PrimerDigital

“Primer Design Options” is general for all given sequences, as background. For individual, selective options and task, sequences need convert to FASTA format with “>”, these options have a highest priority, and they are covering the general (background) options. All these command used optionally and only for advanced tasks. In generally, you can relax and forgot about it. Press F5 show what is FastPCR found in given sequences. The result file (Excel sheet, XML page or Text) contains a list of the best primers and compatible primer combinations. It shows the type of PCR, the left and right flanking regions for primer selection, and the annealing temperature. Each primer combination includes information for primer position and length, optimal melting temperature and product length.

Any of these following commands must be written AFTER the sequence name or “>” (these commands are not case sensitive) and press Enter and the end of line. The commands can occupy any place in the command line.

FastPCR Quick PCR Commands Help

/?	Loading current options of PCR primers design: > /?
-npcr	Standard PCR, example: > -nPCR
-ipcr	Inverted PCR, example: > -iPCR
-lpcr	Long PCR primer design:> -lPCR
-qpcr	Quantitative (real-time) PCR primer design:> -qPCR
-npd  -Fnpd  -Rnpd	No Primer(s) Design, restriction for design primers only for forward or reverse or for both primers; but be shown the primers which added with command -fp= : > -npd
-pcrNo	No PCR primer combinations reporting, but showing all primers: > -pcrNO
(N1-N2)	specify the minimal and maximal size requested for the PCR product, N1 for shortest, N2 for the longest PCR product (N1=N2 is allowed): > (400-500)
-pdN1-N2 -FpdN1-N2 -RpdN1-N2  -pdN1-N2/N3	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 You 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)
-pdN-e -FpdN-e -RpdN-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
-npcN1	Determine the maximum Number of Primers Combinations, for example 10 (0 is allowed): > -npc10
-nprN1  -FnprN1  -RnprN1	Showing maximal Number of designed Primers per task (maximum 200): > -npr10
-ptmsN1	Synchronizing Tm for Primer Pair (±°C): > -ptms10
Instead of using these commands –FpdN1-N2, –RpdN1-N2 or -pdN1-N2, for individual targets selection for Forward or Reverse primers in a particular location, you can apply any multiple combinations of '[ ]' or '] [' inside the sequence(s). Use of these brackets differs from software Primer3, for example:   1. The same location for both Forward and Reverse primers will be designed in the central [nnnnnnnnnn] part (only once '[ ]' is used):   ...AAAAAAAAAA[nnnnnnnnnn]CCCCCCCC...   2. Different locations for Forward and Reverse primers; Forward (red) primers will be chosen inside [1nnnnnn] location and Reverse (blue) primers inside [2nnnnnn] location, (twice '[ ]'):   ...AAAAAAAAAA[1nnnnnn]AAAAAAAA[2nnnnnn]AAAAA...   3. Design primers must flank the central ]nnnnnn[ ; Forward primers will be chosen from 1 to A] bases and Reverse primers will be chosen from [C base to the end of sequence:     ...AAAAAAAAAA]nnnnnn[CCCCCCC...   4. Design primers with overlapping part [nnnnnn] for Forward and Reverse primers; Forward primers will be chosen from [A to n] bases and Reverse primers will be chosen from [n base to C]:   Forward--------------¬    |                 | ...[AAAAAAAAAA[nnnnnn]CCCCCCC]...               |              |                           ---------Reverse Program allow to select up to 1000 independent PCR primers (probe) designing tasks for each sequences using multiple combinations of '[..]' and –FpdN1-N2, –RpdN1-N2 or -TagN1-N2 commands.   Multiplex PCR can be carried out simultaneously within a single sequence with multiple tasks as well as for different sequences or multiple tasks or both cases together. All possible combinations of '[  ]' (Forward) with '[  ]' (Reverse) inside the sequence(s): 1.   [         ]          2.      ]   [ 3.   [  ]   [  ] 4.   [  [   ]  ] 5. ( [  ]   [  ] )n  or(and)   ([  [   ]  ])n
-bst -Fbst -Rbst	design BeST PCR primers for current options design best left or right PCR primers: > -bst
-any -Fany -Rany	design ANY PCR primers: > -any
-lowcg -Flowcg -Rlowcg	design PCR primers for low CG% sequence: > -lowcg
-dfs -Fdfs -Rdfs	design PCR primers for difficult region: > -dfs
-protein -Fprotein -Rprotein	design PCR primers for degenerated sequences: > -protein
-5e -F5e -R5e	Primer 5'-End Tail: additional and an artificial DNA sequence. which will be added to 5' of primers (probes) (any length): > -F5eCGACG -R5eTTTTTT
-c5e -Fc5e -Rc5e	Primer 5'-End Tail, additional, artificial complement DNA sequence to 5' of primers (probes) (any length): > -Fc5eCGACG - program convert to complement 5'-CGTCG (equal to -F5eCGTCG)
-3e -F3e -R3e	Primer 3'-End Tail: additional and an artificial DNA sequence. which will be added to 3' of primers (probes) (any length): > -F3eGG -R3eСС
-c3e -Fc3e -Rc3e	Primer 3'-End Tail, additional, artificial DNA sequence to 3' of primers (probes) (any length): > -Fc3eGG - program convert to complement 5'-CC (equal to -F3eCC)
-lnN1-N2 -FlnN1-N2 -RlnN1-N2	Minimal-Maximal length for Forward or (and) Reverse primers design: > -Fln18-22 -Rln22-22
-tmN1-N2 -FtmN1-N2 -RtmN1-N2	Minimal-Maximal Tm for Forward or (and) Reverse primers design: > -Ftm40-50 -Rtm50-60
-cgN1-N2 -FcgN1-N2 -RcgN1-N2	Minimal-Maximal CG% for Forward or (and) Reverse primers design: > -Fcg45-55 -Rcg50-60
-3tmN1-N2 -F3tmN1-N2 -R3tmN1-N2	Minimal-Maximal Temperature of Melting for last 12 bases at the 3'-end of Forward or Reverse primers: > -F3tm31-35 -R3tm35-45
-qN1 -FqN1 -RqN1	Minimal-Maximal Quality for Forward or (and) Reverse primers: > -Fq5 –Rq90
-oYes|No -FoYes|No -RoYes|No	Primers pverlapping control: if type -FoNo, all forward primers will not overlap; -RoYes all reverse primers will overlap: > -FoNo -RoYes
-x3e -fx3e -Ffx3e -Rfx3e  -fx5e -Ffx5e -Rfx5e	Design PCR primer for a specific sequence on the fixed 5' or 3' ends to selected sequence, for example, if you 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
-fp= -Ffp= -Rfp=	Pre-designed (Fixed) Forward or Reverse one or more primers, always use the actual primer sequence (5'->3') without space with automatically detection the primer's location with mismatches; analyzes more than one primer for both DNA chains: > -fp=attccattccgcgttcga -fp=atcctacgttccgttacc pre-designed Forward and Reverse PCR primer:  > -Ffp=attccattccgcgttcga -Rfp=acgttacggtatttcttgc
-c5=nn! -c3=nn! -Fc5=nn! -Fc3=nn! -Rc5=nn! -Rc3=nn!	Forward or Reverse primers (probes) 3' and 5'-ends nucleotide composition; the program accept the universal degenerate DNA code minimum 2 bases (maximum is primer length) for both ends: nn - for any bases, example for left primers ('-c5RRY!' is equivalent to -c5aat gat agt ggt aac gac agc ggc!)(accepted the sequences with different lengths): > -c5=tss   -c3=wss wssws sswws!
-z3eNameEnzyme -Fz3eNameEnzyme -Rz3eNameEnzyme	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 –3eYCATG^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.
-ctYes|No -FctYes|No -RctYes|No	(Optional)(Copy Test) for PCR primers repetition test repetition test for Forward or Reverse PCR primes design: > -FctYes -RctNo
-FUXpdN1-N2  -FUXpd  -FLUXpdN1-N2 -RLUXpdN1-N2	(LUX Primer Design) design PCR LUX (self-quenched) primers for quantitative PCR, between coordinate N1 and N2 for left or right sequence side. Example, design left PCR LUX primers between coordinate N1 and N2 (N1>N2): > -Fluxpd100-500 -Rpd1000-1200 > -Fluxpd -Rpd1000-1200 > -Fluxpd -ssr/200 -FUX is the same -LLUX –RLUX
-ssr/N  or   -ssr	Design PCR primers to SSR loci software automatically finds Simple Repeats target Sequence and will design primers: N is optional value for distance before (Forward primers) and after (Reverse primers) SSR loci: > -ssr/200

PCR set-up examples

• Example for PCR primer design to Simple Sequence Repeat (SSR) loci (200 bp around SSR):
  > -ssr/200
  
• Example prediction Ta (temperature of annealing) of PCR with one or more existing primers (with -npd command) for current sequence:
  > -fp=gagagtagcttacctcgct -fp=cggtaaggttcttcatgc -npd
• LATE-PCR example, necessary to select forward and reverse primers with a difference in Tm of about 10 degrees:
  > -Ftm50-55 -Rtm60-65 -pTMs10