Specificity result

Done Task link: e11d28e2-bbb3-4523-a0b7-1c6472f389c1 (remember for later use)

Primer list

*PBN and MBN: primer binding number to the plus and minus strand.
Primer ID Sequence (5' → 3') Size (bp) GC (%) Tm (°C) ΔG (kcal/mol) PBN* MBN*
P1 AAGTCAATAGAAGCCGGCGTA 21 47.62 60.04 -22.40 7 6
P2 ACAGGCTCCTCTAGAGGGATAT 22 50.00 59.54 -22.23 7 12
P3 TTTGCAAGGAGAGCCAAAGC 20 50.00 59.83 -21.78 35 30
P4 AGAGGGTTCTGTGGGCAAAT 20 50.00 59.74 -21.42 35 30
P5 CTAGGGATAACAGCGCAATCCT 22 50.00 60.49 -23.10 16 19
P6 TCTCTTGTCCTTTCGTACAGGG 22 50.00 59.96 -22.82 14 13

Hairpin list (0)

No hairpins found.


Dimer list (0)

No dimers found.


Histogram of size of 3 potential amplicons


Description of 3 potential amplicons

Amp ID Product Name Fp x Rp Product Size (bp) Fp Tm (°C) Rp Tm (°C) Fp ΔG (kcal/mol) Rp ΔG (kcal/mol)
 1 chrM P1 x P2 290 61.11 60.34 -23.09 -23.07
 2 chrM P5 x P6 212 60.65 60.15 -23.83 -23.57
 3 chrM P3 x P4 196 59.32 61.04 -21.66 -22.31

Amplicon details

Amp 1: P1 + P2 ==> spec/db/hg38.fa:chrM:917-1207

Size = 290 bp, GC content = 43.45%
F: Tm = 61.11 °C, ΔG = -23.09 kcal/mol
R: Tm = 60.34 °C, ΔG = -23.07 kcal/mol
Binding sites: 918(21/21) ... 1207(22/22)

>>>P1
   1                   21
5' AAGTCAATAGAAGCCGGCGTA 3'
   :::::::::::::::::::::                                                      1207
5' AAGTCAATAGAAGCCGGCGTAaagagtgttttagat...acctggcggtgcttcATATCCCTCTAGAGGAGCCTGT 3'
   918                                                   ::::::::::::::::::::::
                                                      3' ATATCCCTCTAGAGGAGCCTGT 5'
                                                        22                    1
                                                                             P2<<<


>Amp_1 P1 + P2 ==> spec/db/hg38.fa:chrM:917-1207 
AAGTCAATAGAAGCCGGCGTAaagagtgttttagatcaccccctccccaataaagctaaaactcacctgagttgtaaaaa
actccagttgacacaaaatagactacgaaagtggctttaacatatctgaacacacaatagctaagacccaaactgggatt
agataccccactatgcttagccctaaacctcaacagttaaatcaacaaaactgctcgccagaacactacgagccacagct
taaaactcaaaggacctggcggtgcttcATATCCCTCTAGAGGAGCCTGT
Amp 2: P5 + P6 ==> spec/db/hg38.fa:chrM:2928-3140

Size = 212 bp, GC content = 44.81%
F: Tm = 60.65 °C, ΔG = -23.83 kcal/mol
R: Tm = 60.15 °C, ΔG = -23.57 kcal/mol
Binding sites: 2929(22/22) ... 3140(22/22)

>>>P5
   1                    22
5' CTAGGGATAACAGCGCAATCCT 3'
   ::::::::::::::::::::::                                                      3140
5' CTAGGGATAACAGCGCAATCCTattctagagtccata...tacnttcaaattcctCCCTGTACGAAAGGACAAGAGA 3'
   2929                                                   ::::::::::::::::::::::
                                                       3' CCCTGTACGAAAGGACAAGAGA 5'
                                                         22                    1
                                                                              P6<<<


>Amp_2 P5 + P6 ==> spec/db/hg38.fa:chrM:2928-3140 
CTAGGGATAACAGCGCAATCCTattctagagtccatatcaacaatagggtttacgacctcgatgttggatcaggacatcc
cgatggtgcagccgctattaaaggttcgtttgttcaacgattaaagtcctacgtgatctgagttcagaccggagtaatcc
aggtcggtttctatctacnttcaaattcctCCCTGTACGAAAGGACAAGAGA
Amp 3: P3 + P4 ==> spec/db/hg38.fa:chrM:1875-2071

Size = 196 bp, GC content = 45.41%
F: Tm = 59.32 °C, ΔG = -21.66 kcal/mol
R: Tm = 61.04 °C, ΔG = -22.31 kcal/mol
Binding sites: 1876(20/20) ... 2071(20/20)

>>>P3
   1                  20
5' TTTGCAAGGAGAGCCAAAGC 3'
   ::::::::::::::::::::                                                    2071
5' TTTGCAAGGAGAGCCAAAGCtaagacccccgaaac...ttagttcaactttaaATTTGCCCACAGAACCCTCT 3'
   1876                                                 ::::::::::::::::::::
                                                     3' ATTTGCCCACAGAACCCTCT 5'
                                                       20                  1
                                                                          P4<<<


>Amp_3 P3 + P4 ==> spec/db/hg38.fa:chrM:1875-2071 
TTTGCAAGGAGAGCCAAAGCtaagacccccgaaaccagacgagctacctaagaacagctaaaagagcacacccgtctatg
tagcaaaatagtgggaagatttataggtagaggcgacaaacctaccgagcctggtgatagctggttgtccaagatagaat
cttagttcaactttaaATTTGCCCACAGAACCCTCT

Parameters

Database canFam6.fa, hg38.fa, GCF_000005845.2_ASM584v2_genomic.fna
Kvalue 9
MisMatch 0
Product Min Size (bp) 0
Product Max Size (bp) 500
Tm (°C) 47
Concentration of monovalent cations (mM) 50
Concentration of divalent cations (mM) 1.5
Concentration of dNTPs (mM) 0.25
Concentration of annealing oligos (nM) 50
Contact
Request more input?

For bug reports or suggestions, please email us.