RU2013148909A - Способ и продукт для локализованной или пространственной детекции нуклеиновой кислоты в образце ткани - Google Patents

Способ и продукт для локализованной или пространственной детекции нуклеиновой кислоты в образце ткани Download PDF

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RU2013148909A
RU2013148909A RU2013148909/10A RU2013148909A RU2013148909A RU 2013148909 A RU2013148909 A RU 2013148909A RU 2013148909/10 A RU2013148909/10 A RU 2013148909/10A RU 2013148909 A RU2013148909 A RU 2013148909A RU 2013148909 A RU2013148909 A RU 2013148909A
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Йонас ФРИСЕН
Патрик СТОХЛЬ
Йоаким ЛУНДЕНБЕРГ
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Спейшел Транскриптомикс Аб
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Abstract

1. Способ локализованной детекции нуклеиновой кислоты в образце ткани, включающий:(а) предоставление чипа, содержащего подложку, на которой непосредственно или опосредованно иммобилизованы многочисленные разновидности захватывающих зондов, так что каждая разновидность занимает определенное положение на чипе и ориентирована таким образом, что имеет свободный 3′-конец, позволяющий указанному зонду действовать в качестве праймера в реакции удлинения или лигирования праймера, где каждая разновидность указанного захватывающего зонда содержит молекулу нуклеиновой кислоты, имеющую в направлении от 5′ к 3′:(1) позиционную область, которая соответствует положению захватывающего зонда на чипе, и(2) захватывающую область;(б) приведение указанного чипа в контакт с образцом ткани таким образом, что положение захватывающего зонда на чипе может быть сопоставлено с положением в образце ткани, и предоставление возможности нуклеиновой кислоте образца ткани гибридизоваться с захватывающей областью в указанных захватывающих зондах;(в) синтез молекул ДНК на основе захваченных молекул нуклеиновой кислоты с использованием указанных захватывающих зондов в качестве праймеров удлинения или лигирования, где указанные удлиненные или лигированные молекулы ДНК являются мечеными благодаря позиционной области;(г) возможно синтез комплементарной нити указанной меченой ДНК и/или возможно амплификацию указанной меченой ДНК;(д) высвобождение по меньшей мере части меченых молекул ДНК и/или их комплементов или ампликонов с поверхности чипа, где указанная часть включает позиционную область или ее комплемент; и(е) прямой или опосре�

Claims (43)

1. Способ локализованной детекции нуклеиновой кислоты в образце ткани, включающий:
(а) предоставление чипа, содержащего подложку, на которой непосредственно или опосредованно иммобилизованы многочисленные разновидности захватывающих зондов, так что каждая разновидность занимает определенное положение на чипе и ориентирована таким образом, что имеет свободный 3′-конец, позволяющий указанному зонду действовать в качестве праймера в реакции удлинения или лигирования праймера, где каждая разновидность указанного захватывающего зонда содержит молекулу нуклеиновой кислоты, имеющую в направлении от 5′ к 3′:
(1) позиционную область, которая соответствует положению захватывающего зонда на чипе, и
(2) захватывающую область;
(б) приведение указанного чипа в контакт с образцом ткани таким образом, что положение захватывающего зонда на чипе может быть сопоставлено с положением в образце ткани, и предоставление возможности нуклеиновой кислоте образца ткани гибридизоваться с захватывающей областью в указанных захватывающих зондах;
(в) синтез молекул ДНК на основе захваченных молекул нуклеиновой кислоты с использованием указанных захватывающих зондов в качестве праймеров удлинения или лигирования, где указанные удлиненные или лигированные молекулы ДНК являются мечеными благодаря позиционной области;
(г) возможно синтез комплементарной нити указанной меченой ДНК и/или возможно амплификацию указанной меченой ДНК;
(д) высвобождение по меньшей мере части меченых молекул ДНК и/или их комплементов или ампликонов с поверхности чипа, где указанная часть включает позиционную область или ее комплемент; и
(е) прямой или опосредованный анализ последовательности высвобожденных молекул ДНК.
2. Способ по п.1, дополнительно включающий стадию (ж) сопоставления информации из анализа указанной последовательности с изображением указанного образца ткани, где изображение образца ткани получают до или после стадии (в).
3. Способ по п.1, представляющий собой способ определения и/или анализа транскриптома образца ткани, включающий:
(а) предоставление чипа, содержащего подложку, на которой непосредственно или опосредованно иммобилизованы многочисленные разновидности захватывающих зондов, так что каждая разновидность занимает определенное положение на чипе и ориентирована таким образом, что имеет свободный 3′-конец, позволяющий указанному зонду действовать в качестве праймера обратной транскриптазы (ОТ), где каждая разновидность указанного захватывающего зонда содержит молекулу нуклеиновой кислоты, имеющую в направлении от 5′ к 3′:
(1) позиционную область, которая соответствует положению захватывающего зонда на чипе; и
(2) захватывающую область;
(б) приведение указанного чипа в контакт с образцом ткани таким образом, что положение захватывающего зонда на чипе может быть сопоставлено с положением в образце ткани, и предоставление возможности РНК образца ткани гибридизоваться с захватывающей областью в указанных захватывающих зондах;
(в) синтез молекул кДНК на основе захваченных молекул РНК с использованием указанных захватывающих зондов в качестве ОТ-праймеров и возможно амплификацию указанных молекул кДНК;
(г) высвобождение по меньшей мере части молекул кДНК и возможно их ампликонов с поверхности чипа, где указанная высвобожденная молекула может представлять собой первую нить и/или вторую нить молекулы кДНК или ее ампликон и где указанная часть включает позиционную область или ее комплемент;
(д) прямой или опосредованный анализ последовательности высвобожденных молекул ДНК; и возможно
(е) сопоставление информации из анализа указанной последовательности с изображением указанного образца ткани, где изображение образца ткани получают до или после стадии (в).
4. Способ по п.1, где захватывающие зонды представляют собой молекулы ДНК.
5. Способ по п.1, где захватывающие зонды дополнительно содержат универсальную область, которая расположена ближе к 5′-концу относительно позиционной области, где указанная универсальная область содержит:
(1) область амплификации для амплифицирования синтезированных молекул ДНК; и/или
(2) область расщепления для высвобождения синтезированных молекул ДНК с поверхности чипа.
6. Способ по п.1, где позиционная область каждой разновидности захватывающего зонда содержит уникальную штрих-кодовую последовательность.
7. Способ по п.1, где захватывающая область содержит поли(Т)-ДНК-олигонуклеотид, содержащий по меньшей мере 10 остатков дезокситимидина, и/или случайную или вырожденную олигонуклеотидную последовательность.
8. Способ по п.1, где захватывающие зонды иммобилизованы непосредственно на поверхности чипа через их 5′-конец.
9. Способ по п.1, где захватывающие зонды иммобилизованы опосредованно на поверхности чипа в результате гибридизации с поверхностным зондом, где захватывающая область захватывающих зондов содержит расположенную "вверх по течению" последовательность, способную гибридизоваться с 5′-концом поверхностных зондов, которые иммобилизованы на чипе.
10. Способ по п.9, где поверхностные зонды иммобилизованы на поверхности чипа через их 3′-конец.
11. Способ по п.9, где поверхностные зонды содержат последовательность, комплементарную:
(1) по меньшей мере части захватывающей области;
(2) позиционной области; и
(3) по меньшей мере части универсальной области амплификации.
12. Способ по п.1, где указанную комплементарную нить или указанную вторую нить молекулы кДНК синтезируют до или после высвобождения указанных меченых молекул ДНК или указанных молекул кДНК с поверхности чипа.
13. Способ по п.1, где для синтеза нити, комплементарной второй нити кДНК, используют полимеразу замещения нити.
14. Способ по п.1, где для синтеза комплементарной нити или второй нити кДНК используют случайные праймеры.
15. Способ по п.14, где случайные праймеры содержат область амплификации.
16. Способ по п.15, где область амплификации случайных праймеров состоит из нуклеотидной последовательности, отличающейся от области амплификации захватывающего зонда.
17. Способ по п.1, где к одному или обоим концам меченых молекул или их комплементов им нитей или молекул кДНК лигируют адаптеры.
18. Способ по п.1, дополнительно включающий стадию амплификации меченых молекул или молекул кДНК до проведения анализа последовательности.
19. Способ по п.18, где стадию амплификации осуществляют после высвобождения меченых молекул ДНК или кДНК с подложки чипа или где стадию амплификации осуществляют на чипе in situ.
20. Способ по п.18, где стадия амплификации включает ПЦР (полимеразную цепную реакцию).
21. Способ по п.1, где молекулы высвобождают с поверхности чипа посредством:
(1) отщепления нуклеиновой кислоты;
(2) денатурации; и/или
(3) физического способа.
22. Способ по п.21(1), где молекулы высвобождают посредством ферментативного расщепления области расщепления, которая локализована в универсальной области или позиционной области захватывающего зонда.
23. Способ по п.21(2) или (3), где молекулы высвобождают посредством подведения горячей воды или буфера к чипу.
24. Способ по п.1, дополнительно включающий стадию очистки высвобожденных молекул до секвенирования.
25. Способ по п.1, где подложка выбрана из группы, состоящей из стекла, кремния, покрытого поли-L-лизином материала, нитроцеллюлозы, полистирола, сополимеров циклических олефинов (СОС), полимеров циклических олефинов (СОР), полипропилена, полиэтилена и поликарбоната.
26. Способ по п.1, где образец ткани представляет собой срез ткани.
27. Способ по п.26, где срез ткани получают с использованием фиксированной ткани, например фиксированной в формалине залитой парафином (FFPE) ткани, или ткани после глубокого замораживания.
28. Способ по п.1, дополнительно включающий стадию регидратации образца ткани после приведения образца в контакт с чипом и до стадии (в).
29. Способ по п.1, где образец ткани происходит из растения, животного или гриба.
30. Способ по п.1, дополнительно включающий стадию промывки чипа для удаления оставшейся ткани, где указанную стадию осуществляют до высвобождения молекул с чипа.
31. Способ по п.1, где чип содержит по меньшей мере один позиционный маркер, позволяющий осуществлять ориентацию образца ткани на чипе.
32. Способ по п.31, где чип содержит по меньшей мере 10, 50, 100, 200, 500, 1000 или 2000 позиционных маркеров в определенных положениях на поверхности чипа.
33. Способ по п.31, где позиционные маркеры способны гибридизоваться с меченой, предпочтительно меченной флуоресцентной меткой, маркерной молекулой нуклеиновой кислоты.
34. Способ по п.2, где изображение образца ткани получают с использованием световой, светлопольной, темнопольной, фазово-контрастной, флуоресцентной, отражательной, интерференционной или конфокальной микроскопии или их комбинации.
35. Способ по п.34, где изображение образца ткани получают с использованием флуоресцентной микроскопии.
36. Способ по любому из пп.1-35, где стадия анализа последовательности включает стадию секвенирования и, предпочтительно, где стадия секвенирования включает реакцию секвенирования, основанную на обратимых красителях-терминаторах.
37. Способ изготовления или получения чипа (1) для применения в захвате нуклеиновой кислоты из образца ткани, который приводят в контакт с указанным чипом; или (2) для применения в локализованной детекции нуклеиновой кислоты в образце ткани, включающий иммобилизацию многочисленных разновидностей захватывающего зонда непосредственно или опосредованно на подложке чипа, где каждая разновидность указанного захватывающего зонда содержит молекулу нуклеиновой кислоты, имеющую в направлении от 5′ к 3′:
(1) позиционную область, которая соответствует положению захватывающего зонда на чипе; и
(2) захватывающую область.
38. Способ по п.37, где каждая разновидность захватывающего зонда занимает определенное положение на чипе.
39. Способ по п.37, дополнительно включающий любую одну или более чем одну из следующих стадий:
(а) иммобилизация многочисленных поверхностных зондов непосредственно или опосредованно на подложке чипа, где поверхностные зонды содержат:
(1) область, способную гибридизоваться с частью олигонуклеотида захватывающей области (частью, не вовлеченной в захват нуклеиновой кислоты);
(2) комплементарную позиционную область; и
(3) комплементарную универсальную область;
(б) гибридизация олигонуклеотидов захватывающих областей и олигонуклеотидов универсальных областей с поверхностными зондами, иммобилизованными на чипе;
(в) удлинение олигонуклеотидов универсальных областей посредством матричной полимеризации для синтеза позиционной области захватывающего зонда; и
(г) лигирование позиционной области с олигонуклеотидом захватывающей области для получения захватывающего олигонуклеотида.
40. Чип для применения в (1) захвате нуклеиновой кислоты из образца ткани, который приводят в контакт с указанным чипом; или (2) в локализованной детекции нуклеиновой кислоты в образце ткани, содержащий подложку, на которой непосредственно или опосредованно иммобилизованы многочисленные разновидности захватывающих зондов, так что каждая разновидность занимает определенное положение на чипе и ориентирована таким образом, что имеет свободный 3′-конец, позволяющий указанному зонду действовать в качестве праймера удлинения или лигирования, где каждая разновидность указанного захватывающего зонда содержит молекулу нуклеиновой кислоты, имеющую в направлении от 5′ к 3′:
(1) позиционную область, которая соответствует положению захватывающего зонда на чипе, и
(2) захватывающую область для захвата нуклеиновой кислоты образца ткани, который приводят в контакт с указанным чипом.
41. Способ по любому из пп.37-39 или чип по п.40, где захватывающие зонды являются такими, как определено в любом из пп.4-9, поверхностные зонды являются такими, как определено в п.п.10 или 11, подложка является такой, как определено в п.25, образец ткани является таким, как определено в любом из пп.26, 27 или 28, чип является таким, как определено в любом из пп.31-33, и позиционные маркеры являются такими, как определено в п.33.
42. Способ по п.1 или 2, представляющий собой способ локализованной детекции ДНК в образце ткани, включающий:
(а) предоставление чипа, содержащего подложку, на которой непосредственно или опосредованно иммобилизованы многочисленные разновидности захватывающих зондов, так что каждая разновидность занимает определенное положение на чипе и ориентирована таким образом, что имеет свободный 3′-конец, позволяющий указанному зонду действовать в качестве праймера в реакции удлинения или лигирования праймера, где каждая разновидность указанного захватывающего зонда содержит молекулу нуклеиновой кислоты, имеющую в направлении от 5′ к 3′:
(1) позиционную область, которая соответствует положению захватывающего зонда на чипе, и
(2) захватывающую область;
(б) приведение указанного чипа в контакт с образцом ткани таким образом, что положение захватывающего зонда на чипе может быть сопоставлено с положением в образце ткани, и предоставление возможности ДНК образца ткани гибридизоваться с захватывающей областью в указанных захватывающих зондах;
(в) фрагментацию ДНК в указанном образце ткани, которую осуществляют до, в процессе или после приведения чипа в контакт с образцом ткани на стадии (б);
(г) удлинение указанных захватывающих зондов в реакции удлинения праймеров с использованием захваченных фрагментов ДНК в качестве матриц для синтеза удлиненных молекул ДНК или лигирование захваченных фрагментов ДНК с захватывающими зондами в реакции лигирования для синтеза лигированных молекул ДНК, где указанные удлиненные или лигированные молекулы ДНК являются мечеными благодаря позиционной области;
(д) возможно синтез комплементарной нити указанной меченой ДНК и/или возможно амплификацию указанной меченой ДНК;
(е) высвобождение по меньшей мере части меченых молекул ДНК и/или их комплементов и/или ампликонов с поверхности чипа, где указанная часть включает позиционную область или ее комплемент;
(ж) прямой или опосредованный анализ последовательности (например, секвенирование) высвобожденных молекул ДНК; и возможно
(з) сопоставление информации из анализа указанной последовательности с изображением указанного образца ткани, где изображение образца ткани получают до или после стадии (г).
43. Способ по п.42, включающий:
(а) предоставление чипа, содержащего подложку, на которой непосредственно или опосредованно иммобилизованы многочисленные разновидности захватывающих зондов, так что каждая разновидность занимает определенное положение на чипе и ориентирована таким образом, что имеет свободный 3′-конец, позволяющий указанному зонду действовать в качестве праймера в реакции удлинения или лигирования праймера, где каждая разновидность указанного захватывающего зонда содержит молекулу нуклеиновой кислоты, имеющую в направлении от 5′ к 3′:
(1) позиционную область, которая соответствует положению захватывающего зонда на чипе, и
(2) захватывающую область;
(б) приведение указанного чипа в контакт с образцом ткани таким образом, что положение захватывающего зонда на чипе может быть сопоставлено с положением в образце ткани;
(в) фрагментацию ДНК в указанном образце ткани, которую осуществляют до, в процессе или после приведения чипа в контакт с образцом ткани на стадии (б);
(г) обеспечение указанных фрагментов ДНК связывающей областью, которая способна гибридизоваться с указанной захватывающей областью;
(д) предоставление возможности указанным фрагментам ДНК гибридизоваться с захватывающей областью в указанных захватывающих зондах;
(е) удлинение указанных захватывающих зондов в реакции удлинения праймеров с использованием захваченных фрагментов ДНК в качестве матриц для синтеза удлиненных молекул ДНК или лигирование захваченных фрагментов ДНК с захватывающими зондами в реакции лигирования для синтеза лигированных молекул ДНК, где указанные удлиненные или лигированные молекулы ДНК являются мечеными благодаря позиционной области;
(ж) возможно синтез комплементарной нити указанной меченой ДНК и/или возможно амплификацию меченой ДНК;
(з) высвобождение по меньшей мере части меченых молекул ДНК и/или их комплементов и/или ампликонов с поверхности чипа, где указанная часть включает позиционную область или ее комплемент;
(и) прямой или опосредованный анализ последовательности (например, секвенирование) высвобожденных молекул ДНК; и возможно
(к) сопоставление информации из анализа указанной последовательности с изображением указанного образца ткани, где изображение образца ткани получают до или после стадии (е).
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