DNA fragments inserted into host nucleic acids can replicate in host organisms {cloning}|.
hosts: bacteria
Plasmids can insert up to 1000 bases. 50,000-base bacteriophage viruses can infect bacteria and can insert up to 15,000 bases. 300,000-base bacterial artificial chromosome DNA can have all bacterial-chromosome functional regions. Cosmids can hold 45,000 bases between cos sites. Gene-product secretion is preferable to harvesting cells.
In bacteria hosts, eukaryote proteins do not fold properly. Foreign proteins can kill bacteria. Bacteria have no post-translation enzymes.
hosts: yeast
Gene-product secretion is preferable to harvesting cells. In yeast hosts, proteases can destroy generated proteins.
For yeast hosts, replicating nucleic acid can be yeast artificial chromosomes.
Two-micron-circle yeast plasmid has replication origin that makes many copies per cell cycle. Other plasmids that use autonomously replicating sequence, sometimes helped by centromere sequence, make one or two copies per cell cycle. Yeast plasmids {shuttle vector} can work in bacteria.
Yeast vectors {integrating vector} with no replication origin integrate gene into yeast genome.
hosts: plants
For plant hosts, replicating nucleic acid can be Ti plasmid.
hosts: insects
For insect hosts, replicating nucleic acid can be baculovirus. Insect cell cultures have high costs. Gene-product secretion is preferable to harvesting cells.
hosts: mammals
For eukaryotic hosts, replicating nucleic acid can be virus or retrovirus. Mammalian cell culture has highest costs. Gene-product secretion is preferable to harvesting cells.
DNA fragment
DNA fragments can come from foreign organisms by cutting chromosomal DNA into DNA fragments using restriction enzymes. DNA fragments can come from mRNA by making cDNA from mRNA using reverse transcriptase and then making double-stranded DNA from cDNA. Synthesis methods can synthesize DNA.
polylinker
DNA fragments have polylinkers added at both ends, to allow nested cuts by different restriction enzymes.
insertion
DNA fragments can link into replicating nucleic acids using restriction enzymes to cut both nucleic acids and then allowing recombination.
selection
After replicating nucleic acids go into hosts, agents kill hosts if they do not have protecting genes in replicating nucleic acids. For example, bacteria with no plasmids die, because plasmids have genes to protect against antibiotics.
DNA
Host cells that live have DNA fragments, for extraction or secretion. Hybridization can test extracted or secreted DNA for DNA fragments. DNA sequencing can test for DNA fragments. Antibody binding or direct protein assays can test extractions or secretions for DNA-fragment gene products.
Biological Sciences>Genetics>Recombinant DNA>Cloning
4-Genetics-Recombinant DNA-Cloning
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Date Modified: 2022.0224