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:918-1207

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:2929-3140

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:1876-2071

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
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