACCTGGGGC TGTGTTAGAG ATATGGGCTA TAGTCTAGCT CCNGTCTCTG AGGCTGAACT GTACACTCTT TCTTTTACCT GGNAACTGTA TTTCCCTGTG ATTTCACGGG NCTTTCTTAG GCATGCACGC CAGTTTTCTN ATCTTGCAAT TTGGTTAAGG GTAGTAATCC TT We have not specified a particular target for the above sequence, and thus PRIMER will find ANY PCR assay which amplifies a fragment of desired size. If instead, we specifically want to amplify the CA repeat in the above sequence, we could specify: *sequence: seq1 *target: (CA) TNNAGCTCGG TACCCGGGGA TCCTAATAGA ATCTCAGAAT CTGAGAATAA CAGGNTCCAT GAAATGANTG TCAAAGAAAA AGATTCAGAG AAGAGAAAGC GGGTCAGCTG AGTAATAAAC ACCATGCAAA AATGAAACAA ACAAACCCCT TGCCCCCTCA ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACTTACTTCT TAAGGTCCAC ACCCGCAGCA GCAGCAGCAT CACCTGGGGC TGTGTTAGAG ATATGGGCTA TAGTCTAGCT CCNGTCTCTG AGGCTGAACT GTACACTCTT TCTTTTACCT GGNAACTGTA TTTCCCTGTG ATTTCACGGG NCTTTCTTAG GCATGCACGC CAGTTTTCTN ATCTTGCAAT TTGGTTAAGG GTAGTAATCC TT this instructs PRIMER to use the longest tandem repeat of CA found as the target. Alternatively, we could explicitly delimit the target with brackets: *sequence: seq1 *target: [] TNNAGCTCGG TACCCGGGGA TCCTAATAGA ATCTCAGAAT CTGAGAATAA CAGGNTCCAT GAAATGANTG TCAAAGAAAA AGATTCAGAG AAGAGAAAGC GGGTCAGCTG AGTAATAAAC ACCATGCAAA AATGAAACAA ACAAACCCCT TGCCCCCT[CA ACACACACAC ACACACACAC ACACACACAC ACACACACAC ACACACACAC A]CTTACTTCT TAAGGTCCAC ACCCGCAGCA GCAGCAGCAT CACCTGGGGC TGTGTTAGAG ATATGGGCTA TAGTCTAGCT CCNGTCTCTG AGGCTGAACT GTACACTCTT TCTTTTACCT GGNAACTGTA TTTCCCTGTG ATTTCACGGG NCTTTCTTAG GCATGCACGC CAGTTTTCTN ATCTTGCAAT TTGGTTAAGG GTAGTAATCC TT The allowable entries in the full format are described below. Entries below in angle brackets ("<" and ">") indicate that you should fill in the appropriate value. X and Y refer to DNA bases (GATC), and n and m are numbers. *sequence: The name of this sequence, must start a sequence entry. Sequence names should be no more than 8 characters. *target: Specify the target as: (XX) target is a tandem repeat of XXs (XX)(YY) target flanks the largest XX and YY repeats n-m target is base number n through m n target is base number n [] target is enclosed in the sequence by square brackets XXXXXXX target is this subsequence *subsequence: Specify the region of interest as: {} region of interest is enclosed in the sequence by braces n-m region of interest is base number n through m *product: Specify the product size for THIS assay n-m desired product size is n through m bases n-m p-q desired product size is n through m or p through q bases *double-stranded Indicates that double-stranded sequence follows Following the above notations, the raw DNA sequence should be given. PRIMER's format is fairly flexible, and basically looks only for occurrences of the letters A, C, T, and G, upper or lower case. All other characters are ignored, allowing one to easily have PRIMER read the output of sequence management programs. For double stranded sequence, once PRIMER has read a line with any sequence, the next sequence containing line is expected to have the reverse complement of the previous sequence. After the sequence is given, individual PCR primers may be given by adding a line: *primers: Indicating that primers which should be tested are listed on the following lines. Primers may be specified as DNA subsequences, by the base range spanned (e.g. n-m),or by base number and length. Primers must be given one to a line, and each line must start with a ">", indicating a forward primer, or a "<", indicating a reverse primer. For example: *primers: >TCCTAATAGAATCTCAGAAT