Construction of DNA libraries and their screening

Summary

The construction and screening of DNA libraries can be used to: (1) cut the entire genomic DNA of an organism into DNA fragments of a certain length by restriction endonuclease or mechanical force, and then recombine them with suitable vectors in vitro and transform all the positive colonies obtained from the corresponding host cells. (2) Adopting the strategy of "zeroing in" to break down a large number of genes into segments, each of which contains one or a few genes.

Operation method

Construction of DNA libraries and their screening

Principle

A distinctive feature of higher plant genomes is that they contain a large number of repetitive DNA sequences, which are often located in heterochromatin regions and may be related to the structure of chromosomes. Based on the international research on genomes, chromosomes and structural proteins, Ji Jing et al. proposed that there are specific repetitive sequences in chromosomal DNA at an average interval of 30 kb or 250 nm in spirochetes, which is called YR DNA (Yielding repeat DNA).

Materials and Instruments

DNA
Ascending mercury MS medium Agarose EcoR I Hind III BamH I Bg lII Pst I
Electrophoresis Nylon membrane

Move

1. Extraction of rice DNA Rice DNA was extracted by the method of Dellaporta, Wood and Hicks (1983) with slight modification for the isolation of total genomic DNA. Molecular weight was determined by 0.8% agarose gel electrophoresis.2. Gene library construction. The rice genomic DNA was digested with restriction endonuclease γEcoRI for 12 h, and then a 0.5-8 kb fragment was recovered from the gel, ligated into γExCell EcoR I/ CIP vector, and infected with the recipient bacterium NM522 in vitro.3. Identification of gene library. Pick 50 fresh white phage spots, add 150ul of SM buffer, release the plasmid according to the instructions of γExCell kit, and identify the size of genomic DNA insert fragment by EcoRI digestion.4. Non-isotopic labeling of rice genome DNA was performed according to the probe labeling procedure of digoxigenin labeling kit.5. Phage in situ hybridization. Phage transfection and fixation were performed according to the molecular cloning method. Pre-hybridization, hybridization and high sensitivity washing of nylon membranes were performed in a hybridization oven (Biometra). Hybridization signals were detected according to the instructions of Boehringer Mannheim.6. Spot hybridization. 200 clones with strong phage in situ hybridization signals were selected for plasmid release. Refer to the instructions of γExCell kit for the plasmid release procedure. The recombinant plasmids were then extracted in small amounts by alkaline lysis. The 200 recombinant DNAs were dispensed onto a nylon membrane in order of 1ul each, fixed and then spot hybridized with Dig-labeled genomic DNA as probe. 40 clones were selected for hybridization. From them, 40 recombinants with strong hybridization signals were selected and spot hybridization was performed again.7. Southern hybridization. Ten recombinants with strong hybridization signals in the third round of spot hybridization were selected as probes. Genomic DNA was digested by five commonly used restriction endonucleases: EcoRI, H indIII, BamH I, Bg lII and PstI, and separated by electrophoresis on conventional agarose gels, and then the enzyme sections were transferred from the agarose gels to nylon membranes through an electrotransfer device for Southern hybridization.

Caveat

The method of generating DNA fragments requires a high degree of randomization of the cut points so that any gene can be cloned in its entirety, and preferably with sticky ends on both sides to facilitate ligation of the DNA fragments. Mechanical shearing has the advantage of high randomness, but the resulting fragments do not have sticky ends on both sides, which sometimes makes it more difficult to ligate to the vector. The method of endonuclease digestion by restriction endonuclease is less random, but it has sticky ends at both ends, which is convenient for ligating with the vector.

Common Problems

In order to conserve gene libraries efficiently, the number of bacteria containing each specific DNA fragment can be increased by bacterial multiplication. Liquid cultures are not suitable for this purpose because individual bacteria have different abilities to survive and reproduce, and the chances of preserving individual clones are therefore not equal. In solid media, each bacterium forms a separate colony, and the individual bacteria do not interfere or compete with each other, thus facilitating the preservation of all clones. Each colony formed contains about ¹⁰⁷ bacteria, so that almost all the clones in a gene library are amplified ^107-fold. By washing off all the bacteria from the petri dish and preserving them, any clone can be obtained from it when needed.

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