In molecular biology

Molecular biology

Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

, subcloning is a technique used to move a particular gene

Gene

A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...

 of interest from a parent vector

Vector (molecular biology)

In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...

to a destination vector in order to further study its functionality.

Subcloning is not to be confused with molecular cloning

Molecular cloning

Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...

, a related technique.

Procedure

Restriction enzymes are used to excise the gene of interest (the insert) from the parent. The insert is purified in order to isolate it from background junk. A common purification method is gel isolation. The number of copies of the gene is then amplified using Polymerase Chain Reaction

Polymerase chain reaction

The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....

 (PCR).

Simultaneously, the same restriction enzymes are used to digest (cut) the destination. The idea behind using the same restriction enzymes is to create complementary sticky ends, which will facilitate ligation

DNA ligase

In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...

 later on. A phosphatase

Phosphatase

A phosphatase is an enzyme that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group . This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their...

 (commonly Calf Intestinal Alkaline Phosphatase

Alkaline phosphatase

Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...

; CIAP) is also added to prevent self-ligation of the destination vector. The digested destination vector is isolated/purified.

The insert and the destination vector are then mixed together with DNA ligase. A typical ratio of insert genes to destination vectors is 3:1 ; by increasing the insert concentration, self-ligation is further decreased. After letting the reaction mixture sit for a set amount of time at a specific temperature (dependent upon the size of the strands being ligated; for more information see DNA ligase), the insert should become successfully incorporated into the destination plasmid

Plasmid

In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...

.

Amplification of product plasmid

The plasmid is often transformed

Transformation (genetics)

In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...

 into a bacterium

Bacteria

Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...

 like E. coli. Ideally when the bacterium divides the plasmid should also be replicated. In the best case scenario, each bacteria cell should have several copies of the plasmid. After a good number of bacterial colonies have grown, they can be miniprepped

Plasmid preparation

A plasmid preparation is a method used to extract and purify plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria...

 to harvest the plasmid DNA.

Selection

In order to ensure growth of only transformed bacteria (which carry the desired plasmids to be harvested), a marker gene

Marker gene

A marker gene is a gene used in molecular biology to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. There are two types of marker genes: a selectable marker and a marker for screening.-Selectable marker:...

 is used in the destination vector for selection

Genetic screen

A genetic screen is a procedure or test to identify and select individuals who possess a phenotype of interest. A genetic screen for new genes is often referred to as forward genetics as opposed to reverse genetics, the term for identifying mutant alleles in genes that are already known...

. Typical marker genes are for antibiotic resistance

Antibiotic resistance

Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic. While a spontaneous or induced genetic mutation in bacteria may confer resistance to antimicrobial drugs, genes that confer resistance can be transferred between bacteria in a...

 or nutrient biosynthesis

Biosynthesis

Biosynthesis is an enzyme-catalyzed process in cells of living organisms by which substrates are converted to more complex products. The biosynthesis process often consists of several enzymatic steps in which the product of one step is used as substrate in the following step...

. So, for example, the "marker gene" could be for resistance to the antibiotic ampicillin. If the bacteria that were supposed to pick up the desired plasmid had picked up the desired gene then they would also contain the "marker gene". Now the bacteria that picked up the plasmid would be able to grow in ampicillin whereas the bacteria that did not pick up the desired plasmid would still be vulnerable to destruction by the ampicillin. Therefore, successfully transformed bacteria would be "selected."

Example Case: Bacterial plasmid subcloning

In this example, a gene from mammalian gene library will be subcloned into a bacterial plasmid (destination platform). The bacterial plasmid is a piece of circular DNA which contains regulatory elements allowing for the bacteria to produce a gene product (gene expression

Gene expression

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

) if it is placed in the correct place in the plasmid. The production site is flanked by two restriction enzyme cutting sites "A" and "B" with incompatible sticky ends.

The mammalian DNA does not come with these restriction sites, so they are built in by overlap extension PCR

Overlap extension polymerase chain reaction

The overlap extension polymerase chain reaction is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension PCR...

. The primers are designed to put the restriction sites carefully, so that the coding of the protein is in-frame, and a minimum of extra amino acids is implanted on either side of the protein.

Both the PCR product containing the mammalian gene with the new restriction sites and the destination plasmid are subjected to restriction digestion, and the digest products are purified by gel electrophoresis

Gel electrophoresis

Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

.

The digest products, now containing compatible sticky ends with each other (but incompatible sticky ends with themselves) are subjected to ligation, creating a new plasmid which contains the background elements of the original plasmid with a different insert.

The plasmid is transformed

Transformation (genetics)

In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...

 into bacteria and is checked by DNA sequencing

DNA sequencing

DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....

.