Pre-amplification method for trace DNA applied in medicolegal expertise

[0050] Embodiment 1: Primer Mix amplifies the method of 100pg, 50pg and 10pg DNA template under 4 gradients, and specific content is as follows:

[0051] 1. The preparation of the primer Mix mixture, the primer Mix mixture is a mixture of 36 primers from primer1-primer36, prepare 4 gradient primer Mix mixtures, the concentration gradient of the mixture is as follows:

[0052] Primer Mix 1: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.1uM)

[0053] Primer Mix 2: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.05uM)

[0054] Primer Mix 3: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.01uM)

[0055] Primer Mix 4: primer1 (final concentration 3uM) and primer2 (final concentration 3uM), primer3-36 (each primer final concentration 0.005uM)

[0056] 2. Add the followi...

[0066] Embodiment 2: the method for amplifying 100pg, 50pg and 10pg DNA using random primer 9N, primer1 and primer2 double primers, primer1 single primer and Pmier Mix, the specific contents are as follows:

[0067] 1. Random primer 9N amplifies 100pg, 50pg and 10pg DNA, the amplification system is as follows:

[0068] 9N 3ul

[0069] 10×Reaction Buffer 2ul

[0070] 10 (or 50 or 100) pg / ul DNA 1ul

[0071] wxya 2 O 7ul

[0072] Pre-denature at 98°C for 10 minutes, take out the deformed solution, place it on ice quickly for 15 minutes, then add the following reagents to it, and finally amplify at 33°C for 16 hours, 65°C for 15 minutes; 4°C∞;

[0073] Each 2.5mM dNTP 4ul

[0074] 0.5% (w / v) BSA 2ul

[0075] 5000U Phi29 1ul

[0076] figure 2 In "9N", the initial template amount of 1-2 is 100pg, 3-4 is 50pg, 5-6 is 10pg, and 7 is a negative control; no amplification band is shown in the figure.

[0077] 2. Primer 1 (final concentration 3uM) single primer amplifies 100pg,...

[0109] Embodiment 3: Applied to the forensic identification trace DNA pre-amplification method, using Pmier Mix to amplify the method of 100pg, 50pg and 10pg DNA, the specific content is as follows:

[0110] (1) Put 36 kinds of primers from primer1-primer36 into a single PCR tube and mix evenly. The final concentration of primer1 and primer2 is 1uM, and the final concentration of each primer from primer3-primer36 is 0.05μM. Divide into three groups;

[0111] (2) Add 10 μL of 10× reaction buffer, 42 μL of double distilled water and 1 μL of 10, 50 or 100 pg / ul trace DNA template to each group of mixed solutions;

[0112] (3) Place the mixture in step (2) in a PCR instrument for pre-denaturation at 98°C for 5 minutes;

[0113] (4) Take out the denatured solution, place it on ice for 10 minutes, then add 6 μl of dNTP, 3 μl of 0.5% BSA, and 2uL of 5000U Phi29 DNA polymerase, mix well and put it back into the PCR instrument for amplification. 2.5mM, PCR amplification conditions are...