ES2645661T3 - High performance detection of molecular markers based on restriction fragments - Google Patents

High performance detection of molecular markers based on restriction fragments Download PDF

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ES2645661T3
ES2645661T3 ES15158621.1T ES15158621T ES2645661T3 ES 2645661 T3 ES2645661 T3 ES 2645661T3 ES 15158621 T ES15158621 T ES 15158621T ES 2645661 T3 ES2645661 T3 ES 2645661T3
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Michael Josephus Theresia Van Eijk
René Cornelis Josephus Hogers
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Keygene NV
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    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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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

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Abstract

Método para identificar la presencia o ausencia de fragmentos de restricción en una muestra, que comprende las etapas de: (a) proporcionar dos o más muestras de ácidos nucleicos; (b) digerir cada muestra de ácido nucleico con al menos una endonucleasa de restricción para obtener un conjunto de fragmentos de restricción; (c) proporcionar adaptadores sintéticos bicatenarios que comprenden - una sección identificadora específica de la muestra, - al menos un extremo que se puede ligar al extremo romo o que sobresale de un fragmento de restricción; (d) ligar los adaptadores sintéticos bicatenarios a los fragmentos de restricción en el conjunto, para proporcionar un conjunto de fragmentos de restricción ligados al adaptador; (e) determinar la secuencia de al menos la sección identificadora específica de la muestra, y parte de la secuencia del fragmento de restricción situada adyacente a la secuencia derivada del adaptador, (f) comparar dos o más muestras para la presencia o ausencia de fragmentos de restricción ligados al adaptador; (g) identificar la presencia o ausencia de fragmentos de restricción ligados al adaptador en la muestra.Method for identifying the presence or absence of restriction fragments in a sample, comprising the steps of: (a) providing two or more nucleic acid samples; (b) digest each nucleic acid sample with at least one restriction endonuclease to obtain a set of restriction fragments; (c) providing double-stranded synthetic adapters comprising - a specific identifying section of the sample, - at least one end that can be linked to the blunt end or protruding from a restriction fragment; (d) link the double-stranded synthetic adapters to the restriction fragments in the assembly, to provide a set of restriction fragments linked to the adapter; (e) determine the sequence of at least the specific identifying section of the sample, and part of the sequence of the restriction fragment located adjacent to the sequence derived from the adapter, (f) compare two or more samples for the presence or absence of fragments of restriction linked to the adapter; (g) identify the presence or absence of restriction fragments bound to the adapter in the sample.

Description

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reconocimiento de la endonucleasa de restricción y cualquier base selectiva opcional. Cuando se usa un identificador de muestra con 6 pb, los restos son del cortador inusual EcoRI (AACCT), el uso de dos bases selectivas proporciona una secuencia interna del fragmento de restricción de 12 pb que se puede usar para identificar únicamente el fragmento de restricción en la muestra. restriction endonuclease recognition and any optional selective basis. When a sample identifier with 6 bp is used, the remains are from the unusual EcoRI cutter (AACCT), the use of two selective bases provides an internal sequence of the 12 bp restriction fragment that can be used to identify only the restriction fragment In the sample.

[0063] En una realización preferente basada en la tecnología de secuenciación Solexa mencionada anteriormente, la amplificación de los fragmentos de restricción ligados al adaptador se realiza con un cebador que contiene como máximo un nucleótido selectivo en su extremo 3’, preferentemente ningún nucleótido selectivo en su extremo 3’, es decir, el cebador solamente es complementario con el adaptador (un cebador +0). [0063] In a preferred embodiment based on the Solexa sequencing technology mentioned above, amplification of the restriction fragments linked to the adapter is performed with a primer containing at most one selective nucleotide at its 3 'end, preferably no selective nucleotide in its 3 'end, that is, the primer is only complementary to the adapter (a +0 primer).

[0064] En realizaciones alternativas dirigidas a los métodos de secuenciación que se describen en el presente documento, los cebadores usados en la amplificación pueden contener secciones específicas (como alternativa al cebador o secuencias de unión al cebador que se describen en el presente documento) que se usan en la etapa de secuenciación posterior para unir los fragmentos de restricción protegidos con el adaptador o los amplicones con la superficie. Estas se indican generalmente como la región clave o la secuencia compatible con el cebador 5’. [0064] In alternative embodiments directed to the sequencing methods described herein, the primers used in the amplification may contain specific sections (as an alternative to the primer or primer binding sequences described herein) that they are used in the subsequent sequencing step to join the restriction fragments protected with the adapter or amplicons with the surface. These are generally indicated as the key region or sequence compatible with primer 5 ’.

[0065] En una realización de la divulgación, la muestra de ácido nucleico se digiere con al menos una enzima de restricción y al menos se liga un adaptador que comprende una secuencia de reconocimiento para una endonucleasa de restricción de tipo IIs. La digestión posterior del fragmento de restricción ligado al adaptador con una endonuclease de restricción de tipo IIs proporciona, ya que la distancia entre el sitio de reconocimiento y de restricción de una enzima de tipo IIs es relativamente corta (hasta aproximadamente 30 nucleótidos), un fragmento de restricción más corto y uno más largo, al que se puede ligar un adaptador compatible con un sitio de restricción de tipo IIs. Por lo general, se desconoce el saliente del sitio limitado por IIs de modo que se puede usar un conjunto de adaptadores que se degeneran en el saliente. Después de la amplificación (selectiva), los amplicones se pueden secuenciar. La secuencia adaptadora en esta realización por lo general es la que sigue: 5’-sitio de unión al cebador---secuencia identificadora de la muestra---secuencia del extremo cohesivo de tipo IIs degenerado- 3’. El cebador de PCR asociado generalmente es el que sigue: secuencia cegadora--secuencia identificadora de la muestra---secuencia del extremo cohesivo de tipo IIs degenerado---nucleótidos selectivos -3’. El cebador usado para iniciar la secuenciación por síntesis por lo general tiene la estructura: 5’sitio de unión al cebador-3’. Puede ser preferente una etapa de selección del tamaño después de la digestión con la enzima IIs para retirar los fragmentos más pequeños. Al igual que en esta realización, los restos del sitio de restricción son para este tipo de enzima por lo general del orden de 2 a 4 pb, esto resulta en combinación con un identificador de la muestra de 6 pb en la secuenciación de 15 a 17 pb de un fragmento de restricción. [0065] In one embodiment of the disclosure, the nucleic acid sample is digested with at least one restriction enzyme and at least one adapter that comprises a recognition sequence for a type IIs restriction endonuclease is ligated. Subsequent digestion of the restriction fragment bound to the adapter with a type IIs restriction endonuclease provides, since the distance between the recognition and restriction site of a type IIs enzyme is relatively short (up to about 30 nucleotides), a fragment of shorter and longer restriction, to which a compatible adapter can be linked to a type IIs restriction site. In general, the projection of the site limited by IIs is unknown so that a set of adapters that degenerate into the projection can be used. After amplification (selective), the amplicons can be sequenced. The adapter sequence in this embodiment is generally as follows: 5'-primer binding site --- sample identifier sequence --- degenerate type IIs cohesive sequence- 3 '. The associated PCR primer is generally the following: blinding sequence - sample identifying sequence --- degenerated type IIs cohesive end sequence --- selective nucleotides -3 ’. The primer used to initiate the sequencing by synthesis usually has the structure: 5 'primer binding site-3'. A step of size selection may be preferred after digestion with the enzyme IIs to remove the smaller fragments. As in this embodiment, the restriction site residues are for this type of enzyme usually of the order of 2 to 4 bp, this results in combination with a 6 bp sample identifier in sequencing from 15 to 17 bp of a restriction fragment.

[0066] En un aspecto adicional, la divulgación se refiere a kits que comprenden uno o más cebadores, y/o uno o más adaptadores para uso del método, además de componentes convencionales para kits per se. Además, la presente invención encuentra aplicación, entre otros, en el uso del método para la identificación de marcadores moleculares, para genotipificación, análisis de segregación en masa, formación de mapas genéticos, retrocruzamiento asistido por marcador, formación de mapas de sitios de rasgos cuantitativos, formación de mapas de desequilibrio de unión. [0066] In a further aspect, the disclosure refers to kits comprising one or more primers, and / or one or more adapters for use of the method, in addition to conventional components for kits per se. In addition, the present invention finds application, among others, in the use of the method for the identification of molecular markers, for genotyping, mass segregation analysis, formation of genetic maps, marker assisted backcrossing, formation of quantitative feature site maps. , formation of union imbalance maps.

Ejemplo Example

[0067] Se aisló ADN de 2 padres y 88 descendencias usando métodos convencionales. Los padres (2 x) y la descendencia (= 4 x) estaban en dúplex con diferentes índices para someter a ensayo la reproducibilidad. Se usaron marcas para distinguir las muestras entre sí diferían por lo menos en 2 nucleótidos de cualquier otra marca usada en los experimentos. La calidad se está sometiendo a ensayo en las distintas etapas usando geles de agarosa y PAA. [0067] DNA from 2 parents and 88 offspring was isolated using conventional methods. The parents (2 x) and the offspring (= 4 x) were in duplex with different indexes to test the reproducibility. Marks were used to distinguish the samples from each other differing at least 2 nucleotides from any other brand used in the experiments. Quality is being tested at different stages using agarose gels and PAA.

Ejemplo 1 Example 1

[0068] Para cada muestra de ADN, se realiza una etapa de restricción-ligación usando EcoRI y MseI como enzimas. Los adaptadores se basan en las secuencias de hibridación situadas en la superficie del sistema de secuenciación de alto rendimiento Solexa, más en particular, un adaptador de EcoRI contiene la secuencia P5 (parte del cebador de la secuencia) y el adaptador de MseI contiene la secuencia P7 (secuencia de cebador de PCR unido por puente). El adaptador de EcoRI contiene adicionalmente la muestra que identifica la marca. Se usan 96 adaptadores de EcoRI diferentes y un adaptador de MseI. Es posible usar un adaptador de EcoRI degenerado. La preparación del molde incluye una etapa de selección del tamaño mediante incubación de la mezcla durante 10 minutos a 80 grados Celsius después de la etapa de restricción (EcoRI + MseI) pero antes de la etapa de ligación al adaptador. Los fragmentos menores de 130 nt se retiran (en una muestra de maíz). La complejidad de la mezcla se reduce mediante una amplificación previa selectiva usando +1 cebadores (es decir, que contiene un nucleótido aleatoriamente selectivo en el extremo 3’, usando 96 cebadores de EcoRI+1 si un cebador de MseI+1 (o un cebador de EcoRI+1 con marca degenerada y un cebador de MseI +1). Se realiza amplificación selectiva para reducir la complejidad de la mezcla hasta el tamaño deseado usando [0068] For each DNA sample, a restriction-ligation step is performed using EcoRI and MseI as enzymes. The adapters are based on the hybridization sequences located on the surface of the Solexa high performance sequencing system, more particularly, an EcoRI adapter contains the P5 sequence (part of the sequence primer) and the MseI adapter contains the sequence P7 (bridged PCR primer sequence). The EcoRI adapter additionally contains the sample that identifies the brand. 96 different EcoRI adapters and an MseI adapter are used. It is possible to use a degenerated EcoRI adapter. The mold preparation includes a size selection stage by incubation of the mixture for 10 minutes at 80 degrees Celsius after the restriction stage (EcoRI + MseI) but before the ligation stage to the adapter. Fragments less than 130 nt are removed (in a corn sample). The complexity of the mixture is reduced by selective prior amplification using +1 primers (i.e., it contains a randomly selective nucleotide at the 3 'end, using 96 EcoRI + 1 primers if an MseI + 1 primer (or a primer EcoRI + 1 with degenerate label and a MseI + 1 primer) Selective amplification is performed to reduce the complexity of the mixture to the desired size using

11 eleven

cebadores EcoRI+2 (= P5 lateral) y MseI+3 (= P7 lateral) que necesitan el uso de 96 cebadores de EcoRI+2 y un cebador de MseI+3. Se realiza PCR de cola usando un cebador de EcoRI con la secuencia del cebador de PCR con puente en P5 como la cola. Los productos se codifican usando columnas de Sephadex™. Las concentraciones se determinan y se normalizan y se crean grupos. Los grupos se someten a secuenciación Primers EcoRI + 2 (= lateral P5) and MseI + 3 (= lateral P7) that require the use of 96 EcoRI + 2 primers and an MseI + 3 primer. Tail PCR is performed using an EcoRI primer with the PCR primer sequence with P5 bridge as the tail. Products are encoded using Sephadex ™ columns. Concentrations are determined and normalized and groups are created. The groups undergo sequencing

5 masiva en paralelo basándose en tecnología de Solexa que comprende amplificación de PCR por puente y secuenciación seguido de análisis de datos para determinar los genotipos de los padres y la descendencia. 5 in parallel based on Solexa technology comprising bridge PCR amplification and sequencing followed by data analysis to determine parental genotypes and offspring.

[0069] Un escenario alternativo no usa PCR de cola, pero usa cebadores de EcoRI+2 fosforilados. Debido a la falta de coincidencia con el adaptador original, la temperatura de hibridación en el perfil de pacificación se [0069] An alternative scenario does not use tail PCR, but uses phosphorylated EcoRI + 2 primers. Due to the mismatch with the original adapter, the hybridization temperature in the pacification profile is

10 reduce en 3 grados Celsius para 13 ciclos de contacto hacia abajo desde 62-53 grados Celsius seguido de 23 ciclos a 53 grados Celsius. Después de la ligación del adaptador con la secuencia de PCR con puente de P5, se realiza PCR con cebadores de PCR con puente de P5 y P7. 10 reduces by 3 degrees Celsius for 13 cycles of contact down from 62-53 degrees Celsius followed by 23 cycles at 53 degrees Celsius. After ligation of the adapter with the P5 bridge PCR sequence, PCR is performed with P5 and P7 bridge PCR primers.

[0070] Un segundo escenario alternativo se basa en la preparación de moldes convencionales como se ha [0070] A second alternative scenario is based on the preparation of conventional molds as has been

15 descrito anteriormente en el presente documento, (pre)amplificación selectiva para reducir la complejidad. La amplificación selectiva se realiza con cebadores que contienen los sitios de restricción de EcoRI y MseI reconstituidos. Esto permite la retirada de las secuencias adaptadoras antes de la secuenciación, reduciendo de este modo la cantidad de datos a analizar. Se realiza purificación de los productos con columnas de SephadexTM para retirar restos de ADN Taq polimerasa. Se preparan moldes en los que las secuencias 15 described hereinbefore, (pre) selective amplification to reduce complexity. Selective amplification is performed with primers containing the reconstituted EcoRI and MseI restriction sites. This allows the removal of the adapter sequences before sequencing, thereby reducing the amount of data to be analyzed. Purification of the products with SephadexTM columns is performed to remove Taq polymerase DNA residues. Molds are prepared in which the sequences

20 adaptadoras (sitio reconstituido) se reemplazan con adaptadores de Solexa usando un aumento de diez veces de adaptador de EcoRI y enzima de EcoRI para compensar el aumento del número de sitios de EcoRI para el ADN genómico. Los adaptadores de EcoRI de Solexa también contienen las marcas, por lo tanto, se necesitan 96 adaptadores de EcoRI de Solexa marcados. La hebra de la parte final del adaptador se bloquea en el extremo 3’ (en este caso con grupo amino 3’) para bloquear la extensión con una polimerasa. Se realiza PCR 20 adapters (reconstituted site) are replaced with Solexa adapters using a tenfold increase in EcoRI adapter and EcoRI enzyme to compensate for the increase in the number of EcoRI sites for genomic DNA. Solexa EcoRI adapters also contain the brands, therefore, 96 Solexa EcoRI adapters are needed. The strand of the end of the adapter is blocked at the 3 ’end (in this case with amino group 3’) to block the extension with a polymerase. PCR is performed

25 con cebadores de PCR con puente en P5 y P7. Los productos se purifican con columnas de Qiagen. 25 with PCR primers with bridge in P5 and P7. The products are purified with Qiagen columns.

Ejemplo 2 Example 2

[0071] Se realizó detección de fragmentos de AFLP basada en secuencias usando tecnología de Matriz de [0071] Sequence-based AFLP fragment detection was performed using Matrix technology.

30 Molécula Individual Clonal (CSMATM) de Solexa, una plataforma de Secuenciación por Síntesis capaz de analizar hasta 40 millones de fragmentos individuales en una sola ejecución de la secuencia. 30 Clonal Individual Molecule (CSMATM) of Solexa, a Synthesis Sequencing platform capable of analyzing up to 40 million individual fragments in a single sequence execution.

[0072] La secuencia experimental implica la preparación de moldes de AFLP, amplificación (AFLP) selectiva, amplificación por puente de una sola molécula y secuenciación de millones de marcas de la secuencia a partir [0072] The experimental sequence involves the preparation of AFLP molds, selective amplification (AFLP), single-molecule bridge amplification and sequencing of millions of sequence tags from

35 de un extremo de la enzima de restricción de los fragmentos de AFLP. Se usaron las líneas precursoras B73 y Mo17 y 87 de maíz. Se usaron Líneas de la Misma Estirpe Recombinantes (RILs) y se secuencia non en 8,9 millones de extremos de fragmentos de EcoRI para AFLP para proporcionar una prueba del principio para detección con AFLP basada en secuencias. 35 of one end of the restriction enzyme of the AFLP fragments. The precursor lines B73 and Mo17 and 87 of corn were used. Recombinant Strain Same Line (RILs) Lines were used and non-sequenced on 8.9 million ends of EcoRI fragments for AFLP to provide a principle test for sequence-based AFLP detection.

40 [0073] Se seleccionaron líneas precursoras B73 y Mo17 y 87 RILs. Se prepararon moldes para AFLP usando la combinación de enzima de restricción EcoRI/MseI. Se realizó amplificación selectiva usando cebadores +2/+3 para AFLP. Se prepararon fragmentos de molde para amplificación por puente de CSMA de Solexa realizando una segunda restricción/ligación usando adaptadores de EcoRI que contienen secuencias únicas de marca de identificación (ID) de la muestra de 5 pb. Se incluyeron líneas precursoras y tres muestras de RIL dos veces 40 [0073] Precursor lines B73 and Mo17 and 87 RILs were selected. Molds for AFLP were prepared using the EcoRI / MseI restriction enzyme combination. Selective amplification was performed using primers + 2 / + 3 for AFLP. Mold fragments for Solexa CSMA bridge amplification were prepared by performing a second restriction / ligation using EcoRI adapters containing unique identification tag (ID) sequences of the 5 bp sample. Precursor lines and three RIL samples were included twice

45 usando diferentes marcas de ID de la muestra de 5 pb para medir la reproducibilidad dentro del experimento. 45 using different ID tags of the 5 bp sample to measure reproducibility within the experiment.

[0074] Se identificaron marcadores de AFLP basados en secuencia mediante extracción de marcas de secuencias de 27 pb observadas a diferentes frecuencias en B73 y Mo17, que se segregan en la descendencia de RIL. [0074] Sequence-based AFLP markers were identified by extracting 27 bp sequence tags observed at different frequencies in B73 and Mo17, which are segregated in the RIL offspring.

50 [0075] Se compararon datos de marcadores de AFLP basados en secuencia con puntuaciones de marcadores de AFLP obtenidas con identificación genética de AFLP convencional usando detección basada en la longitud de las cuatro combinaciones de EcoRI/MseI +3/+3 cebadorcorrespondiente. [0075] Sequence-based AFLP marker data were compared with AFLP marker scores obtained with genetic identification of conventional AFLP using length-based detection of the four combinations of EcoRI / MseI + 3 / + 3 corresponding primer.

55 Estadística de la ejecución de la secuencia en 5 flujos celulares 55 Sequence execution statistics in 5 cell flows

[0076] [0076]

# marcas de la secuencia generadas 8.941.407 # sequence tags generated 8,941,407

# marcas de la secuencia con IDs de la muestra desconocidas 8.029.595 # sequence tags with unknown sample IDs 8,029,595

# marcas de la secuencia diferentes con IDs de la muestra conocidas 206.758 # different sequence marks with known sample IDs 206,758

# datos de la secuencia en Mbp generados por 241,4# sequence data in Mbp generated by 241.4

marcas de la secuencia por muestra total del intervalo de la frecuencia 55.374 - 112.527  sequence marks per total sample of the frequency range 55.374 - 112.527

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