Basic Process of Molecular Cloning
Molecular cloning is a method to purify and expand specific DNA fragments at the molecular level. Use the in-vitro recombination method to insert the target gene into a cloning vector to form a recombinant cloning vector. Introduce it into a suitable recipient cell and make it be replicated and amplified through transformation or transduction. Then separate and purify the required cloning vector from the screened recipient cells to obtain many copies of inserted DNA. It enables the target gene to be amplified and the host cell to acquire new genetic characteristics.
Insert DNA fragment
The DNA fragment you want to clone is also called Insert. It may come from a prokaryotic cell or a eukaryotic cell, genomic DNA, a part of another plasmid or a linear DNA fragment, a DNA fragment synthesized or a cDNA produced by reverse transcription of mRNA. When we insert DNA fragments, the first step is restriction enzyme digestion to generate matching sticky ends for subsequent insertion of the fragment into the vector.
Vector
The vector is a tool that can carry foreign genes (or DNA fragments) into cells for replication, integration or expression. The vector is generally a plasmid, but it can also be a phage.
Conditions of a cloning vector:
Ø It is transferable to
recipient cells and can carry exogenous genes into host cells.
Ø It can replicate autonomously in host cells and realize the proliferation of exogenous genes.
Ø It has a multiple cloning
site (MCS) composed of sites recognized by a single restriction enzyme.
Ø It has marker genes for
selecting clones, such as antibiotic resistance. If the host integrates the
plasmid, it will survive in the antibiotic-containing medium. So that we select
cells that have been successfully transformed.
Ø The cloning vector must be safe and contain no genes harmful to the recipient cells. And it will not be arbitrarily transferred to cells of other organisms, especially human cells.
Traditional Cloning Workflow
（credit: neb.com）
PCR
PCR is generally the first step of molecular cloning to obtain vast target genes. PCR is consists of three basic reaction steps: denaturation - annealing - extension. The template DNA, primers, deoxynucleotides, DNA polymerase, and buffer are basic components of the PCR reaction.
Digestion and Ligation
A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.
A diagram of restriction enzymes and DNA ligase (Credit: khanacademy.org)
Our goal in cloning is to insert a target gene (e.g., for human insulin) into a plasmid. Using a carefully chosen restriction enzyme, we digest:
l The plasmid, which has a
single cut site
l The target gene fragment, which has a cut site near each end
Then, we combine the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene.
Diagram depicting
restriction digestion and ligation in a simplified schematic (Credit: khanacademy.org)
We
start with a circular bacterial plasmid and a target gene. On the two ends of
the target gene are restriction sites, or DNA sequences recognized by a
particular restriction enzyme. In the plasmid, there is also a restriction site
recognized by that same enzyme, right after a promoter that will drive
expression in bacteria.
Both the plasmid and the target gene are (separately) digested with the restriction enzyme. The fragments are purified and combined. They have matching "sticky ends," or single-stranded DNA overhangs, so they can stick together.
The enzyme DNA ligase joins the fragments with matching ends together to form a single, unbroken molecule of DNA. This produces a recombinant plasmid that contains the target gene.
Transformation
Plasmids and other DNA can be introduced into bacteria, such as the harmless E. coli used in labs, in a process called transformation. During transformation, specially prepared bacterial cells are given a shock (such as high temperature) that encourages them to take up foreign DNA.
(Credit: khanacademy.org)
A plasmid typically contains an antibiotic resistance gene, which allows bacteria to survive in the presence of a specific antibiotic. Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic. Bacteria without a plasmid will die, while bacteria carrying a plasmid can live and reproduce. Each surviving bacterium will give rise to a small, dot-like group, or colony, of identical bacteria that all carry the same plasmid.
(Credit: khanacademy.org)
Not all colonies will necessarily contain the right plasmid. That’s because, during a ligation, DNA fragments don’t always get “pasted” in exactly the way we intend. Instead, we must collect DNA from several colonies and see whether each one contain the right plasmid. Methods like restriction enzyme digestion and PCR are commonly used to check the plasmids.
Reference
1. Overview: DNA cloning
https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/overview-dna-cloning
2. Foundations of Molecular Cloning - Past, Present and Future
https://international.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future
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