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Home > ÀüÁ¦Ç°º¸±â > PCR > Overview > [Cancer Research] HLA typing

[Cancer Research] HLA typing

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[Reference data] DNA polymerase

Takara LA Taq¢ç
1) Lan, James H., et al. "Impact of three Illumina library construction methods on GC bias and HLA genotype calling." Human Immunology 76.2-3 (2015): 166-175. ³» ¹ßÃé
NGS´Â ±¸ºÐÀÌ ¾î·Á¿î ´ë¸³ À¯ÀüÀÚ¸¦ È®ÀÎÇÏ°í ³ôÀº Á¤È®µµ·Î HLA typingÀÌ °¡´ÉÇÑ °ÍÀ¸·Î Á¡Â÷ Àνĵǰí ÀÖ´Ù. HLA À¯ÀüÀÚ´Â ´ÙÇü¼ºÀÇ Æ¯Â¡À» °¡Áö°í ÀÖ¾î, NGS¸¦ ÅëÇØ À̸¦ ºÐ¼®ÇÏ´Â °æ¿ì Á¾Á¾ ±ÕÀÏÇÏÁö ¾ÊÀº reads°¡ ºÐÆ÷ÇÔÀ¸·Î½á º¯ÀÌ °ËÃâÀÇ ½Å·Úµµ¸¦ ³·Ãâ ¼ö ÀÖ´Ù. ÃÖ±Ù, Coverage bias¸¦ À¯¹ßÇÏ´Â ¿äÀÎÀÌ library Á¦ÀÛ °úÁ¤¿¡¼­ À¯¹ßµÈ´Ù´Â »ç½ÇÀÌ È®ÀÎµÈ ¹Ù ÀÖ´Ù. HLA genotyping °úÁ¤¿¡¼­ÀÇ ¿µÇâÀ» È®ÀÎÇϱâ À§ÇØ, º» ³í¹®ÀÇ ÀúÀÚ´Â ¸ÕÀú, 12°³ »ùÇÃÀ» ´ë»óÀ¸·Î Takara LA Taq¢ç À» ÀÌ¿ëÇØ long-range PCRÀ» ÁøÇàÇÏ¿´´Ù. °¢ »ùÇÿ¡¼­ HLA-A, -B, -C, -DRB1, -DQB1¿¡ ´ëÇÑ À¯ÀüÀÚ¸¦ ÁõÆøÇÑ µÚ, TruSeq Nano, Nextera, Nextera XT, ÃÑ ¼¼°³ÀÇ Illumina¢ç library Á¦ÀÛ Á¦Ç°À» ÀÌ¿ëÇÏ¿© °á°ú¸¦ ºñ±³ÇÏ¿´´Ù. ÀÌ ¿¬±¸ °á°ú¸¦ ÅëÇØ, ±ÕÀÏÇÏÁö ¾ÊÀº read depth°¡ HLA typing¿¡ ºÎÁ¤ÀûÀÎ ¿µÇâÀÌ ÀÖÀ½À» È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù.


±×¸² 1. Comparison of three Illumina library construction protocols. Covaris M220 was used to fragment DNA in TruSeq Nano.

2) Mayor, Neema P., et al. "HLA typing for the next generation." PLoS One 10.5 (2015): e0127153. ³» ¹ßÃé
´ë¸³ À¯ÀüÀÚ ¼öÁØ¿¡¼­ÀÇ HLA typingÀ» Çѹø¿¡ ÁøÇàÇÏ´Â °ÍÀº Á¶Á÷ ȣȯ¼ºÀ» È®ÀÎÇÏ´Â ¿¬±¸¿¡¼­ ¸Å¿ì ÀÌ»óÀûÀÌ´Ù. ´ë·®À¸·Î ÁøÇàµÇ´Â ´ëºÎºÐÀÇ molecular HLA typing Á¢±Ù¹ýÀÇ °á°ú·Î ºÐ¼®Çϱ⠾î·Á¿î DNA ¼­¿­ÀÇ ´ÙÇü¼º °ËÃâÀ» ¾î·Æ°Ô ÇÑ´Ù. º¸´Ù Á¤È®ÇÑ ¹æ¹ýÀ» ÀÌ¿ëÇÏ´Â °æ¿ì¿¡´Â Á¾Á¾ ½Ã°£°ú ºñ¿ë¿¡ ÇÑ°è°¡ ÀÖ´Ù Pacific Bioscience´Â Single Molecule Real Time (SMRT) DNA sequencing¸¦ ÀÌ¿ëÇÏ¿© ´ë·®À¸·Î º¸´Ù Á¤È®ÇÑ HLA typingÀ» ÇÒ¼ö ÀÖÀ½À» È®ÀÎÇÏ¿´´Ù. SMRT sequencing ¹æ¹ýÀº 3 kb ÀÌ»óÀÇ HLA class ¥° À¯ÀüÀÚ ¼­¿­À» Takara LA Taq¢ç À» ÀÌ¿ëÇØ long-range PCRÇÑ ÈÄ, ±ä ±æÀÌÀÇ reads¸¦ ¿¹¿Ü ¾øÀÌ ºÐ¼®ÇÔÀ¸·Î½á ´ÙÇü¼ºÀ» ¶ç´Â ¼­¿­À» È®ÀÎÇÒ ¼ö ÀÖ´Ù.


±×¸² 1. Basic stages of the Single Molecule Real-Time (SMRT) DNA sequencing method.

PrimeSTAR¢ç GXL DNA Polymerase
1) Liu, Chang, et al. "Accurate typing of human leukocyte antigen class I genes by Oxford Nanopore sequencing." The Journal of Molecular Diagnostics 20.4 (2018): 428-435. ³» ¹ßÃé
HLA typingÀº Àå±â ±âÁõÀÚ¿Í ¼öÇýÀÚ °£ÀÇ ¸é¿ª ¹ÝÀÀ È®ÀÎÀ» À§ÇØ ¸Å¿ì Áß¿äÇÏ´Ù. ¹°·Ð ºü¸£°í Á¤È®ÇÑ HLA typing ¹æ¹ýÀ» ÀÌ¿ëÇÏ´Â °ÍÀÌ Àå±â ±âÁõÀ» À§ÇØ °¡Àå ÀÌ»óÀûÀ̳ª, Sanger sequencingÀ̳ª NGSÀ» ÀÌ¿ëÇÏ´Â °ÍÀº ±â¼úÀûÀÎ ÇÑ°è°¡ ÀÖ¾ú´Ù. ´ëºÎºÐÀÇ Illumina ȤÀº ion-torrentÀÇ HLA typing ¹æ¹ýÀº long-range PCR product¸¦ enzyme ó¸®ÇÑ ÈÄ ÁõÆøÀ» Çѹø ´õ ÁøÇàÇÏ¿©, ÃÑ ºÐ¼® ½Ã°£ÀÌ ±æ »Ó¸¸ ¾Æ´Ï¶ó GC ¼­¿­À» ´Ù¼ö Æ÷ÇÔÇÏ´Â exon ºÎÀ§¿¡¼­ ÆíÇ⼺ÀÌ È®À뵃 ¼ö ÀÖ´Ù. º» ³í¹®Àº Àΰ£ DNA »ùÇ÷κÎÅÍ Á¤È®ÇÑ class ¥° HLA À¯ÀüÀÚ¸¦ ºÐ¼®Çϱâ À§Çؼ­´Â Oxford nanopore sequencingÀÌ ÃÖÀûÈ­µÇ¾ú´Ù´Â °ÍÀ» Áõ¸íÇÏ°íÀÚ ÇÏ¿´´Ù. À̸¦ À§ÇØ, PrimeSTAR¢ç GXL DNA polymerase¸¦ ÀÌ¿ëÇØ ¸ÕÀú Class ¥° HLA À¯ÀüÀÚ Áß HLA-A, -B, -C ¼­¿­À» long-range PCR·Î ÁõÆøÇÏ¿´À¸¸ç, sequencing adaptor¸¦ ligationÇÑ µÚ MinION sequencingÀ» ÁøÇàÇÏ¿´´Ù.

2) Ozaki, Yuki, et al. "Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing." BMC genomics 16.1 (2015): 318. ³» ¹ßÃé
NGS¸¦ ÀÌ¿ëÇÑ HLA genotypingÀº PCR, NGS, allele assignment ÃÑ ¼¼ °úÁ¤À¸·Î ÁøÇàµÈ´Ù. PCR-sequence specific oligonucleotide primers (SSOP)³ª sequence based typing (SBT) ¹æ¹ý°ú ºñ±³ÇßÀ» ¶§, PCR-NGS´Â ¸Å¿ì ³ëµ¿ Áý¾àÀûÀÌ°í, ½Ã°£ÀÌ ¸¹ÀÌ ¼Ò¿äµÈ´Ù. ¿©·¯ DNA »ùÇÿ¡¼­ÀÇ NGS ±â¹Ý HLA genotyping ¹æ¹ýÀ» °£¼ÒÈ­ÇÏ°í °¡¼ÓÈ­ Çϱâ À§Çؼ­, º» ³í¹®ÀÇ ÀúÀÚµéÀº ¸ÕÀú, ´ÙÇü¼ºÀÇ exonÀ» genotypingÇÒ ¼ö ÀÖ´Â HLA loci Ÿ°ÙÀÇ middle ranged PCR primer set¸¦ »õ·Î µðÀÚÀÎÇÏ¿´°í PrimeSTAR¢ç GXL DNA polymerase¸¦ ÀÌ¿ëÇØ multiplex·Î PCR ÁõÆøÇÏ¿´´Ù. À̸¦ ÀÌ¿ëÇØ HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, HLA-DPB1À» Æ÷ÇÔÇÏ´Â ÃÑ 9°³ÀÇ HLA loci¸¦ ´ë»óÀ¸·Î genotypingÀ» À§ÇÑ multiplex PCR ¹æ¹ýÀ» °³¹ßÇÏ°í Æò°¡ÇÏ¿´´Ù.


±×¸² 5. Outline of the targeted PCR regions in nine HLA loci. Black, gray and white boxes indicate promoter regions, highly polymorphic exons and the other exons, respectively.

3) Yin, Yuxin, et al. "Application of high-throughput next-generation sequencing for HLA typing on buccal extracted DNA: results from over 10,000 donor recruitment samples." PLoS One 11.10 (2016): e0165810.³» ¹ßÃé
Hematopoietic stem cell transplantation (HSCT)¿¡¼­ÀÇ Á¤È®ÇÑ HLA typingÀ» ÇÏ´Â °ÍÀº HLA Áúº´ ¹× Àå±â ÀÌ½Ä ¿¬±¸¿¡¼­ ¸Å¿ì Áß¿äÇÏ´Ù. ÇÏÁö¸¸, ÃÖ±ÙÀÇ Molecular typing ¹æ¹ýÀº HLA À¯ÀüÀÚÀÇ Ç׿ø ÀÎ½Ä ºÎÀ§ (antigen recognition site, ARS) ¸¸À» È®ÀÎÇϱ⠶§¹®¿¡, °á°úÀûÀ¸·Î ¸¹Àº cis-trans ÇüÅ°¡ ºÒºÐ¸íÇÏ¿© Ãß°¡ÀûÀÎ typing ¹æ¹ýÀ¸·Î À̸¦ È®ÀÎÇؾ߸¸ Çß´Ù. º» ³í¹®ÀÇ ÀúÀÚµéÀº National Marrow Donar Program (NMDP) °ñ¼ö ±âÁõÀÚ 10,063¸íÀ» ´ë»óÀ¸·Î HLA typingÀ» ÁøÇàÇÏ¿´´Ù. ¸ÕÀú ȹµæÇÑ Swap DNA¸¦ Á¤Á¦ÇÑ ÈÄ, PrimeSTAR¢ç GXL DNA polymerase¿Í HLA class ¥° À¯ÀüÀÚ Àüü ¼­¿­À» Ÿ°ÙÇÏ´Â PCR primer·Î long-range PCRÀ» ÁøÇà ÈÄ, NGS·Î À̸¦ ºÐ¼®ÇÏ¿© °á°ú¸¦ µµÃâÇÏ¿´´Ù.


±×¸² 1. HLA typing strategy.
The primer design captures full-length HLA class I genes (HLA-A, -B, -C) and exons 2-4 of DRB1 and DQB1 genes. There are two multiplex primer sets: Class I primer mix includes HLA-A, -B, -C, and Class II primer mix includes DRB1 and DQB1.