Pcr hot start by magnesium sequestration
一种耐热、合成肽的技术，应用在微生物的测定/检验、生物化学设备和方法、肽等方向，能够解决测定间数据再现性和数据比较复杂等问题
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Embodiment 1
[0199] A peptide with the sequence H-DIETDIET-NH2 was synthesized, purified by HPLC, lyophilized and dissolved in 30 mM Tris-HCl, pH 8.5. PCR reactions were performed in a volume of 50 μl containing 50 ng, 25 ng, 10 ng, 5 ng or 1 ng of human genomic DNA, 2.5 units of Taq polymerase (Roche Applied Science catalog number 11146165001), 0.4 mM forward primer (aga cag tac agc cag cct ca) (SEQ ID No: 1), 0.4mM reverse primer (gacttc aaa ttt ctg ctc ctc) (SEQ ID NO: 2), 0.2mM dATP, dCTP, dGTP and dCTP, Taq PCR reaction buffer (Roche Applied Science catalog number 11146165001), with or without H-DIETDIET-NH2-peptide. PCR was performed as follows: 2 minutes at 94°C, 35 cycles of 10 seconds at 94°C, 30 seconds at 60°C and 30 seconds at 72°C, and a final extension step of 7 minutes at 72°C. PCR products were analyzed by agarose gel electrophoresis.
[0200] Such as figure 1 As can be seen in , the addition of 2.0 mM of the disclosed peptide resulted in a significant reduction in the p...
Embodiment 2
[0202] Analysis of the peptide with the sequence H-FDGDFDGD-NH2. PCR reactions were carried out in a volume of 50 μl containing 25 ng or 10 ng of human genomic DNA, 2.5 units of Taq polymerase (Roche Applied Science catalog number 11146165001), 0.4 mM forward primer (aga cag tacagc cag cct ca) (SEQ ID No: 1), 0.4 mM reverse primer (agt atg ccc ccgcac agg a) (SEQ ID NO: 3), 0.2 mM dATP, dCTP, dGTP and dCTP, Taq PCR reaction buffer (Roche Applied Science catalog number 11146165001), with or without H -FDGDFDGD-NH2-peptide. PCR cycling conditions were as follows: 2 minutes at 94°C, 35 cycles of 10 seconds at 94°C, 30 seconds at 60°C and 30 seconds at 72°C, and a final extension step of 7 minutes at 72°C. PCR products were analyzed by agarose gel electrophoresis.
[0203] Such as figure 2 As seen in , the addition of 3 mM H-FDGDFDGD-NH2 resulted in reduced primer / dimer product formation. An increase in specific product formation was observed in parallel.
Embodiment 3
[0205] Peptides with the sequence H-DIETDIET-NH2 were analyzed in real-time RT-PCR. The PCR reaction was performed in a volume of 20 μΐ, containing 10 cells available in the LightCycler h-G6PDH Housekeeping Gene Kit from Roche Applied Science (Cat. No.: 3261883). 2 、10 3 、10 4 、10 5 and 10 6 Copies of RNA standard, 2.4 units of Transcriptor and 1.6 units of FastStart polymerase, reaction buffer from LightCycler RNA Amplification Kit (Amplification Kit) SYBR Green (Roche Applied Science catalog number 12015137001), 0.5 mM forward primer (ggg tgc atc ggg tga cct g) (SEQ ID NO: 4), 0.5 mM reverse primer (agc cac tgt gag gcg gga) (SEQ ID NO: 5), with or without 1 mM H-DIETDIET-NH2-peptide. PCR was performed in LightCycler 2.0 as follows: 45 cycles of 10 minutes at 55°C, 10 minutes at 95°C, 10 seconds at 95°C, 10 seconds at 55°C and 13 seconds at 72°C.
[0206] The results of this example are shown in FIG. 3 . It exemplifies that magnesium-binding peptides reduce primer-dimer...
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