Composition for biosample treatment and method for nucleic acid amplification using the same
A technology for biological samples and nucleic acid amplification reactions, which is applied in the field of compositions that simplify the processing of biological samples, and can solve problems such as sample contamination
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Embodiment 1
[0044] [Example 1] Comparison with commercially available reagents
[0045] (1) Use the composition of this case for nucleic acid purification
[0046] (1-1) Preparation of composition (1)
[0047] Take 12.5 μl of perfluorohexane (FluorinertTM), 10 μl of polytetramethylene glycol (polytetramethylene glycol) and 0.1 μl of Triton X-100, and mix them uniformly to form composition (1).
[0048] (1-2) Mouse blood amplification reaction
[0049] Take 10 μl of mouse blood and 10 μl of serum, add the composition (1) prepared above, mix evenly, let stand for 3 minutes, and then mix with 12.5 μl nucleic acid amplification reaction reagent (10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, 0.1%Triton X-10020mM, Tris-HCl pH8.8, polymerase enzyme) were evenly mixed for the following PCR amplification reaction.
[0050] (1-3) PCR amplification reaction
[0051] Perform a polymerase chain reaction (PCR) amplification reaction under the following conditions to amplify the target gene GAPDH.
[0052] ...
Embodiment 2～5
[0079] [Embodiment 2~5] the sample volume scope of processing
[0080] Mix the volume of the composition (1) prepared in Example 1 shown in Table 1 below with an equal volume of sample blood, and then mix it with the nucleic acid amplification reaction reagent. Afterwards, with the primer pair 1mM (Fermentas The kit GAPDH standard primer) was denatured at 95°C on a PCR machine (ABI9700); 58°C was bonded, and the PCR reaction with a cycle number of 40 was used to amplify the DNA sample of the labeled primer in the blood. The solution after the above PCR reaction was subjected to 75V electrophoresis on TAE gel, and the electrophoresis picture shown in Figure 2 was obtained.
[0081] [Table 1]
[0082]
[0083] Column L in Figure 2 represents the sample band (ladder), columns 1-4 represent the amplified DNA sample of Example 5, columns 5-8 represent the amplified DNA sample of Example 4, column 9 It is a blank column. Columns 10-12 represent the amplified DNA samples of Ex...
Embodiment 6
[0085] [Example 6] Application in real-time PCR
[0086] The composition (1) prepared in Example 1 was mixed with an equal volume of 10 μl of blood from the sample, and then mixed with the nucleic acid amplification reaction reagent. Afterwards, 1 mM (ABI PCRcontrol18S / GAPDH standard primer) was denatured at 95°C in a PCR machine (ABI7500) with primers for amplifying 18S / GAPDH; 58°C was bonded, and the PCR reaction with a cycle number of 40 was used to amplify the labeled primers in the blood. DNA / RNA samples, the results are shown in Figures 3 and 4.
[0087] Figure 3 shows the RNA 18S amplification map obtained using the ABI standard 18S primer and carbon needle set. Figure 4 shows a diagram of the GAPDH interval obtained using the ABI standard 18S primer and carbon needle set.
[0088] As shown by the results of this example, the composition of this case can be applied to the application of real-time PCR, which meets the requirements of nucleic acid amplification reaction...
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