Introduction to Molecular Cloning Methods

What is Molecular Cloning?

In the broadest sense, cloning describes the method for creating exact genetic copies of cells, tissues, or animals. When applied to molecular biology, cloning refers to the creation of a DNA molecule with origins from multiple sources (i.e. recombinant DNA) to be replicated within host organisms. Through the cloning process, the host cell makes multiple copies of the DNA fragment, allowing scientists to use it in many types of molecular biology experiments.

Scientists use molecular cloning for a variety of applications, including:

• Expressing proteins at high levels for medical or research purposes
• Mutating a gene to study the effects of the mutation on the cell
• Introducing a gene into an organism to give it a specific characteristic (ex: degrading chemicals in the environment, improving growth, etc.)

Before highlighting various molecular cloning techniques available to scientists today, we’ll first look at the terminology used in molecular cloning and the general cloning process.

Common Terms in Molecular Cloning

• Insert: The piece of DNA that will be added to the cloning vector. This DNA is any DNA sequence of interest that the researcher wants to clone, amplify, and/or manipulate.
• Cloning vector (plasmid): A piece of DNA that can be maintained and replicated in an organism and often contains one or more antibiotic resistance genes and multiple cloning sites.
• Construct: An artificially-designed piece of DNA created using a cloning method and consisting of the insert and cloning vector
• Clone (clonal population): Collection of genetically identical cells (ex: bacterial colony) that have originated from a single ancestor (parent cell)
• Antibiotic resistance gene: Gene that allows bacteria to survive in the presence of a specific antibiotic
• Multiple cloning site: A short segment of DNA on the vector that contains multiple restriction sites, which help scientists introduce new genes and DNA fragments into the vector
• Host cells: Cells, commonly bacteria, used to replicate and maintain the cloned DNA

Overview of the Cloning Process

The cloning process generally involves joining insert DNA and a cloning vector together, inserting them into bacteria, identifying the bacteria that contain the correct construct, and verifying the sequence of the cloned DNA. Let’s examine the steps in more detail:

Isolate and purify DNA fragments

The exact DNA fragment(s) you need depends on your cloning strategy (discussed below). These fragments originate from PCR reactions or other isolation methods. Once created, various enzymes, such as restriction enzymes or Taq polymerase, modify the fragment ends to be compatible with the vector DNA before ligation.

Prepare vector DNA

Vector DNA can be purchased from commercial vendors or prepared using PCR or restriction digests of existing vectors.

Ligate fragments together

The ligation step uses enzymes (ex: DNA ligase) to join the insert DNA fragment(s) to the vector.

Transform into bacteria

This step introduces the ligation mixture into the bacterial host using either heat-shock or electroporation, two methods that allow DNA to cross the cell membrane. After this step, some bacteria will contain the correct clone, while others do not.

Identify correct clones

Scientists use various methods to help distinguish and/or identify the clones with the correct constructs. These methods involve growing the bacterial mixture in the presence of antibiotics so that only cells containing the vector backbone survive. Other methods use vectors with a gene that gives the bacteria a specific color (ex: blue-white screening). Inserting a DNA fragment within that gene inactivates it so scientists can visually distinguish clones where the vector contains the insert.

Isolate plasmids from individual clones

At this stage, bacterial colonies that likely contain the correct construct can be grown so scientists can harvest plasmid from them. After isolating the plasmid, it can be further analyzed using restriction digests to check whether it produces DNA fragments of the expected size. 

Verify plasmid sequence

To truly verify that the isolated construct contains the correct insert and that there are no errors, the constructs can be sequenced.

All cloning methods generally follow the above steps. The main difference between the methods occurs in how you prepare the insert and vector, the ligation step, and how to identify the correct clones. 

Choosing a Molecular Cloning Method

With a plethora of cloning methods available, it can be difficult to decide which method to use. In designing a cloning experiment consider:

• Number of inserts: Some methods, such as restriction cloning, are straightforward when inserting one fragment into the vector. However, other methods like Gibson assembly are better suited for a larger number of inserts.
• Size of insert - Is the method suitable for the size of insert? For example, inserts that are particularly small or large may not work for specific methods.
• Speed/complexity of method - Does the reaction require one step or multiple steps? Some methods also require more careful primer design than others.
• Flexibility - Do you need to move your insert to other vectors? How easy is it to subclone into another vector?
• Cost: Different cloning methods have different costs associated with them. For example: do you need to purchase multiple enzymes, proprietary reagents, or vectors? Can you clone into a vector that you currently have in the lab?
• Efficiency: Do you have to screen many clones to find the right one or repeat your experiment?

The table below serves as a general guide to help you choose a cloning strategy for your experiment. However, please note each cloning experiment can behave differently due to the sequence of the insert or whether the gene expressed may be toxic to the cell. Therefore, there may be cases where the cloning method exceeds or doesn’t meet the guidelines below.

Restriction Enzyme Cloning

Restriction enzyme cloning was the first cloning method developed and is still widely used today. This method uses restriction enzymes to cut the insert DNA and the vector at specific sites, leaving either overhangs or “sticky ends,” where one strand of DNA is longer than the other, or blunt ends, where both strands of DNA are the same length. If the sequences of the sticky ends are compatible with one another, then the vector and insert can be pieced together. Regardless whether the fragments have sticky ends or blunt ends, fragments are joined together using an enzyme called DNA ligase to create the recombinant piece of DNA (Figure 1).

This method is ideal when cloning single fragments, or a small number of fragments, into the vector.

Figure 1. Restriction enzyme cloning.

Pros and cons of restriction enzyme cloning

Pros

• A widely used and established method with many options for cloning vectors and restriction sites
• Many tools available to design restriction enzyme cloning projects

Cons 

• Can be time consuming as it is a multi-step process (digest first, then ligate)
• Need to ensure enzyme does not cut within insert sequence 
• Cut vector may recircularize on itself if it is not ligated to the insert
• If you’re creating many different plasmids for your experiment, you might need multiple restriction enzymes as one enzyme may not be suitable for all inserts

Resources for restriction cloning in Geneious Prime

➤ Restriction Cloning: Video series covering how to find and analyze restriction sites and how to simulate restriction cloning

➤ Restriction Cloning Tutorial: Written tutorial on using the restriction cloning tool in Geneious Prime

TOPO Cloning

Unlike restriction enzyme cloning, which uses a restriction enzyme and a DNA ligase, TOPO cloning accomplishes DNA cleavage and ligation with one enzyme: topoisomerase I. Topoisomerase I has two activities which make it ideal for cloning:

• It cuts DNA at the sequence 5´-(C/T)CCTT-3'
• It ligates DNA fragments when bound to the 3’ phosphate group in thymidine

Topoisomerase I enables three types of TOPO cloning:

TA cloning

TA cloning, or sticky-end TOPO cloning, uses an A overhang on the 3' end of a PCR product to place it into a TOPO vector containing a 3’ T overhang with a covalently attached topoisomerase I enzyme (Figure 2). As many high fidelity enzymes do not introduce a 3’ A overhang, use Taq polymerase to generate the PCR product. Otherwise, a short incubation with Taq after a PCR reaction with Hifi polymerase will add the necessary 3’ A overhang.

Figure 2. TA TOPO cloning.

Blunt end cloning

Inserts are created either with high-fidelity polymerases that leave blunt ends or with restriction enzymes that form blunt ends (Figure 3). When mixed with a blunt TOPO vector and topoisomerase I, ligation occurs.

Figure 3. Blunt end TOPO cloning.

Directional cloning

Insert contains a 5’ CACC overhang and a 3’ blunt end to give directionality (Figure 4). Topoisomerase I ligates the insert with a vector that contains a 5’ GTGG and a 3’ blunt end

Figure 4. Directional TOPO Cloning

Pros and cons of TOPO Cloning

Pros

• Routine process without the need to design restriction digest protocol and purchase restriction enzymes specific for the exact cloning experiment
• Can offer directionality depending on method

Cons

• Insert size limited to 2-3kb
• Limited vector choices and may not be suitable for custom vectors

Resources for TOPO cloning in Geneious Prime

➤ TOPO Cloning: Video series on simulating blunt, directional, and TA TOPO cloning in Geneious Prime

➤ TOPO Cloning Tutorial: Written tutorial with practice exercises on simulating TOPO cloning methods in Geneious Prime

Gateway Cloning

Gateway cloning takes advantage of site-specific recombination enzymes that recognize specific sequences on DNA to swap out the fragments between two strands (i.e. vector and insert). These sequences are known as recombination sites and the sites used in Gateway cloning are called attB, attP, attL, and attR.

Gateway cloning proceeds in two steps (Figure 5):

1. BP reaction: The BP reaction generates the entry clone based on recombination of attB and attP sites on fragment and donor vector. The enzyme BP clonase catalyzes this reaction and creates the entry clone containing the DNA fragment with attL recombination sites.
2. LR reaction: The LR reaction generates the expression clone via recombination of the attL site of the entry clone and the attR site of the destination vector. The enzyme LR clonase catalyzes this reaction to move the insert DNA into the destination vector.

Figure 5. Gateway cloning BP and LR reactions.

Gateway cloning is particularly useful if you need to move your insert between multiple destination vectors. To do this, you’d create one entry clone using the BP reaction and then use that clone to create multiple expression clones using the LR reaction with different destination vectors. Gateway cloning can also assemble multiple fragments in one tube by taking advantage of the specificity of the recombination sites. For example, attB1 only recombines with attP1, so designing inserts with the recombination sites in a specific order can give your construct directionality.

Pros and cons of Gateway Cloning

Pros

• Faster than restriction cloning (BP and LR reactions can be done in one step)
• Libraries of cloned and sequenced gene fragments available for purchase

Cons

• Assembles only up to four fragments
• Scars are left in plasmids
• Can be more difficult to execute

Resources for Gateway cloning in Geneious Prime

➤ Gateway Cloning: Video series describing how to clone one or more fragment with Gateway cloning.

➤ Gateway Cloning Tutorial: Written guide with practice exercises on simulating Gateway cloning in Geneious Prime.

Gibson Cloning

Gibson assembly uses T5 exonuclease, Phusion polymerase, and Taq ligase to assemble up to 15 overlapping DNA fragments (Figure 6). These fragments are created using PCR with primers that contain both template specific sequences and sequences that overlap with the adjacent fragment (either another insert or the vector) in the final construct. Before assembly begins, the vector is linearized to produce overhangs using either restriction digest or inverse PCR.

Gibson assembly begins with T5 exonuclease chewing back the 5’ ends of the DNA to create overhangs. When compatible, overhangs from two fragments bind to one another and DNA polymerase extends exposed DNA. Finally, DNA ligase repairs the nicks in between the fragments.

Figure 6. Gibson assembly of two DNA fragments with overlapping regions.

Pros and cons of Gibson assembly

Pros

• A “one pot” assembly
• Fast
• Can be used to join up to 15 fragments (but success rate decreases over 5 fragments)

Cons 

• Can be more expensive than other methods
• Not suitable for small fragments (<200 bp)

Resources for Gibson assembly in Geneious Prime

➤ Gibson Assembly: Video on how to perform gibson assembly in Geneious Prime.

➤ Gibson Assembly Tutorial: Practice exercises on simulating single fragment or multiple fragment Gibson assembly in Geneious Prime.

In-Fusion Cloning

In-Fusion cloning is similar to Gibson assembly methods in that a linearized vector is mixed with overlapping PCR product(s). The difference: In-Fusion cloning uses one enzyme instead of three. This cloning method takes advantage of the DNA polymerase from the vaccinia virus, which contains a 3’ -> 5’ exonuclease activity. During cloning, the In-Fusion enzyme first chews back the 3’ ends of the fragments to create overhangs. This complex is transformed into E. coli where it is subsequently ligated.

In-Fusion cloning has many advantages as it’s suitable when you need scarless assembly, directional cloning, and the capacity to insert multiple fragments at once.

Figure 7. In-Fusion cloning uses In-Fusion enzymes to chew back DNA ends before they are repaired and ligated in E. coli.

Pros and cons of In-Fusion Cloning

Pros

• No restriction enzymes or ligase required
• Directional cloning
• Seamless cloning
• Fast

Cons

• Requires careful primer design
• Any secondary structure in the overlapping regions can be difficult to deal with

Resources for In-Fusion cloning in Geneious Prime

➤ In-Fusion Cloning: Video on how In-Fusion cloning works and how to perform In-Fusion cloning in Geneious Prime.

Golden Gate Cloning

Golden Gate cloning uses Type IIS restriction enzymes that cleave DNA outside of the recognition site. Different Type IIS restriction enzymes cut the DNA at different distances from the recognition site and thus create overhangs with different lengths. As the cut site is not sequence dependent, scientists can create overhangs with any possible sequence. This has two advantages: (1) scientists can design constructs with multiple overlapping inserts and (2) destination vectors won’t circularize on itself because their sticky ends are incompatible. These features allow Golden Gate cloning to proceed in just one step where the digestion and ligation occurs within one mixture.

Figure 8. Golden Gate cloning eliminates the Type IIS restriction sites from the final product.

Pros and cons of Golden Gate cloning

Pros 

• One step, one pot process in less than 30 min
• Assemble multiple fragments
• Destination vector cannot recircularize after digestion

Cons

• Type IIS recognition sites cannot be present in the insert. If the insert has the recognition site, eliminate it by introducing a point mutation before cloning.

Resources for Golden Gate cloning in Geneious Prime

➤ Golden Gate Cloning: Video series on simulating Golden Gate cloning in Geneious Prime.

➤ Golden Gate Cloning Tutorial: Written exercises on simulating Golden Gate cloning in Geneious Prime.

Parts Cloning (Combinatorial Cloning)

Parts cloning, or combinatorial cloning, uses libraries of genetic elements (ex: promoters, coding regions, terminators, etc.) and various cloning techniques (ex: Golden Gate, Gateway, etc.) to create multi-part constructs. Synthetic biology and engineering applications use parts cloning to create biological systems from standardized, modular components. Thus, parts cloning can only occur when using well characterized or standardized components that are found within a modular toolkit of parts.

Pros and cons of parts cloning

Pros

• Easy to mix and match modules
• Parts are well characterized so how they will behave in the construct is predictable

Cons

• Only available for standardized parts
• May be difficult to integrate non-modular parts
• Cost associated with obtaining the modular parts toolkit

Resources for parts cloning in Geneious Prime

➤ Parts Cloning: Video on simulating parts cloning and concatenate libraries of genetic elements outputting all possible combinations.