Information about Cloning Methods

Restriction Digest Approaches:
1.  Standard RE Digests 
2.  CpoI-based Directional Cloning

Recombinational Approaches:
1.  Cre/Lox (P1 phage) approach (e.g. Clontech  Creator System)
2.  Recombinational Approaches, att lambda phage  approach (e.g.  Invitrogen  Gateway System)

Bacterial Mating Approaches: 
MAGIC approach (Elledge Lab, HMS)

 

Standard RE Digests

Description
In standard restriction enzyme (RE) cloning, specific enzymes are used to cleave DNA at specific DNA sequences, and fragments with compatible ends (or blunt-ended fragments) are ligated to one another to form a circular plasmid, which can be transformed and propagated in bacteria. The method can be used to add, remove or otherwise manipulate DNA sequences.

Learn More
To learn more about this method, please consult a molecular biology textbook or protocol manual.

Advantages
A significant advantage of RE cloning is that because it has been in use for many years, reagents and protocols are readily available to most researchers.

In PlasmID
Virtually any plasmid can be manipulated using restriction enzyme cloning methods. Particularly useful are those plasmids that contain a multiple cloning site (MCS), also called a polylinker.

CpoI-based Directional Cloning

Description
The CpoI enzyme (available from Fermentas and other vendors) recognizes the sequence cggtccg or cggaccg. The enzyme cuts after the first cg, leaving a three base-pair three-prime overhang. These properties make it possible to use CpoI to clone specially designed PCR-amplified fragments in-frame and directionally into a CpoI-digested vector. The CpoI approach can be used to add inserts to a vector with aCpoI cloning site.

Learn More
To learn more about this method, see papers that use the method, such as Petrucco et al. (2002) "A Nick-sensing 3' DNA Repair Enzyme from Arabidopsis" (PMID: 11948185), Izumiya et al. (2003) "Cell cycle regulation ..." (PMID: 12915577 ) or Izumiya et al. (2005) "Kaposi's sarcoma-associated ..." (PMID: 16014952 ). In addition, it is helpful to draw out the vector and PCR product ends to see how the method can work.

Advantages
Significant advantages of the CpoI-based directional cloning method over conventional restriction digest methods include (1) as the overhang is three base-pairs, it's easy to keep things in-frame, and (2) a single enzyme can be used in a directional approach (most directional approaches require that one end is cut with one restriction enzyme and the other end is cut with a different restriction enzyme).

In PlasmID
Several plasmids shared with the repository by J. Kamil (Laboratory of D. Coen, BCMP Department at Harvard Medical School) that are useful for adding N-terminal and C-terminal tags (e.g. 6xHis, T7 eptiope tags). Try "advanced search" with "Kamil" as author or try "search by vector" and choose "CpoI-based cloning" as the cloning method.

Cre/Lox (P1 phage) approach (e.g. Clontech Creator System

Description
Clontech's Creator (TM) system and other LoxP site-containing plasmids can be used to add, replace or otherwise manipulate DNA using the Cre enzyme, which catalyzes site-specific recombination at LoxP sites. Please note that not every vector containing LoxP is appropriate for Cre/Lox-based cloning approaches. Sometimes the LoxP is present to make possible some experimental approach that is unrelated to cloning (for example, LoxP-containing constructs are sometime introduced into cells and then in vivo expression of Cre is used to bring about changes).

Learn More
To learn more about this method, please see the Clontech website www.clontech.com and appropriate references, which include Hoess et al. (1982) "P1 site-specific recombination: nucleotide sequence of the recombining sites" (PubMed ID 6954485.).

Advantages
A significant advantage of the system is that an insert can be cloned into a single 'master' or 'entry' vector and readily sub-cloned into many different expression vectors without the need to digest with restriction enzymes, gel purify, ligate, etc.

In PlasmID
The many sequence-verified open reading frame (ORF) or cDNA clones in pDNR-Dual shared with the repository by the Harvard Institute of Proteomics (HIP) at Harvard Medical School. Some of these clones are "closed" format such that the normal stop codon is present and others are "fusion" format in which the stop codon is replaced by a leu codon so that a C-terminal tag can be added.

att Lambda Phage Recombination approach (e.g. Invitrogen Gateway System)

Description
Invitrogen's Gateway (TM) system and other lambda phage (att) recombination site-containing plasmids can be used to add, replace or otherwise manipulate DNA using an enzyme that catalyzes site-specific recombination at att-type sites.

Learn More
To learn more about this method, please see the Invitrogen website www.invitrogen.com and appropriate references, which include Landy (1989) "Dynamic, structural and regulatory aspects of lambda site-specific recombination" (PubMed ID 2528323).

Advantages
Similar to the Cre/Lox approach, a significant advantage of the system is that an insert can be cloned into a single 'master' or 'entry' vector and readily sub-cloned into many different expression vectors without the need to digest with restriction enzymes, gel purify, ligate, etc.

In PlasmID
Examples include the very many sequence-verified open reading frame (ORF) or cDNA clones in pDONR201 or pDONR221 shared with the repository by the Harvard Institute of Proteomics (HIP) at Harvard Medical School. Some of these clones are "closed" format such that the normal stop codon is present and others are "fusion" format in which the stop codon is replaced by a leu codon so that a C-terminal tag can be added.

Bacterial Mating Approach (e.g. MAGIC, Elledge Lab, HMS)

Description
"Mating-Assisted Genetically Integrated Cloning" or MAGIC was developed by Li & Elledge (HHMI/Harvard Medical School) and relies on in vivo DNA cleavage and homologous recombination events that occur during bacterial mating to combine specifically designed 'donor' and 'recipient' plasmids (e.g. insert-containing donors and specific recipient expression vectors).

Learn More
To learn more about this method, please see Li & Elledge (2005) "MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules" (PubMed ID 15731760).

Advantages
A significant advantage of the method is that the bacterial do the work of combining inserts and vectors and thus, constructs can be generated without the need for restriction enzyme digest, gel purification, etc. or use of recombination-promoting enzymes.

In PlasmID
Examples include the set of pPRIME-(marker)-recipient clones for shRNA-based approaches that was shared with the repository by the Elledge lab (HHMI/Harvard Medical School).