FastPCR Quick PCR Commands Help


PCR

Element nameDescriptionFastPCR online Support
 /?Loading current options of PCR primers design: > /?
no
 -npcrStandard PCR, example: > -nPCR

yes
 -ipcrInverted PCR, example: > -iPCR

yes
 -lpcrLong PCR primer design: > -lPCR

no
 -lamp"Loop-mediated Isothermal Amplification" primer design:
{-LAMP } - LAMP assay design without using Loop Primers
{-LAMP2 } - LAMP assay design with Loop Primers


yes
 -qpcrQuantitative (real-time) PCR primer design:> -qPCR

no
  -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

yes
 -pcrNoPCR primers design without PCR primer combinations reporting:
> -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=N2 is allowed):
> (400-500)

yes
 -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

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
-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
yes
 -marray
 -Beacon
 -TaqMan
Probe design in DNA sequence:
> -marray

design TaqMan both direction:
> -taqman 
no
 -LUXpdN1-N2
-FLUXpd
-RLUXpd
(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):
> -Fluxpd -Fpd100-500 -Rpd1000-1200
> -LUXpd1000-1200
> -Fluxpd -ssr/200

no
 -npcN1Determine the maximum Number of Primers Combinations, for example 10 (0 is allowed):
> -npc10
yes
  -nprN1
 -FnprN1
 -RnprN1
Showing maximal Number of designed Primers per task:
> -npr10

yes
 -ptmsN1Synchronizing Tm for Primer Pair (±°C):
> -ptms10
yes
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, user 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 -pdN1-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) within the sequence(s):
1.  [        ]
2.     ]  [
3.  [  ]  [  ]
4.  [  [  ]  ]
5. ([  ]  [  ] )n  or(and)   ([ [   ] ])n



Primers desing options control

Element nameDescriptionjPCR Support
  -bst
 -Fbst
 -Rbst
design BeST PCR primers for current options design best left or right PCR primers:
> -bst

no
  -any
 -Fany
 -Rany
design ANY PCR primers:
> -any

no
  -lowcg
 -Flowcg
 -Rlowcg
design PCR primers for low CG% sequence:
> -lowcg

no
  -any
 -Fany
 -Rany
design ANY PCR primers:
> -any

no
  -dfs
 -Fdfs
 -Rdfs
design PCR primers for difficult region:
> -dfs

no
  -protein
 -Fprotein
 -Rprotein
design PCR primers for degenerated sequences:
> -protein

no
  -lnN1-N2
 -FlnN1-N2
 -RlnN1-N2
Minimal-Maximal length for Forward or (and) Reverse primers design:
> -FlN18-22 -RlN22-22
yes
  -tmN1-N2
 -FtmN1-N2
 -RtmN1-N2
Minimal-Maximal Tm for Forward or (and) Reverse primers design:
> -Ftm40-50 -Rtm50-60

yes
  -cgN1-N2
 -FcgN1-N2
 -RcgN1-N2
Minimal-Maximal CG% for Forward or (and) Reverse primers design:
> -Fcg45-55 -Rcg50-60

yes
  -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

yes
  -qN1
 -FqN1
 -RqN1
Minimal-Maximal Quality for Forward or (and) Reverse primers:
> -Fq5 –Rq90


yes
  -dmrN1
 -FdmrN1
 -RdmrN1
The level of rigor detection primer dimer, where N1 from 0 to 5. Level 1 is the default and is the most stringent criterion, the value of 5 for less sensitive detection. A value of 0 to ignore the detection of primer dimer:
> -dmr0


yes
  -opYes|No
 -FoYes|No
 -RoYes|No
Primers overlapping control: if type -FoNo, all forward primers will not overlap; -RoYes all reverse primers will overlap:
> -FoNo -RoYes

yes
 -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

yes
   -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 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]
 -Fc3[NNN NNN]
 -Rc3[NNN NNN]
  -c5[NNN NNN]
 -Fc5[NNN NNN]
 -Rc5[NNN NNN]

 -cc3[NNN NNN]
-Fcc3[NNN NNN]
-Rcc3[NNN NNN]
 -cc5[NNN NNN]
-Fcc5[NNN NNN]
-Rcc5[NNN NNN]

Certain sequence(s) in the direct or complementary (second c) oriantation 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 lenght 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 wssws sswws]
> -c3[ACCCTTCG]

example, SNP site for Reverse primers with site 5'-Mcatatc, program will convert to complementary sequence 5'gatatgK:

> -Rcc3[Mcatatc]
yes
  -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: –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
  -N3eN1
 -FN3eN1
 -RN3eN1
Shift by 0 to N1 nucleotides from the 3'-end of the template: -c3[NN] and -z3eNameEnzyme.

For example: -c3CAGG -N3e3 the programs to choose primers which include the template CAGG within the 3-end primer up to 3 nucleotides from the end:
5'aaaaaaaaaaCAGG-3'
5'aaaaaaaaaCAGGa-3'
5'aaaaaaaaCAGGaa-3'
5'aaaaaaaaCAGGaaa-3'

The template -c3[NN] and -z3eNameEnzyme can be located anywhere from 5 'end to 3' end, if user specify a length greater than the length of the primer: -N3e500
yes
  -5eNNN
 -F5eNNN
 -R5eNNN

 -c5eNNN
-Fc5eNNN
-Rc5eNNN
Primer 5'-End Tail: additional, not from current DNA sequence. which will be added to 5' of primers (probes) (any length):
> -F5eCGACG -R5eTTTTTT


> -Fc5eCAACG - program convert to complement 5'-CGTTG (equal to -F5eCGTTG)

yes
  -3eNNN
 -F3eNNN
 -R3eNNN

 -c3eNNN
-Fc3eNNN
-Rc3eNNN
Primer 3'-End Tail: additional, not from current DNA sequence. which will be added to 3' of primers (probes) (any length):
> -F3eGG -R3eСС


> -Fc3eGG - program convert to complement 5'-CC (equal to -F3eCC)

yes
  -eNNN
 -ceNNN
Generally, both primer 3' and 5'-Ends Tails, additional, not from current DNA sequence to 3' and 5' of primers (probes) (any length):
> -eGGA
yes


Special cases

Element nameDescriptionjPCR Support
  -pr[NNN NNN]
 -Fpr[NNN NNN]
 -Rpr[NNN NNN]
Pre-designed Forward or Reverse one or more primers, at 5'->3', with space between primers, detection the primer's location with mismatches; analyzes more than one primer for both DNA chains:

> -pr[attccattccgcgttcga tcctacgttccgttacc]

pre-designed Forward and Reverse PCR primer: 
> -Fpr[attccattccgcgttcga] -Rpr[cgttacggtatttcttgc]

yes
  -ssr/N
  -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
yes






PCR set-up examples