Multiple dna typing method and kit for hla gene
A DNA typing, HLA-A technology, applied in the direction of recombinant DNA technology, DNA / RNA fragments, biochemical equipment and methods, etc., can solve the problems that the PCR multiplex method has not yet been achieved, and the PCR conditions have not been completely unified.
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Embodiment 1）
[0161] [Purpose]
[0162] The amplification status of each HLA gene was confirmed 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 for 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 e...
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), PCR was performed using the extracted genomic DNA as a template, and each gene-specific primer set for 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.
[0174](1) Add 10 μL of 2x Gflx 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] It is necessary to check the difference in PCR status between the general-purpose PCR equipment, GeneAmp PCR System 9700 (Life Technologies) and the high-speed amplification equipment specifically for PCR, PCR Thermal Cycler Fast (TaKaRa). time.
[0182] [method]
[0183] Using Tks Gflex DNA Polymerase (TaKaRa), PCR was performed using the extracted genomic DNA as a template, and each gene-specific primer set for 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.
[0184] (1) Add 10 μL of 2x Gflex 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 with sterilized water 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 9...
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