Element name | Description | FastPCR online Support |
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/? | Loading current options of PCR primers design: /? | no |
-iPCR | Inverted PCR: { -ipcr } | yes |
-npd | Exclude primers design, but external primers can be added with the -fpr[] command: { -Fnpd -Fpr[attccattccgcgttcga/cgttacggtatttcttgc] } | yes |
-pcrNo | PCR primers design only, no PCR primer combinations reporting (is equivalent to "-npc0" command): > -pcrNO | yes |
(N1-N2) | specify the minimal and maximal size requested for the PCR product, N1 for shortest, N2 for the longest PCR product; (N1) or (N1-N2) is allowed: > (400-500) | yes |
-pdN1-N2 | Primer Design to define position of the target DNA on initial N1 and final N2 position (N2>N1) in DNA sequence: > -pd350-700 design PCR primers between coordinate N1 and N2 (N2>N1) for Forward or Reverse: > -Fpd100-500 -Rpd1000-1200 User can specify the area in which to search for primers around the area (-pdN1-N2/N3), for example, interested area 400-500, and we want to pick up primers surrounding the site within 100 bases, whereas the primers for Forward will be between 300-400, and 500-600 for Reverse: > -pd400-500/100 this command is equal to -Fpd300-400 -Rpd500-600 generally: -pdN1-N2/N3 is equal to -Fpd(N1-N3)-N2 -RpdN2-(N2+N3) | yes |
-pdN-e | Design PCR primers in area: from the end of sequence minus N bases design left or right PCR primers from the end of sequence minus N bases: > -Fpd200-e > -Rpd200-e | yes |
-excludeN1-N2 | The Excluded Region List(s) denotes locations where primers and probe must not bind. Multiple excluded regions may be defined per sequence. This feature can be used to avoid unwanted regions (ex. intron, SNP etc). For example, if the excluded region is bases 500-1000 for the target selection need use the command: > -exclude500-1000 An alternative way is to use two sign '/' for the start and end of such excluded region (possible multiple times): >example1 [gtcccgagaacctgagtatgcatcacccggatcgcttcttcc/gggaggtgttggggg/ ctatctcggtgttttctgactgcttggcttccgcgagtcattgccatgctagcgta] [attgcaataaccggagcgagatgatgcacc/ccccc/ccttgacaagcgccaataccacgcactattaagagtaaaaaaaa] | yes |
-pr[NNN/NNN] | External primer(s) for use in PCR analysis, at 5'->3', with a "/" to separate the primers, automatic detection of the location of the primer with mismatches;: > -pr[attccattccgcgttcga/tcctacgttccgttacc] pre-designed Forward PCR primer: > -Fpr[attccattccgcgttcga/cgttacggtatttcttgc] | yes |
-npcN | Determine the maximum Number of Primers Combinations, for example 10 (-npc0 is equivalent to "-pcrNo" command): > -npc10 | yes |
-nprN | Showing maximal Number of designed Primers per task: > -npr10 | yes |
-ptmsN | Synchronizing Tm for Primer Pair (±°C): > -ptms10 | yes |
The user can specify individually for each sequence location for both Forward and Reverse primers design using ‘[‘ and ‘]’ inside each sequence. Optionally - use two ‘/.../’ signs for the start and end of the excluded region (this is possible multiple times).
All possible combinations of '[ Forward ]' with '[ Reverse ]' within the sequence(s):
1. [ ]
2. [ ] [ ]
3. [ [ ] ]
Element name | Description | FastPCR online Support |
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-lnN1-N2 | Determines the range of primer length (12-500 nt), N1(Minimal)-N2(Maximal) length of primers (ex., between 18 to 32 nt: -ln18-35) or probes (ex., between 12 to 28 nt: -pln12-28): > -FlN18-22 -RlN22-22 | yes |
-tmN1-N2 | Determines the range of primer Tm, N1(Minimal)-N2(Maximal) Tm of primers or probe: > -Ftm40-50 -Rtm50-60 | yes |
-3tmN1-N2 | Determines the range of primer Tm at 3’end, N1(Minimal)-N2(Maximal) Tm for last 12 bases at the 3'-end of primers: > -F3tm31-35 -R3tm35-45 | yes |
-qN | Primer Quality (PQ) (virtual PCR efficiency) is the theoretical primer PCR efficiency value, it is recommended closer to 90%. By default -q70, but it can be reduced to -q50, in situations where primers are cannot be designed with the default option. > -Fq50 -Rq90 | yes |
-lcN | Linguistic sequence complexity (LC), this value is in percentage (20-100%), it is recommended closer to 90%. By default -lc70, but it can be reduced to -lc50, in situations where primers are cannot be designed with the default option. > -lc60 -Pq70 | yes |
-dmr=N | The level of sensitivity for primer-dimer detection, default value is 1 (as the strictest criterion), the higher the value, the lower the detection sensitivity, if the value is 0, the program will not assess for the presence of primer-dimers, the optimal value of this parameter is 2, as the most realistic. N from 0 to 5. Level 1 is the default and is the most stringent criterion, the value of 5 for insensitive detection. > -dmr=0 | yes |
-opYes|No | Primers overlapping control: if type -FopNo, all forward primers will not overlap; -RopYes all reverse primers will overlap: > -FopNo -RopYes | yes |
-ctYes|No | (Copy Test) Non-specific priming control is avoiding primer design on repeated sequences in the DNA template. By default, this command is -ctYes, checking the secondary (non-specific) binding test. > -FctYes -RctNo | yes |
-x3e | Design PCR primer for a specific sequence on the fixed 5' or 3' ends to selected sequence, for example, if user need to link all primers to 5'end of sequence, use -fx5e, program will show all primers with the same location but different length. The same situation is for a linkage the 3'end of primers to a certain location: > -fx3e | yes |
-c3[NNN/NNN] | Specifies for primers 3’ends with these patterns with three bases per pattern. Certain sequence(s) in the direct or complementary (second c) orientation for Forward or Reverse primers (probes) 3' or 5'-ends nucleotide composition or an indication of a specific sequence location; the program accept the universal degenerate DNA code (IUB/IUPAC), and minimum 2 bases in length and maximum for the primer length: [NNN/NNN] - for any bases, example for Forward primers -c3[RRY] is equivalent to -c3[aat/gat/agt/ggt/aac/gac/agc/ggc]; accepted the sequences with different lengths: > -c3[wss/wss/ssw] > -c3[ACCC/TTCG] | yes |
-z3eNameEnzyme | Primers (probes) directly designed to the restriction enzyme site at 3' end.“NameEnzyme” is the restriction enzyme name: example, this is as the alternative command: –c3YCATG^R is the same as –z3eXceI: > -Fz3eXceI Result: 3’end of all primers contains sequences: YCATGR For not included enzymes in FastPCR database, this command will be ignored. | no |
-5e[NNN] or -5e[NNN/NNN] | Adding non-template DNA sequences to primer ends: adding sequence to the 5’end with command: ‘-5e[NN]’ or adding sequence to the 3’end with command: ‘-3e[NN]’, where ‘NN’ is a sequence of one or more bases, for example: ‘-F5e[CGACG] -R5e[TTTTTT]’, adds sequence ‘CGACG’ to forward primers and sequence ‘TTTTTT’ to reverse primers at 5’ends. For tasks in which it is necessary to specify several allelic variants with individual added tail sequences, which can be indicated with a separator, the forward slash character "/" is used to denote each tail variant. For example, for bi-allelic scoring of single nucleotide polymorphisms (SNPs) and insertions with deletions (Indels) at specific loci in relation to the development of competitive allele specific PCR (AS-PCR) genotyping assays, the two allele-specific primers will be added with the standard FAM (5′-GAAGGTGACCAAGTTCATGCT-3′) and HEX (5′-GAAGGTCGGAGTCAACGGATT-3′) tails at the 5′ end: > -F5e[gaaggtgaccaagttcatgct/gaaggtcggagtcaacggatt] | yes |
-3e[NNN] or -3e[NNN/NNN] | Adding non-template DNA sequences to primer ends: adding sequence to the 3’end with command: ‘-3e[NN]’, where ‘NN’ is a sequence of one or more bases, for example: ‘-F3e[CGACG] -R3e[TTTTTT]’, adds sequence ‘CGACG’ to forward primers and sequence ‘TTTTTT’ to reverse primers at 3’ends. > -F3e[GG] -R3e[CC] | yes |
-excludeIUPACN | Avoidance of degenerate (mixed, ambiguous) IUPAC nucleotides within primer/probe sequences. N - allowed number of degenerate nucleotides in the primer sequence; the default is 0; no more than 2 degenerate bases in the primer sequence: -nomxbpr2 | no |
Element name | Description | FastPCR online Support | -LampN | Loop-mediated Isothermal Amplification (LAMP) primer design: { -Lamp1 } - LAMP assay design without using Loop Primers { -Lamp2 } - LAMP assay design with Loop Primers { -ln18-25 -tm55-57 -c3[sww sws wws wss www] } >example1 -Lamp1 cctgcgatcgcacgcacgaagtaagggtattccatgcgtctgggtgggcacagtacaaaggggcaggattcgcctgaagatataccttgaatgtta >example using Loop Primer: -Lamp2 cctgcgatcgcacgcacgaagtaagggtattccatgcgtctgggtgggcacagtacaaaggggcaggattcgcctgaagatataccttgaatgtta | yes |
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-AsPCRN | Allele-specific polymerase chain reaction (AS-PCR): KASP™ (Kompetitive Allele Specific PCR) or PACE™ (PCR Allele Competitive Extension) based genotyping assay design for biallelic (or up to four variants) discrimination of single nucleotide polymorphisms (SNPs) and insertions and deletions (Indels) at specific loci. N - the number (length) of polymorphic nucleotides from the 3'end; the default is 2 (-asPCR2), where the SNP is located at the penultimate base from the 3'-end of primer. Recommended minimum 2, in this case, SNP is located at the penultimate base from the 3'-end of primer; for InDels variants, this number is equal to polymorphic nucleotides at the 3'-end. If user do not specify the number: -asPCR, in this case the program will automatically determine the optimal length for effective discrimination of single nucleotide polymorphisms. In this example, the command (-P5e[gaaggtgaccaagttcatgct/gaaggtcggagtcaacggatt]) is indicated of adding of 2 variants of 5'-tail sequences for each allelic variant and the amplicon size is not more than 150 nucleotides (40-150): { -aspcr2 -P5e[gaaggtgaccaagttcatgct/gaaggtcggagtcaacggatt] (40-150) } >example1 cttagatcgacaggtctaagagctgaagagctagctattaaagtcgagc [C/G] agctgctagacgtcgcagtcgacacagctagcctaggacaaagtctcgtg >example2 cttagatcgacaggtctaagagctgaagagctagctattaaagtcgagc [S] agctgctagacgtcgcagtcgacacagctagcctaggacaaagtctcgtg Advanced example - 3 InDels variants: > -aspcr3 -P5e[gaaggtgaccaagttcatgct/gaaggtcggagtcaacggatt/gaaggtcggagtcaacggacc] TAAGAGCTGAAGAGCTAGCTGGCTAGCTGATTAA [ATAT/CC/A] AGCTGCTAGACGTCGCAGTCGACACTGACGTCCTAGGACAAAGTCTCGTG | yes |
-ASqPCRN | Allele-specific quantitative (real-time) polymerase chain reaction (AS-qPCR) - genotyping assays are based on competitive allele-specific qPCR and enable specific-allelic scoring of single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) at specific loci. The AS-qPCR Assay mix contains assay-specific non-labeled oligos: allele-specific forward primers and one common reverse primer with one common TaqMan/MGB-probe as a reporter. N - the number (length) of polymorphic nucleotides from the 3'end; the default is 2 (-asqPCR2), where the SNP is located at the penultimate base from the 3'-end of primer. Recommended minimum 2, in this case, SNP is located at the penultimate base from the 3'-end of primer; for InDels variants, this number is equal to polymorphic nucleotides at the 3'-end. If user do not specify the number: -asqPCR, in this case the program will automatically determine the optimal length for effective discrimination of single nucleotide polymorphisms. >MGB-example: -ASqPCR2 -ln16-28 -Tm54-56 -pln12-28 -pTm53-57 gagagtgaccgattgcgtatacctgaacaactaacccccatcggccatcgcagtggcgctcccggcactgagaaaacgtccctaaccttgtgccccg ctcacatagacagcgataacaattttatgagatatcggctgtttaggaagtgcaaatgttaacgactgtct [ATAGACGTCGATCGT/] tacgatataagtgccaagataagacttcggaattaaatacttgggtttctacccgcaatacgaggtgcatgtgcaccatttcgatatcatgaggtgg ttgtggactagatgcatagctctttctattattgtgctgccattcacgctccaggagaccttacccagttt >TaqMan-example: -ASqPCR1 -ln16-28 -Tm54-56 -pln12-28 -pTm59-63 gagagtgaccgattgcgtatacctgaacaactaacccccatcggccatcgcagtggcgctcccggcactgagaaaacgtccctaaccttgtgccccg ctcacatagacagcgataacaattttatgagatatcggctgtttaggaagtgcaaatgttaacgactgtct [ATAGACGTCGATCGT/] tacgatataagtgccaagataagacttcggaattaaatacttgggtttctacccgcaatacgaggtgcatgtgcaccatttcgatatcatgaggtgg ttgtggactagatgcatagctctttctattattgtgctgccattcacgctccaggagaccttacccagttt | no |
-castPCRN |
The castPCR® - competitive, allele-specific TaqMan PCR utilizes an allele-specific primer for mutant allele detection that competes with an MGB blocker oligonucleotide to suppress the wild-type background and enable specific-allelic scoring of single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) at specific loci. N - the number (length) of polymorphic nucleotides from the 3'end; the default is 2 (-castPCR2), where the SNP is located at the penultimate base from the 3'-end of primer. Recommended minimum 2, in this case, SNP is located at the penultimate base from the 3'-end of primer; for InDels variants, this number is equal to polymorphic nucleotides at the 3'-end. If user do not specify the number: -castPCR, in this case the program will automatically determine the optimal length for effective discrimination of single nucleotide polymorphisms. >CastPCR-example1: -CastPCR -ln16-28 -Tm54-56 -pln12-28 -pTm53-57 gagagtgaccgattgcgtatacctgaacaactaacccccatcggccatcgcagtggcgctcccggcactgagaaaacgtccctaaccttgtgccccg ctcacatagacagcgataacaattttatgagatatcggctgtttaggaagtgcaaatgttaacgactgtct [S] tacgatataagtgccaagataagacttcggaattaaatacttgggtttctacccgcaatacgaggtgcatgtgcaccatttcgatatcatgaggtgg ttgtggactagatgcatagctctttctattattgtgctgccattcacgctccaggagaccttacccagttt >CastPCR-example2: -CastPCR -ln16-28 -Tm54-56 -pln12-28 -pTm53-57 gagagtgaccgattgcgtatacctgaacaactaacccccatcggccatcgcagtggcgctcccggcactgagaaaacgtccctaaccttgtgccccg ctcacatagacagcgataacaattttatgagatatcggctgtttaggaagtgcaaatgttaacgactgtct [CC/AA] tacgatataagtgccaagataagacttcggaattaaatacttgggtttctacccgcaatacgaggtgcatgtgcaccatttcgatatcatgaggtgg ttgtggactagatgcatagctctttctattattgtgctgccattcacgctccaggagaccttacccagttt | no |
-SNaPshot |
The SNaPshot® Multiplex System is a primer extension-based method for genotyping known SNP positions through the automated DNA analyzer invented by Applied Biosystems. Through its multiplexing capability, up to 10 SNPs can be analyzed in a single reaction by using unlabeled, user-defined primers. { -snapshot -pTm52-54 -pln16-35 -popyes } >example1 cttagatcgacaggtctaagagctgaagagctagctattaaagtcgagc [W] agctgctagacgtcgcagtcgacacagctagcctaggacaaagtctcgtg >example2 cttagatcgacaggtctaagagctgaagagctagctattaaagtcgagc [S] agctgctagacgtcgcagtcgacacagctagcctaggacaaagtctcgtg | no |
-MLPA |
A Multiplex Ligation Assay (MLA) or a digital MLA (dMLA) - MLPA® Multiplex Ligation-dependent Probe Amplification is a technology designed to detect copy number changes (deletions and duplications) in genomic DNA invented by MRC Holland that allows detection of DNA copy number changes of up to 40 sequences in a single reaction. MLPA has a variety of applications including detection of mutations and single nucleotide polymorphisms, analysis of DNA methylation, relative mRNA quantification, chromosomal characterisation of cell lines and tissue samples, detection of gene copy number, detection of duplications and deletions and aneuploidy determination. { -mlpa -pTm62-65 -pln21-50 -P5e[GGGTTCCCTAAGGGTTGGA/TCTAGATTGGATCTTGCTGGCAC] } >example1 cttagatcgacaggtctaagagctgaagagctagctattaaagtcgagc [A] agctgctagacgtcgcagtcgacacagctagcctaggacaaagtctcgtg >example2 cttagatcgacaggtctaagagctgaagagctagctattaaagtcgagc [CC] agctgctagacgtcgcagtcgacacagctagcctaggacaaagtctcgtg | no |
-LUXpdN1-N2 | LUX (Light Upon eXtension) primer design. LUX fluorogenic (self-quenched) Forward primers desig for quantitative PCR, between coordinate N1 and N2 for left or right sequence side. The design factors are the presence of either a C or G as the terminal 3′ nucleotide of the primer, the fluorophore being attached to the second or third base (T) from the 3′ end, the presence of one or more Gs within the 3 nt flanking the labeled nucleotide and, for hairpin primers, the existence of a 5′ tail that is complementary to the 3′ end of the primer. The 5′ tail forms a blunt-end hairpin at temperatures below its melting point. The stem of the hairpin primers have a ΔG ranging from –1.6 to –5.8 kcal/mol. Example, design forward LUX primers: > -Fluxpd [ctccccagacctatgttgtaagtggtcatgatacatgactagggtagagagaggagtgtctgtcaaaaaaga][ctgcgtgccctgagcttagctaccggttgaggtatctgagggactaccagtaacagatccccaggaattagccagaaaaaa] > -LUXpd100-200 cttagatcgacaggtctaagagctgaagagctagctattaaagtcgagcagctgctagacgtcgcagtcgacacagctagcctaggacaaagtctcgtg | no |
-tiling[±N] | Design of overlapping [<0] and non-overlapping [>0] DNA amplicons in a single-tube multiplex PCR for targeted next-generation sequencing (DNA-Based Molecular Tagging System): -tiling[-120] is negative value of N, meaning overlapping amplicons at about 120 bp; -tiling[20] value of N is positive (or 0) for non-overlapping amplicons. no | |
-TaqMan |
An example using MSA as a target for genotyping assay design. Allele-specific quantitative PCR with TaqMan-MGB probe - genotyping assays are based on competitive allele-specific TaqMan-MGB probe and enable specific-allelic scoring of single nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) at specific loci. The AS-qPCR Assay mix contains assay-specific non-labeled oligos: forward and reverse primer with allele-specific MGB. { -mgb -ln16-28 -Tm54-56 } alignment
10 20 30 40 50 60
| no |
-ssr/N | Design PCR primers to Simple sequence repeat (SSR) loci, the software automatically finds SSR loci and will design primers: N is optional value for distance before (Forward primers) and after (Reverse primers) SSR loci: > -ssr/200 | yes |