各隣接配列のエントロピー、エンタルピーを考慮したTmを利用してプライマー配列候補を自動計算するプログラムを書きます。
目次
最近接塩基対法
以下の式を利用して、Tm値を求めます。
$Tm=\frac{\Delta H}{A + \Delta S + R\ln \frac{C}{4}}-273.15+16.6\log [Na^+]$
この時の各種熱力学的パラメータは以下の通りです。
parameters = {
"deltaH":{
"AA":-9.1,
"AT":-8.6,
"TA":-6.0,
"CA":-5.8,
"GT":-6.5,
"CT":-7.8,
"GA":-5.6,
"CG":-11.9,
"GC":-11.1,
"GG":-11.0,
"TT":-9.1,
"AT":-8.6,
"TA":-6.0,
"TG":-5.8,
"AC":-6.5,
"AG":-7.8,
"TC":-5.6,
"CG":-11.9,
"GC":-11.1,
"CC":-11.0,
},
"deltaS":{
"AA":-0.024,
"AT":-0.0239,
"TA":-0.0169,
"CA":-0.0129,
"GT":-0.0173,
"CT":-0.0208,
"GA":-0.0135,
"CG":-0.0278,
"GC":-0.0267,
"GG":-0.0266,
"TT":-0.024,
"AT":-0.0239,
"TA":-0.0169,
"TG":-0.0129,
"AC":-0.0173,
"AG":-0.0208,
"TC":-0.0135,
"CG":-0.0278,
"GC":-0.0267,
"CC":-0.0266,
},
}上記を利用して実際に配列のTm値を求める関数を作成します。
def Tm(seq: str) -> float:
parameters: dict[str,dict[str,float]] = {
"deltaH":{
"AA":-9.1,
"AT":-8.6,
"TA":-6.0,
"CA":-5.8,
"GT":-6.5,
"CT":-7.8,
"GA":-5.6,
"CG":-11.9,
"GC":-11.1,
"GG":-11.0,
"TT":-9.1,
"AT":-8.6,
"TA":-6.0,
"TG":-5.8,
"AC":-6.5,
"AG":-7.8,
"TC":-5.6,
"CG":-11.9,
"GC":-11.1,
"CC":-11.0,
},
"deltaS":{
"AA":-0.024,
"AT":-0.0239,
"TA":-0.0169,
"CA":-0.0129,
"GT":-0.0173,
"CT":-0.0208,
"GA":-0.0135,
"CG":-0.0278,
"GC":-0.0267,
"GG":-0.0266,
"TT":-0.024,
"AT":-0.0239,
"TA":-0.0169,
"TG":-0.0129,
"AC":-0.0173,
"AG":-0.0208,
"TC":-0.0135,
"CG":-0.0278,
"GC":-0.0267,
"CC":-0.0266,
},
}
deltaH,deltaS = 0, 0
for i in range(len(seq)-1):
deltaH += parameters["deltaH"][seq[i:i+2]]
deltaS += parameters["deltaS"][seq[i:i+2]]
return (deltaH)/(-0.0108+deltaS+0.00199*np.log(0.0000005/4)) - 273.15+16.6*np.log10(0.05)最適プライマーの探索
以下の制約条件のもと、最適なプライマー候補を探索します。
・塩基配列サイズは25bp以上、35bp以下であること
・GC rateは0.45から0.6であること
・Tm値は63以上であること
・ATリッチな配列(５連続以上)でないこと
・GC3'末端を持つことこの条件のもと、以下の配列について検証してみます。
CGATAAGCTAGCTTCACGCTGCCGCAAGCACTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGGATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCCTCGCGGACGTGCTCATAGTCCACGACGCCCGTGATTTTGTAGCCCTGGCCGACGGCCAGCAGGTAGGCCGACAGGCTCATGCCGGCCGCCGCCGCCTTTTCCTCAATCGCTCTTCGTTCGTCTGGAAGGCAGTACACCTTGATAGGTGGGCTGCCCTTCCTGGTTGGCTTGGTTTCATCAGCCATCCGCTTGCCCTCATCTGTTACGCCGGCGGTAGCCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCCGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAAGCGCTGCTTCCCTGCTGTTTTGTGGAATATCTACCGACTGGAAACAGGCAAATGCAGGAAATTACTGAACTGAGGGGACAGGCGAGAGACGATGCCAAAGAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCtgacgttctagACCCTACCTCTCCAAGATTACCGGCGTGCCCATGGTGGCCCTGGCCACCAACGTCATGCTGGGCAAAAGCCTGCCGGAGCAGGGCTACCGGGGCGGCTTAATGCCGCCGCCGGATTTTACCGCCGTAAAGGTCCCCGTCTTTTCCTTCGGCAAGCTGTTGCAGGTGGACACCTCCCTGGGACCGGAGATGAAGTCCACCGGCGAGGTAATGGGGATTGATCCCGTCTTCGAACGCGCCCTCTATAAAGGCCTGGTAGCCGCCGGCTGCTCCATCCCCCATCACGGCACCCTGCTGGCGACCATCGCCGATAAGGACAAGGCGGAAGCAGTGCCCATCATCAAGGGCTTTGCCGAACTGGGCTTCCAGGTGGTGGCTACCGCCGGCACCGCCGGCGCCCTGGCCGCAGCGGGACTCTTCGTAGAGAGGGTGGGGAAGATCCGCGAGGGTTCGCCCCACATTATCGACTATATCCGGGAAGGGAAGGTCCACTTTGTCCTCAACACCCTCACCAGGGGCAAGATGCCCGGCCGGGACGGTTTTAAGATCCGCCGCGCCGCGGCCGAACTGGGCATCCCCTGCCTGACTTCCCTGGATACGGCCCGGGCCCTGCTCAAAGTCCTCCAGTCCCTGAAGTCCGGCGACGGGTTTAACCTCAAACCCCTGCAGGAGTATGTACCCCTTTCCCGTCCTTAACGGAGGAGCGCCAAATCGCCTCCGCCCCACCCCGGCAGGAGCAGCAGCCCGCGGCTGCACCGGCCGGGCGGTTTCCCGGCCGGCCCTTCAAGCACCAGGCGAGATGGCCGGGCCGCCGCCATTTAGCATATCAAGAGCGCCGGAAGGGAAGGGCTTTTCCGGTTTTTACCGGTCGGGGTTAAGCCTGACTTAAGGGCCGGTACCGGACCCTCCCCATATTCACTCCGCTTACACTCCGTTTTTTGAACTATAAGATCATAAAGCGATATTTAAGGGCTTCTGGCCTGCTTGCCAACACTAATGTACCTGCAGGAGATGATCCGCATGCATGCCAAGGACAAAATAATCGTCGCCCTGGATGTTCCCGACCTGGCTGCCGGGGAAAAGCTGGTGGACCGGCTTTCCCCCTACGCCGGCATGTTTAAAGTCGGCCTGGAGTTTTTCACCGCCGCCGGGCCGGCGGCCGTCCGGATGGTAAAGGAGCGTGGTGGCCGGGTATTTGCCGACCTGAAGTTCCACGACATCCCCAACACCGTGGCCGGAGCGGCGCGGGCCCTGGTGCGCCTGGGCGTGGATATGCTCAACGTTCACGCCGCCGGCGGCAAGGCCATGCTGCAGGCTGCCGCCGCCGCCGTCCGGGAGGAGGCCGCGGCCTTAAACCGCCCGGCGCCGGTAATAATCGCGGTCACTGTTTTGACCAGCCTGGACAGGGAAGCTCTACGCTGCGAGGTGGGTATCGAGCGAGAGGTAGAAGAACAGGTGGCCCGCTGGGCGCTCCTGGCCCGGGAGGCCGGCCTGGACGGCGTAGTAGCCTCGCCCCGGGAGATCCGGGCCATCCGGGAGGCCTGCGGGCCGGAGTTCGTCATCGTGACCCCGGGCGTGCGCCCGGCTGGGTCCGACCGGGGCGACCAGCGCCGGGTCATGACCCCGGCCGAGGCCCTGCGGGAGGGCGCCTCCTACCTGGTCATCGGCCGGCCCATCACCGCGGCCCCCGACCCCGTCGCCGCCGCCCGGGCCATCGCGGCGGAAATAGAGATGGTGAAATAATAACTGGACGGTTGCCAAGTACCGGGACGAGCAGGGCATCCCGGCGGCGGCTAAAAGAAAACGATATTAGTTAAGAAGGATTTTGACCATTTGTGTTGAATAGATAGTGTTTGACGGTACAATCTCCGGCAATTAGCAATATATCATAATAAATCCTGATTGGGTTAGGAATAATATCAAAAGCCAAGGAGCCTGAAAGCGGTGGGGGTTGACGCTGCAGGAATTTAACCCTTGCCGTTACAATAAATATAAGGAGGAGTACATAATGAACTTCAACAAGATCGATCTGGACAACTGGAAACGCAAGGAAATCTTCAACCATTATCTGAACCAGCAGACCACCTTCTCCATCACCACGGAGATCGACATCTCCGTGCTGTACCGGAACATCAAGCAAGAAGGCTACAAGTTCTACCCCGCCTTCATCTTTCTGGTCACGCGGGTCATCAACAGCAACACCGCCTTCCGCACCGGGTACAACTCCGACGGCGAGCTCGGCTACTGGGACAAGCTGGAACCGCTCTACACCATCTTCGACGGCGTGAGCAAGACCTTTAGCGGCATCTGGACGCCCGTGAAGAACGACTTCAAGGAGTTCTACGATCTGTACCTCTCCGACGTGGAAAAGTACAACGGCTCCGGCAAGCTGTTCCCGAAAACCCCCATTCCCGAAAACGCCTTTTCCCTCTCCATCATCCCGTGGACCAGCTTCACCGGCTTTAATCTGAACATTAACAACAACAGCAACTATCTGCTGCCGATCATCACCGCCGGCAAGTTCATCAACAAGGGCAACAGCATCTACCTCCCGCTGAGTCTGCAAGTCCACCACAGCGTCTGCGATGGCTACCACGCCGGGCTGTTTATGAACAGCATCCAAGAACTGAGCGACCGCCCGAATGACTGGCTGCTGTAATCGGCCTGCTTTCATGCTTGATAATTTTTGTCATGTAGGGCTACAATGATAGTAACAGGTGATGACACGATGGAACGAATTAACTTTATCAATACCCGGGAGTTTAAAAATAGAGCAACCCAAATCTTGAGGCAGGTACAAAAAGACCAGGTTATTATTATAACCAATCGCGGTAAACCTGTAGCCACTTTAAAAGGTTTCAATCCACGTGACCTGGTTGTTGCAGAAGATAGACATGATAGCCTTTACCAGCATTTGCGGCAACAAATTTTAAAAGAAAGTCCAGAACTGGCTGCCAGGGATACCAGGCAAATCGCCACTGATTTTGAAAAGATAACAGCTAAAATGAGAAAACAGATTGCCTACAGGACCTGGGAAGAAATGGACCGGCACTTAAAAGGGGATCCTTATGATCTTACTGGATACTAATATTTTTATAATCGATCGTTTTTTTCCAGGGGATAGTCATTACGCTATAAACAAGGAATTCATTCAAGAGTTATCGCGGCTCGAGGCGGGGTTTTCTATTTTTTCGTTATTAGAACTTACCGGCATTGCTTCTTTTAACCTTTCAGCCAAAGAATTGCAGCAATGGTTGTTTGATTTTGCCTCCGTTTATCCTATTCGTATTCTTGATCCCTATGATTTAAAGATTGATTCTGCCAAGGAATGGTATACTAAATTTTTGCAGGAACTAATGGCAAAAGTTACCCACCAAATGACTTTTGGCGACGCTATTTTTTTACGTGAAGCTGAAGGTTATCAAGTAGAGTATATTATTAGCTGGAATAAGAAACATTTTCTTTCACGTACGACAATCAAAGTGCTCAACCCTGAAGAGTTTTTGACAATATGGAAACCGCAATAATTCCTTTGGTCATAAGCAAGGCGTGGGCTTTTTTAATCTCGTTGGAATGGGTAACTTTATGGGGTTAGCTCCCGGCAACCTAAATCGGAAGGTGCATAAGCTTGGACagatatcaggtcaGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCG
先ほどのTm関数を使用して、候補配列を取り出します。
primer_candidates:list[str] = [seq[i:bp+i] for bp in range(25,36) for i in range(0,len(seq) - bp)
if 0.45<get_GC_rate(seq[i:bp+i])<0.6 and
Tm(seq[i:bp+i])> 63 and
((seq[i:bp+i][0] == "G" and seq[i:bp+i][-1] == "C") or (seq[i:bp+i][0] == "C" and seq[i:bp+i][-1] == "G")) and
"AAAA" not in seq[i:bp+i] and
"TTTT" not in seq[i:bp+i]
]