Turbot fluorescence labeling microsatellite sextuple PCR family tree recognizing method

Abstract

The invention relates to a turbot fluorescence labeling microsatellite sextuple PCR family tree recognizing method which comprises the following steps: using three kinds of fluorescence (6FAM, HEX andNED) for pairwise labeling six pairs of primers according to the sizes of the PCR amplifying products of the primers; using primer design software for excluding the possibility that the six pairs ofprimers form dipolymer in the PCR reaction process, and combining the six pairs of primers in the same PCR reaction system which comprises ROX fluorescence molecule internal labels; adjusting the PCRreaction system, and establishing parameters which can enable the six pairs of primers to be simultaneously amplified in the same PCR reaction system and a proper PCR reaction procedure; and analyzingPCR products in six microsatellite positions of the turbot through ABI3130 or a similar gene analyzer according to the fluorescence labels. The sextuple PCR technology can improve the detection efficiency of the turbot microsatellite labeling, wherein the single parent excluding ratio is 99.63%, and the individual recognizing ratio is 99.99%; and the sextuple PCR technology can distinguish different family trees of the turbot or recognize and identify individuals and parent children.

Description

Technical field: [0001] The invention belongs to molecular marker auxiliary technology of genetic breeding of molecular biology, and relates to a method for identification of turbot fluorescent marker microsatellite six-fold PCR family. Background technique: [0002] Turbot (Scophthalmus maximus L.), also known as turbot, is a high-quality mariculture species native to Europe. my country introduced aquaculture in 1992 and is an important marine aquaculture object in my country. The use of molecular marker-assisted breeding is one of the important ways of turbot breeding, and the most widely used molecular marker is microsatellite. Conventional microsatellite detection technology is PCR amplification of a single site with a single pair of primers, and its products are separated and detected by polyacrylamide gel electrophoresis, which is inefficient and takes a long time. Microsatellite multiplex PCR is based on conventional PCR technology. Multiple pairs of specific micros...

AI Technical Summary

Problems solved by technology

The advantage is that triple PCR improves the efficiency; the disadvantage is that the efficiency of triple PCR is limited, and the PCR products of different target fragments are difficult to distinguish by visual inspection

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