Method for extensively screening scallop SNP

A large-scale, screening technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of low separation efficiency of whole genome sequencing, and achieve the effect of excellent effect, safe and reliable principle, and reliable operation.

Inactive Publication Date: 2010-09-29
OCEAN UNIV OF CHINA
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Problems solved by technology

However, due to the large genome of scallop with 1.2G and many repetitive sequences, the separation efficiency of whole genome sequencing is relatively low. Analysis software is used to screen SNP markers. On the one hand, it avoids repetitive sequences and improves screening efficiency; on the other hand, it can

Method used

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Embodiment

[0030] The specific operation steps of this embodiment are as follows:

[0031] 1. Use guanidinium isothiocyanate, phenol, and chloroform to extract individual RNA from the blastula, gastrula, trochophore, D-shaped larva, and adult shellfish tissues of Chlamys farreri, and mix them in proportion to measure them by UV spectrophotometry. meter to measure its concentration for later use;

[0032] 2. Construction of full-length cDNA samples of Chlamys farreri:

[0033] Refer to the SMARTTM cDNA Library Construction Kit, but the sequence of the cDNA synthesis primer needs to be replaced with 5'-AAGCAGTGGTATCAACGCAGAGTCGCAGTCGGTACTTTTTCTTTTTTV-3'.

[0034] (1) cDNA first-strand synthesis:

[0035] For the reverse transcription reaction, use 1ug RNA as a template, add RNase-Free water to 4ul, add 1ul 10uM cDNA synthesis primers, 1ul 10uM SMART IVTM Oligonucleotide (5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3'), incubate at 70°C for 3min, and place on ice immediately Cool for 1 min; then a...

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Abstract

The invention belongs to a method for extensively screening scallop SNP in the technical field of the scallop DNA molecular genetic marker, which comprises the following steps that: solution D and phenol-chloroform are firstly used for extracting a plurality of Chlamys farreri RNA, and a ultraviolet specrophotometer is used for measuring the concentration of the mixture of solution D and the phenol-chloroform which are uniformly mixed; a scallop full-length cDNA sample is re-established and comprises the synthesis of a cDNA first chain and the synthesis and augmentation of a cDNA second chain; then homogenization of full-length cDNA is performed, and ultrasonic breaking is performed; then sequence measuring joints are connected, and terminal repair, joint connection and sample augmentation are included; finally a biological software is used for analyze the data to obtain an SNP marker; then an SNP marker to be screened is selected to design a primer; and DNA of individual scallop is extracted to be equivalently mixed to be used as a template, conventional colony sequence measuring is undertaken after the PCR augmentation, and the position point with the occurring frequency of single base difference being 10 percent or more is defined as an SNP marker. The method has safe and reliable principle, simple and controllable flow procedures, reliable running of large-scale screening, excellent effect and strong practicability.

Description

Technical field: [0001] The invention belongs to the technical field of scallop DNA molecular genetic markers, and relates to a method for large-scale screening of scallop SNP markers. Background technique: [0002] Due to their large number and stable heritability, SNP markers are widely used in the construction of high-density genetic linkage maps and the QTL mapping of trait-related functional genes. As one of the most important marine economically cultured shellfish in my country, scallop occupies an extremely important position in the marine aquaculture industry. Large-scale screening of scallop SNP markers is crucial for the construction of high-density genetic maps of scallops, the fine mapping of QTLs related to economic traits, and further molecular design breeding. At present, large-scale screening of SNP markers is widely used in humans and model organisms with rich genomic information. However, no efficient and low-cost large-scale screening methods have been re...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 包振民王珊胡景杰胡晓丽李纪勤侯睿付晓腾陆维
Owner OCEAN UNIV OF CHINA
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