Hla gene multiplex dna typing method and kit
A technology of DNA typing and HLA-A, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems that the PCR conditions are not completely unified, and the PCR multiplex method has not yet been achieved. The effect of speed and simplicity
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Embodiment 1）
[0161] [Purpose]
[0162] The amplification status of each HLA gene was checked using PrimeSTAR GXL DNA Polymerase (TaKaRa) as an enzyme.
[0163] [method]
[0164] PCR was performed using PrimeSTAR GXL DNA Polymerase (TaKaRa), using the extracted genomic DNA as a template, and each gene-specific primer set of HLA class I and HLA class II (refer to Table 1 to Table 3: SEQ ID NO: 1 to 31). The specific steps are as follows.
[0165] (1) Add 4 μL of 5xPrimeSTAR GXL buffer solution, 1.6 μL of dNTP solution, 1-3 μL (4 pmol / μL) of PCR primers, and 0.4 μL of Prime STAR GXL polymerase to 50 ng of genomic DNA solution. The total volume was adjusted to 20 μL with sterile water.
[0166] (2) After keeping this at 94° C. for 2 minutes, two steps of 98° C. for 10 seconds and 70° C. for 5 minutes were repeated 30 times as one process. In this PCR, GeneAmp PCR System 9700 (Life Technologies) was used. After PCR, the amplification status of PCR products was confirmed by agarose gel elec...
Embodiment 2）
[0170] [Purpose]
[0171] The purpose is to confirm the amplification status of each HLA gene using Tks Gflex DNA Polymerase (TaKaRa) as an enzyme.
[0172] [method]
[0173] Using Tks Gflex DNA Polymerase (TaKaRa), the extracted genomic DNA was used as a template, and PCR was performed using HLA class I and HLA class II gene-specific primer sets (refer to Table 1 to Table 3: SEQ ID NO: 1 to 31). The specific steps are as follows.
[0174](1) Add 10 μL of 2xGflx PCR Buffer, 1-3 μL (4 pmol / μL) of PCR primers, and 0.2 μL of Tks Gflex DNA Polymerase to 50 ng of genomic DNA solution, and adjust the total amount of the reaction solution with sterilized water 20 μL.
[0175] (2) After heat-retaining at 94° C. for 2 minutes, two steps of 98° C. for 10 seconds and 68° C. for 5 minutes were repeated 10 times as one process. Then, two steps of 10 seconds at 98° C. and 5 minutes at 65° C. were repeated 20 times as one process. In this PCR, GeneAmp PCR System 9700 (Life Technologies)...
Embodiment 3）
[0180] [Purpose]
[0181] Confirm the difference in PCR status between the general-purpose PCR equipment, GeneAmp PCR System 9700 (Life Technologies) and the high-speed amplification equipment specifically used for PCR, PCR Thermal Cycler Fast (TaKaRa), and study the time required for PCR .
[0182] [method]
[0183] Using Tks Gflex DNA Polymerase (TaKaRa), the extracted genomic DNA was used as a template, and PCR was performed using HLA class I and HLA class II gene-specific primer sets (refer to Table 1 to Table 3: SEQ ID NO: 1 to 31). The specific steps are as follows.
[0184] (1) Add 10 μL of 2xGflex PCR Buffer, 1-3 μL (4 pmol / μL) of PCR primers, and 0.2 μL of Tks Gflx DNA Polymerase to 50 ng of genomic DNA solution, and adjust the total amount of the reaction solution to 20 μL.
[0185] (2) After heat-retaining at 94° C. for 2 minutes, two steps of 98° C. for 10 seconds and 68° C. for 5 minutes were repeated 10 times as one process. Then, two steps of 10 seconds at ...
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