# Multiple cloning site

A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites—a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of an MCS in a plasmid is to allow a piece of DNA to be inserted into that region.
An MCS is found in a variety of vectors, including cloning vectors to increase the number of copies of target DNA, and in expression vectors to create a protein product. In expression vectors, the MCS is located downstream of the promoter.

## Creating a multiple cloning site

In some instances, a vector may not contain an MCS. Rather, an MCS can be added to a vector. The first step is designing complementary oligonucleotide sequences that contain restriction enzyme sites along with additional bases on the end that are complementary to the vector after digesting. Then the oligonucleotide sequences can be annealed and ligated into the digested and purified vector. The digested vector is cut with a restriction enzyme that complements the oligonucleotide insert overhangs. After ligation, transform the vector into bacteria and verify the insert by sequencing. This method can also be used to add new restriction sites to a multiple cloning site.

## Uses

Multiple cloning sites are a feature that allows for the insertion of foreign DNA without disrupting the rest of the plasmid which makes it extremely useful in biotechnology, bioengineering, and molecular genetics. MCS can aid in making transgenic organisms, more commonly known as a genetically modified organism (GMO) using genetic engineering. To take advantage of the MCS in genetic engineering, a gene of interest has to be added to the vector during production when the MCS is cut open. After the MCS is made and ligated it will include the gene of interest and can be amplified to increase gene copy number in a bacterium-host. After the bacterium replicates, the gene of interest can be extracted out of the bacterium. In some instances, an expression vector can be used to create a protein product. After the products are isolated, they have a wide variety of uses such as the production of insulin, the creation of vaccines, production of antibiotics, and creation of gene therapies.

## Structural features in vector types

MCSs have distinct structural features depending on the type of vector in which they are used.
In cloning vectors, MCSs are often embedded within a selection marker, such as the lacZα gene in pUC vectors. This design enables efficient identification of recombinant plasmids, as the insertion of foreign DNA into the MCS disrupts the marker gene, allowing for blue-white screening or other selection methods.
In expression vectors, MCSs are positioned between a promoter and a terminator to regulate gene expression. The upstream promoter can be either constitutive or inducible, responding to specific chemical inducers, while the downstream terminator ensures proper transcriptional termination and enhances plasmid stability.
In reporter vectors, an MCS is typically located near a reporter gene, such as a fluorescent protein (GFP), luciferase, or lacZ. This configuration allows for the insertion of promoter sequences into the MCS, facilitating studies on promoter activity and gene regulation by measuring reporter gene expression.

## Historical Background
### Early Developments of Cloning Vectors

In 1973, Stanley N. Cohen and Herbert W. Boyer conducted experiments demonstrating that genes from one organism could be transferred into bacterial cells and expressed. They utilized the plasmid pSC101, a naturally occurring plasmid from the bacterial species Salmonella panama, which they engineered to include a single EcoRI restriction site and a tetracycline resistance gene. The pSC101 plasmid did not contain a formal multiple cloning site, but its design highlighted the utility of restriction sites for gene insertion, laying the groundwork for the later development of dedicated MCS regions in vectors.

### Introduction of pBR322 and Enhanced Cloning Sites

Building upon previous work, in 1977, researchers Francisco Bolivar and Raymond L. Rodriguez, under the guidance of Herbert W. Boyer, developed the pBR322 plasmid. Although it did not harbor a formal multiple cloning site, this plasmid was one of the first vectors to incorporate multiple unique restriction sites. These sites were strategically placed within its sequence, including within antibiotic resistance genes, enabling insertional inactivation for the identification of recombinant clones. This design allowed for greater flexibility in inserting foreign DNA fragments and marked an important step towards the development of dedicated MCS regions in future vectors, such as the pUC series.

### Development of pUC Vectors and Formalization of MCS

In 1982, Jeffrey Vieira and Joachim Messing introduced the pUC series of plasmids, derived from M13mp7. These vectors included a designated region containing numerous unique restriction sites, formally establishing the concept of the MCS. The pUC vectors allowed for efficient sequencing and cloning using universal primers, significantly advancing molecular cloning techniques.

## Modern design and optimization of the MCS

Modern advancements in multiple cloning site design have enhanced cloning efficiency, flexibility, and convenience, contributing to their widespread use in molecular biology.

### Design of MCS

Strategic selection and arrangement of restriction sites within MCSs maximize flexibility and compatibility, reducing potential cloning complications. Furthermore allowing greater workability and versatility to accommodate many experiments and applications. Inside of an MCS, the incorporation of multiple unique restriction sites in tandem allow for versatile enzyme selection. This arrangement enables precise enzymatic cuts at specific locations, facilitating targeted modifications of MCSs for various applications. Commonly used plasmids, such as pUC19 and other pUC vectors, incorporate strategically positioned restriction sites to facilitate efficient cloning, further enhancing MCS versatility in molecular applications.

### Reduction of unwanted restriction sites

Advancements in bioinformatics and molecular techniques enable identification and elimination of undesirable restriction sites, streamlining the cloning process. Bioinformatic tools assist in scanning and identifying unwanted restriction sites within the gene of interest or vector backbone. These sites can cause significant variation in protein expression depending on their position, with some leading to dramatic reductions in expression when present within the MCS. By targeting and removing these problematic sites, researchers can optimize MCSs to ensure consistent and efficient protein expression.

### Modularity and flexibility

Modern MCSs are designed with modularity in mind, allowing for seamless integration and exchange to genetic elements, which is particularly beneficial in synthetic biology applications. Standardized flanking genetic sequences in MCS design enable easy swapping of genetic components. This modular approach supports the rapid construction and customization of vectors for specific research needs. The MoClo system enables the efficiency assembly of DNA fragments into multigene constructs by utilizing a modular approach. This method allows for seamless joining of DNA fragments (without unwanted sequences) making the process both size-effective and efficient. For example, the MoClo system can be used to efficiently join two genes, such as a promoter and a coding sequence, each sourced from different MSCs, into a single multigene construct without introducing unwanted sequences.

### Compatibility with advanced cloning techniques

Contemporary MCSs are optimized for compatibility with advanced cloning methods, enhancing the efficiency and precision of genetic manipulations. Modern MCS designs accommodate techniques such as Gibson Assembly and Golden Gate cloning, which offer advantages over traditional restriction enzyme-based methods, including the simultaneous assembly of multiple DNA fragments. This compatibility enhances the versatility and effectiveness of the MCS region. This compatibility is crucial for synthetic biology and genetic engineering, where efficient and precise assembly of multiple genetic elements is essential for constructing complex genetic circuits or metabolic pathways.

### Optimizing sequence context

Attention to sequence context surrounding MCSs ensures efficient cloning and accurate gene expression. Minimizing secondary structures that can form between DNA elements within the MCS (such as promoters and open reading frames) prevents interference with restriction enzyme activity and optimizes MCS function. For instance, secondary structures in the 5' untranslated region can hinder ribosome binding, reducing translation efficiency. Additionally, the careful placement of stop codons and maintenance of reading frames help prevent unintended mutations or translational errors, such as premature stop codons that could truncate the protein product, further enhancing MCS reliability.

### Challenges and limitations

Despite optimizations and advancements, certain challenges persist in MCS design, necessitating ongoing research and innovation. One issue is the presence of internal restriction sites within genes or vectors, which can interfere with restriction enzyme activity and cloning efficiency. Additionally, the structural context of MCSs may introduce unintended regulatory effects. For instance, MCSs positioned too far from the promoter regions can lead to the formation of secondary structures, impacting the expression of a gene inside of the MCS. Furthermore, variability in sequence context (such as differences in promoter proximity or adjacent regulatory elements) can result in inconsistent gene expression. These inconsistencies arise because variations in surrounding sequences may influence transcription efficiency, mRNA stability, or translation initiation. To minimize such variability, further innovations are needed to develop standardized MCS designs that ensure consistent performance across diverse genetic constructs.

## Example

One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18. Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster (the polylinker). It has restriction sites for various restriction enzymes, including EcoRI, BamHI, and PstI. Another vector used in genetic engineering is pUC19, which is similar to pUC18, but its polylinker region is reversed. E.coli is also commonly used as the bacterial host because of the availability, quick growth rate, and versatility. An example of a plasmid cloning vector which modifies the inserted protein is pFUSE-Fc plasmid.
In order to genetically engineer insulin, the first step is to cut the MCS in the plasmid being used. Once the MCS is cut, the gene for human insulin can be added making the plasmid genetically modified. After that, the genetically modified plasmid is put into the bacterial host and allowed to divide. To make the large supply that is demanded, the host cells are put into a large fermentation tank that is an optimal environment for the host. The process is finished by filtering out the insulin from the host. Purification can then take place so the insulin can be packaged and distributed to individuals with diabetes.