RU2018138848A - Неинвазивная диагностика путем секвенирования 5-гидроксиметилированной бесклеточной днк - Google Patents
Неинвазивная диагностика путем секвенирования 5-гидроксиметилированной бесклеточной днк Download PDFInfo
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- RU2018138848A RU2018138848A RU2018138848A RU2018138848A RU2018138848A RU 2018138848 A RU2018138848 A RU 2018138848A RU 2018138848 A RU2018138848 A RU 2018138848A RU 2018138848 A RU2018138848 A RU 2018138848A RU 2018138848 A RU2018138848 A RU 2018138848A
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- bcdna
- hydroxymethylation
- hydroxymethylated
- sequence
- dna
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- 238000007031 hydroxymethylation reaction Methods 0.000 claims 15
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
- C12Q1/6855—Ligating adaptors
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Claims (42)
1. Способ секвенирования гидроксиметилированной бесклеточной ДНК (бкДНК), включающий:
(a) добавление адаптерных последовательностей на концы бкДНК;
(b) инкубацию лигированной с адаптерами бкДНК с ДНК-β-глюкозилтрансферазой и UDP-глюкозой, модифицированной хемоселективной группой, при этом происходит ковалентное мечение молекул гидроксиметилированной ДНК в бкДНК хемоселективной группой;
(c) присоединение биотинового компонента к хемоселективно модифицированной бкДНК по реакции циклоприсоединения;
(d) обогащение биотинилированных молекул ДНК путем связывания продукта из стадии (c) с носителем, связывающимся с биотином;
(e) амплификацию обогащенной ДНК с помощью праймеров, связывающихся с адаптерами; и
(f) секвенирование амплифицированной ДНК с получением множества прочтений последовательности,
причем способ не включает высвобождения биотинилированных молекул ДНК из носителя после стадии (d), перед стадией (e).
2. Способ анализа образцов, включающий:
(a) идентификацию гидроксиметилированных последовательностей в образце бесклеточной ДНК (бкДНК) пациента путем:
(i) лигирования адаптерных последовательностей с бкДНК; (ii) модификации остатков гидроксиметилцитозина (5hmC) в бкДНК так, чтобы они содержали метку захвата; (iii) обогащения бкДНК, содержащей остатки гидроксиметилцитозина, путем связывания бкДНК с носителем через метку захвата, получая при этом обогащенную композицию, содержащую связанную с носителем гидроксиметилированную бкДНК; (iv) амплификации молекул бкДНК без высвобождения гидроксиметилированной бкДНК из носителя, получая амплифицированную ДНК; (v) секвенирования продуктов амплификации с получением множества прочтений последовательности; и (vi) идентификации гидроксиметилированных последовательностей в прочтениях последовательности;
(b) сравнение идентифицированных гидроксиметилированных последовательностей с набором сигнатурных гидроксиметилированных последовательностей, коррелирующих с фенотипом; и
(c) составление отчета, показывающего корреляцию с фенотипом.
3. Способ по п. 2, при этом захватная метка включает биотиновый компонент.
4. Способ по п. 3, при этом захватная метка вводится по остаткам гидроксиметилцитозина в бкДНК на стадии (a)(ii) способом, включающим: инкубацию лигированной с адаптерами бкДНК с ДНК-β-глюкозилтрансферазой и UDP-глюкозой, модифицированной хемоселективной группой, при этом хемоселективная группа ковалентно связывается с остатками 5hmC в бкДНК; и проведение реакции биотинового компонента с хемоселективной группой, получая биотинилированную, гидроксиметилированную бкДНК.
5. Способ по п. 4, при этом бкДНК подвергается амплификации на стадии (a)(iv) с помощью праймеров, связывающихся с адаптерами.
6. Способ по п. 5, дополнительно включающий, после стадии (a)(iii) и перед стадией (a)(iv), отмывку носителя и постановку реакции амплификации, содержащей носитель, без высвобождения биотинилированных молекул ДНК из носителя.
7. Способ по п. 4, при этом UDP-глюкоза, модифицированная хемоселективной группой, включает UDP-6-N3-Glu, биотиновый компонент включает биотин, модифицированный дибензоциклооктином, а носитель включает авидин или стрептавидин.
8. Способ по п. 2, при этом фенотипом является заболевание, состояние или клинический исход.
9. Способ по п. 2, при этом стадия (a)(vi) является количественной.
10. Способ по п. 9, при этом стадия (a) дополнительно включает определение уровня гидроксиметилирования у каждой идентифицированной гидроксиметилированной последовательности, получая первый профиль гидроксиметилирования, включающий уровень гидроксиметилирования у каждой идентифицированной последовательности.
11. Способ по п. 10, дополнительно включающий получение второго образца бкДНК от пациента в другой момент времени и повторение стадии (a) со вторым образцом, получая второй профиль гидроксиметилирования.
12. Способ по п. 11, дополнительно включающий сравнение второго профиля гидроксиметилирования с первым профилем гидроксиметилирования для выявления изменений в гидроксиметилировании с течением времени.
13. Способ по п. 12, при этом сравнение приводит к получению карты изменений гидроксиметилирования в ходе заболевания или при лечении заболевания.
14. Способ по п. 10, при этом набор сигнатурных последовательностей гидроксиметилирования включает карту целевых локусов в контрольном профиле гидроксиметилирования, а стадия (b) включает сравнение уровня гидроксиметилирования для каждой гидроксиметилированной последовательности с уровнем гидроксиметилирования в соответствующем целевом локусе.
15. Способ по п. 14, дополнительно включающий определение того, является ли каждая идентифицированная гидроксиметилированная последовательность по уровню гидроксиметилирования чрезмерно представленной или недостаточно представленной относительно уровня гидроксиметилирования в соответствующем целевом локусе.
16. Способ по п. 15, при этом способ дополнительно включает (e) получение диагноза, решения о лечении или прогноза, исходя из результатов по идентичности тех гидроксиметилированных последовательностей, которые чрезмерно или недостаточно представлены относительно соответствующих целевых локусов.
17. Способ по п. 16, при этом диагноз, решение о лечении или прогноз составляет диагностику рака.
18. Способ по п. 17, при этом целевые локусы включают в себя одно или несколько тел следующих генов: ABRACL, ADAMTS4, AGFG2, ALDH1A3, ALG10B, AMOTL1, APCDD1L-AS1, ARL6IP6, ASF1B, ATP6V0A2, AUNIP, BAGE, C2orf62, C8orf22, CALCB, CC2D1B, CCDC33, CCNL2, CLDN15, COMMD6, CPLX2, CRP, CTRC, DACH1, DAZL, DDX11L1, DHRS3, DUSP26, DUSP28, EPN3, EPPIN-WFDC6, ETAA1, FAM96A, FENDRR, FLJ16779, FLJ31813, GBX1, GLP2R, GMCL1P1, GNPDA2, GPR26, GSTP1, HMOX2, HOXC5, IGSF9B, INSC, INSL4, IRF7, KIF16B, KIF20B, LARS, LDHD, LHX5, LINC00158, LINC00304, LOC100128946, LOC100131234, LOC100132287, LOC100506963, LOC100507250, LOC100507410, LOC255411, LOC729737, MAFF, NPAS4, NRADDP, P2RX2, PAIP1, PAX1, PODXL2, POU4F3, PSMG1, PTPN2, RAG1, RBM14-RBM4, RDH11, RFPL3, RNF122, RNF223, RNF34, SAMD11, SHISA2, SIGLEC10, SLAMF7, SLC25A46, SLC25A47, SLC9A3R2, SORD, SOX18, SPATA31E1, SSR2, STXBP3, SYT11, SYT2, TCEA3, THAP7-AS1, TMEM168, TMEM65, TMX2, TPM4, TPO, TRAM1, TTC24, UBQLN4, WASH7P, ZNF284, ZNF423, ZNF444, ZNF800, ZNF850 и ZRANB2.
19. Способ по п. 18, при этом адаптерные последовательности содержат молекулярный баркод.
20. Способ по п. 19, при этом молекулярный баркод включает последовательность идентификатора образца и последовательность идентификатора молекулы.
21. Способ по п. 2, при этом перед стадией (a) в образец вносится композиция добавляемого контроля.
22. Способ по п. 21, при этом композиция добавляемого контроля включает три ампликона, синтезированных с помощью коктейля из dATP, dGTP, dTTP и (1) dCTP, (2) dmCTP или (3) dhmCTP и dCTP.
23. Набор для анализа бкДНК, включающий:
ДНК-β-глюкозилтрансферазу;
UDP-глюкозу, модифицированную хемоселективной группой;
адаптер, содержащий по меньшей мере один молекулярный баркод; и
добавляемый контроль, включающий три ампликона, синтезированных с помощью коктейля из dATP, dGTP, dTTP и (1) dCTP, (2) dmCTP или (3) dhmCTP и dCTP.
24. Набор по п. 23, при этом по меньшей мере один баркод включает последовательность идентификатора образца и последовательность идентификатора молекулы.
25. Образец, включающий:
пул молекул бесклеточной ДНК, лигированной с адаптерами, которые получены из нескольких разных источников и содержат один или несколько модифицированных гидроксиметилцитозинов, содержащих метку захвата, причем молекулы ДНК дополнительно включают в себя молекулярные баркоды, указывающие на их источники и позволяющие отличить последовательности из разных источников после анализа; и
добавленный контроль, включающий три ампликона, синтезированных с помощью коктейля из dATP, dGTP, dTTP и (1) dCTP, (2) dmCTP или (3) dhmCTP и dCTP.
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