Method for constructing Copepoda full-length cDNA (complementary deoxyribonucleic acid) library

Abstract

The invention discloses a method for constructing a Copepoda full-length cDNA (complementary deoxyribonucleic acid) library. The method comprises the following steps: extracting high-quality RNA (ribonucleic acid) from a small amount of Copepoda individual, and carrying out reverse transcription to obtain a first chain cDNA by using Modified oligo dT as a primer; and on the basis of the sequence of a messenger RNA (ribonucleic acid) transsplicing precursor spliced leader (SL for short) of the Copepoda, designing a forward degenerate primer Copepod SL, carrying out PCR (polymerase chain reaction) amplification for 25-35 cycles by using a Modified oligo dT upper adaptor sequence as a reverse primer and the first chain cDNA as a template, and carrying out the conventional connection-transformation-cloning process to obtain the full-length cDNA library of Copepoda. The method has the advantages of small required sample quantity and high efficiency, is simple and easy to operate, is suitable for Copepoda molecular ecology research, and facilitates and promotes the quick and accurate screening of the functional gene.

Description

[0001] technical field [0002] The invention relates to the field of biotechnology, in particular to a method for constructing a full-length cDNA library of copepods. Background technique [0003] Copepods are small crustaceans widely found in marine and freshwater environments. As an important part of secondary productivity, copepods are an important link in the food web of aquatic ecosystems, and play a connecting role between primary producers and higher trophic levels; in addition, copepods through feeding, breathing and Processes such as excretion become an important part of the microfood cycle. For oceanographers, it is a great challenge to study and predict the response of marine organisms to environmental changes on an individual and larger scale, and to understand the responses of individuals and groups at the molecular level is to explore the adaptation mechanism of organisms to environmental changes. key. Copepods are characterized by their small size, short l...

AI Technical Summary

Problems solved by technology

Conventional methods for constructing full-length cDNA libraries generally have the characteristics of large amounts of materials, time-consuming, and cumbersome steps

Due to the small size of small crustaceans such as copepods and the low RNA content of individual individuals, the conventional full-length cDNA library construction method usually requires thousands of individuals, and it is difficult to obtain a sufficient amount through field sampling.

In addition, copepods often have parasitic ciliates, fungi, algae and other organisms attached to their bodies, and copepods themselves also eat a variety of prey organisms. Conventional cDNA library construction methods cannot rule out the existence of these organisms. Contamination by Pod cDNA Library Sequences

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