Preparation method of capture probe of large-fragment genome as well as kit

Abstract

The invention provides a preparation method of a capture probe of a large-fragment genome. The preparation method comprises the following steps: designing a PCR primer and performing PCR reaction amplification on a to-be-captured genome; mixing PCR products generated by the PCR reaction into a mixture and crushing the mixture into regulated fragments; connecting joints containing RNA polymerase identification sequences at the two ends of the regulated fragments to construct a library; transcribing the fragments in the obtained library into RNA products by using RNA polymerase; adding DNA digestive enzyme and digesting DNA chains in the RNA products; and performing purification treatment on the digested products to obtain the captured gene probe. Amplification products of the to-be-capturedarea gene are crushed into the regulated fragments, and the gene probe (RNA probe) with proper length is obtained through the subsequent reaction. The RNA with the length range is not liable to selfannealing to form a secondary structure, and the joint efficiency is high when the probe is used. Therefore, higher fault tolerance is achieved while the specificity is guaranteed, and higher sensitivity is achieved.

Description

technical field [0001] The present invention relates to the preparation and processing of biological samples, and more specifically, to a preparation method and a kit of a capture probe of a large fragment genome. Background technique [0002] Since the birth of high-throughput sequencing technology in 2005, it has shown broad application prospects. Through sequencing, scientists can see important information such as single nucleotide polymorphisms, mutation hotspots, genome structural variations, and population polymorphisms. In many studies, it is not necessary to sequence the whole genome, and the data requirements for whole genome sequencing are large, which is not suitable for all application fields. Therefore, in the field of biochemical methods, many capture methods for target region sequencing have been produced, among which two methods are mainly probe capture and multiplex PCR. Probe capture is suitable for making long and large genomic regions, while multiplex P...

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Problems solved by technology

However, some of these RNA probes are too long to self-anneal to form secondary structures in an excessive environment, which reduces the binding efficiency of the probes during use.

However, in the above-mentioned patent documents, it is necessary to construct a vector for the amplified product, and then use the constructed cloning vector as an object to prepare gene probes, which is not suitable for capturing a large number of regions.

[0005] In addition, there are also some documents disclosing other gene probe preparation methods, including the following: (1) directly adding adapters after PCR, which is also a small region fragment, and does not remove residual DNA after transcription; (2) requires Synthetic DNA probes, so the synthesis cost will be higher, and unidirectional amplification is required when preparing single strands. This amplification method belongs to linear amplification, and the efficiency is usually poor; (3) Preparation of single strands requires multiple experiments. Exonuclease hydrolysis, many steps, will produce certain deviations, and the enzyme efficiency will also affect the quality of probe preparation

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